clinical chemistry and laboratory medicine

2010·VOLUME 48·SUPPLEMENT 1 ISSN 1437-8523 · e-ISSN 1437-4331

CLINICAL CHEMISTRY AND LABORATORY MEDICINE2010 · VOLUME 48 · SUPPLEMENT 1 · PP. S1-S128

CLINICAL CHEMISTRY AND LABORATORY MEDICINE 10TH EFCC CONTINUOUS POSTGRADUATE COURSE IN CLINICAL CHEMISTRY: ‘‘NEW TRENDS IN CLASSIFICATION, DIAGNOSIS AND MANAGEMENT OF THROMBOPHILIA’’, OCTOBER 23-24, 2010, DUBROVNIK, CROATIA Edited by Ana-Maria Simundic, László Muszbek, Elizabeta Topic and Andrea Rita Horvath

EDITOR-IN-CHIEF

Mario Plebani, Padova

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Clinical Chemistry and Laboratory Medicine Published in Association with the International Federation of Clinical Chemistry and Laboratory Medicine and the European Federation of Clinical Chemistry and Laboratory Medicine

CCLM is the official journal of the Association of Clinical Biochemists in Ireland (ACBI), the Belgian Society of Clinical Chemistry (BVKC/SBCC), the German United Society for Clinical Chemistry and Laboratory Medicine (DGKL), the Greek Society of Clinical Chemistry-Clinical Biochemistry, the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC), the Slovenian Association for Clinical Chemistry and the Spanish Society for Clinical Biochemistry and Molecular Pathology (SEQC).

Editor-in-Chief

Mario Plebani Padova, Italy

Associate Editors

Giuseppe Lippi Reviews Editor Parma, Italy Philippe Gillery Reims, France Steven Kazmierczak Portland, USA Karl J. Lackner Mainz, Germany Bohuslav Melichar Olomouc, Czech Republic Gérard Siest Perspectives Editor Nancy, France John B. Whitfield Brisbane, Australia

Editorial Board

Marianne Abi Fadel Beirut, Lebanon Francisco V. Alvarez Oviedo, Spain Hassan M.E. Azzazy Cairo, Egypt Arnold von Eckardstein Zurich, Switzerland Andrea Griesmacher Innsbruck, Austria Jean-Louis Guéant Vandoeuvre-les-Nancy, France Wolfgang Herrmann Homburg/Saar, Germany Johannes J.M.L. Hoffmann Nuenen, Netherlands Herbert Hooijkaas Rotterdam, Netherlands Kiyoshi Ichihara Ube, Japan Ellis Jacobs New York, USA Naziha Kaabachi Tunis, Tunisia Jeong-Ho Kim Seoul, Korea

Wolfgang Korte St. Gallen, Switzerland Christos Kroupis Athens, Greece Leslie Charles Lai Kuala Lumpur, Malaysia W.K. Christopher Lam Shatin, Hong Kong Janja Marc Ljubljana, Slovenia Eiji Miyoshi Osaka, Japan Michael Neumaier Mannheim, Germany Tomris Özben Antalya, Turkey Vladimir Palicka Hradec Králové, Czech Republic Mauro Panteghini Milan, Italy José M. Queraltó Barcelona, Spain Marileia Scartezini Curitiba, Brazil Gerd Schmitz Regensburg, Germany

Hong Shang Shenyang, China Ziyu Shen Beijing, China Ana-Maria Simundic Zagreb, Croatia Bart Staels Lille, France Elizabeth Topic´ Zagreb, Croatia Gregory J. Tsongalis Lebanon, USA Pierre Wallemacq Brussels, Belgium Shengkai Yan Beijing, China K.T. Jerry Yeo Chicago, USA Ian S. Young Belfast, UK Managing Editor

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Clin Chem Lab Med 2010;48(Suppl.1):S1–S128, © 2010 by Walter De Gruyter • Berlin • New York

Contents EDITORIAL Special issue of the 10th EFCC Continuous Postgraduate Course in Clinical Chemistry: ‘‘New Trends in Classification, Diagnosis and Management of Thrombophilia’’, October 23-24, 2010, Dubrovnik, Croatia

Ana-Maria Simundic, László Muszbek, Elizabeta Topic and Andrea Rita Horvath S1

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game

Zsuzsanna Bereczky, Kitti B. Kovács and László Muszbek S53 Antithrombin deficiency and its laboratory diagnosis

László Muszbek, Zsuzsanna Bereczky, Bettina Kovács and István Komáromi S67 Factor V Leiden and FII 20210 testing in thromboembolic disorders

REVIEWS Platelet physiology and antiplatelet agents

Tim Thijs, Benedicte P. Nuyttens, Hans Deckmyn and Katleen Broos S3 Hypercoagulable state, pathophysiology, classification and epidemiology

Tadej Pajic´ S79 Hyperhomocysteinemia and thrombophilia

Mojca Bozˇic´ -Mijovski S89 Pediatric thrombosis

Alenka Trampus-Bakija S97

Zrinka Alfirevic´ and Igor Alfirevic´ S15

Thrombophilia screening – at the right time, for the right patient, with a good reason

Diagnostic algorithm for thrombophilia screening

Methodological issues of genetic association studies

Sandra Margetic S27 Genetic basis of thrombosis

Valeria Bafunno and Maurizio Margaglione S41

Mojca Stegnar S105

Ana-Maria Simundic S115 Pharmacogenetics guided anticoagulation

Raute Sunder-Plassmann and Christine Mannhalter S119

Article in press - uncorrected proof Clin Chem Lab Med 2010;48(Suppl 1):S1 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2010.373

Editorial

Special issue of the 10th EFCC Continuous Postgraduate Course in Clinical Chemistry: ‘‘New Trends in Classification, Diagnosis and Management of Thrombophilia’’, October 2010, Dubrovnik, Croatia

Ana-Maria Simundic, La´szlo´ Muszbek, Elizabeta Topic and Andrea Rita Horvath

This Special issue of the Journal is dedicated to the papers delivered by the highly esteemed speakers from the 10th EFCC Continuous Postgraduate Course in Clinical Chemistry entitled ‘‘New Trends in Classification, Diagnosis and Management of Thrombophilia’’, which took place in Dubrovnik, Croatia on October 23–24, 2010. This postgraduate course was established by the Committee for Education and training (C-ET) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC). During the last 9 years, it has been co-organized by the Croatian Society for Medical Biochemists, Slovenian Association for Clinical Chemistry and Inter-University Centre (IUC) in Dubrovnik. Dubrovnik courses are aimed at providing high level postgraduate education, and have covered new trends and classification and diagnosis and management of various diseases, such as cardiovascular diseases, autoimmune diseases, kidney diseases, diabetes mellitus, thyroid diseases etc. The EFCC is very proud to have this opportunity to offer such a high level educational event each year, which attracts more and more participants from all over Europe. In order to strengthen its relationship with other clinical and professional European organizations, which is one of its major strategic aims, this year the EFCC also invited the European Thrombosis Research Organization (ETRO) to join us in the organization of this 10th anniversary event. Until now, course lectures were published as whole text articles in the course Handbook, and were also made available as free full-text articles in the eJIFCC at the IFCC and EFCC web sites. We are very thankful to the CCLM Editorial Board for offering us such a wonderful opportunity to publish articles from the Dubrovnik course within this special issue of the journal, which is the official journal of the EFCC. We would also like to acknowledge our special thanks to Mrs. Heike Jahnke, the Journal Managing Editor for her help and assistance in putting this issue together. The purpose of this course was to review and highlight the current understanding of the pathophysiology, epidemiology and clinical and molecular characteristics of throm-

bophilia. The course brought together 60 participants and 14 outstanding invited speakers. In this issue, we present reviews covering the topics of the course: the structure and function of platelets, the pathophysiology, genetic basis, classification and epidemiology of hypercoagulable states and diagnostic algorithms for thrombophilia screening. The course also covered some specific topics, such as pediatric thrombosis, deficiency of antithrombin, protein C and S, as well as the association of hyperhomocysteinemia and thrombophilia. The last session of the course focused on genetic association studies in thrombophilia research and pharmacogenetics guided anticoagulation therapy. We hope that this Special issue proves a useful and a valuable resource to all who are involved in thrombophilia research, as well as to all specialists in clinical chemistry and laboratory medicine who are involved with laboratory hemostasis on a daily basis, providing support to the care of patients. Ana-Maria Simundic1,* La´szlo´ Muszbek2 Elizabeta Topic3 Andrea Rita Horvath4 1 Clinical Institute of Chemistry, Emergency Laboratory Department, University Hospital ‘‘SESTRE MILOSRDNICE’’, Zagreb, Croatia 2 Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary, Past-president of the European Thrombosis Research Organization (ETRO) 3 President of the Croatian Society of Medical Biochemists, Zagreb, Croatia 4 SEALS North, Department of Clinical Chemistry, Sydney, Australia; President of the European Federation of Clinical Chemistry and Laboratory Medicine (EFCC) *Corresponding author: Ana-Maria Simundic Clinical Institute of Chemistry Emergency Laboratory Department University Hospital ‘‘SESTRE MILOSRDNICE’’ Vinogradska 29 Zagreb 1000 Croatia

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Article in press - uncorrected proof Clin Chem Lab Med 2010;48(Suppl 1):S3–S13 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2010.363

Review

Platelet physiology and antiplatelet agents

Tim Thijs, Benedicte P. Nuyttens, Hans Deckmyn* and Katleen Broos Laboratory for Thrombosis Research, KU Leuven campus Kortrijk, Kortrijk, Belgium

Abstract Apart from the central beneficial role platelets play in hemostasis, they are also involved in atherothrombotic diseases. Here, we review the current knowledge of platelet intracellular signal transduction pathways involved in platelet adhesion, activation, amplification of the activation signal and aggregation, as well as pathways limiting platelet aggregation. A thorough understanding of these pathways allows explanation of the mechanism of action of existing antiplatelet agents, but also helps to identify targets for novel drug development. Clin Chem Lab Med 2010;48:S3–13. Keywords: antiplatelet agents; platelet activation; platelet adhesion; platelet aggregation; platelet inhibition.

Introduction Platelets play a central role in maintaining hemostasis, but are also involved in atherothrombotic diseases. Genetic polymorphisms result in large variability in platelet responsiveness to activation signals. Recently, large scale platelet transcriptome and proteome studies have been undertaken to better understand how these variations influence platelet function (1–3), as well as identification of loci for risk of myocardial infarction and coronary artery disease (4). In addition, comparative transcriptome analysis identified new platelet proteins with structural features suggesting a role in platelet function (5). In parallel, new approaches combining proteomic profiling and computational analysis will create novel insight into mechanisms of platelet activation and will in the future lead to the discovery of new protein-protein interaction network dynamics (6). Finally, all the information arising from these various approaches can be functionally validated in newly developed functional genomic models, such as the zebrafish morpholino oligonucleotide knock-out model (7, 8) or the NOD/SCID xenotransplantation model *Corresponding author: Hans Deckmyn, Laboratory for Thrombosis Research, KU Leuven campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium Phone: q32 56 246422, Fax: q32 56 246997, E-mail: [email protected] Received June 7, 2010; accepted September 5, 2010; previously published online November 6, 2010

(9). Taken together, these new findings will lead to a whole new era in understanding platelet function. This review focuses on the current knowledge of platelet signal transduction pathways, involving platelet adhesion, activation, aggregation and signals limiting platelet aggregation, in combination with known genetic defects and antiplatelet agents that have proven efficiency in primate models or in humans.

Adhesion When the blood vessel wall is damaged, subendothelial structures are exposed to flowing blood. Collagen is the most abundant thrombogenic protein present in the subendothelial matrix. It occurs in more than 20 isoforms, with fibrillar collagen type I and III being the most abundant components present in the matrix lining the vessel wall. Platelets can bind directly to exposed collagen through two major receptors namely integrin a2b1 and glycoprotein (GP) VI (Figure 1). The initial tethering and binding of the platelet is called adhesion. However, under high shear conditions, such as that found in arterioles, neither a2b1, GP VI, nor a combination of both is sufficient to mediate platelet adhesion, which now requires the GP Ib-V-IX receptor complex, and its main ligand, von Willebrand Factor (vWF). vWF binds to exposed collagen fibers through its A3 domain, resulting in a shearmediated structural change in the molecule that allows GP Ib to bind to the vWF A1 domain (Figure 1). This is considered to be primarily an adhesive interaction characterized by a fast dissociation rate, slowing down platelets and allowing stronger bonds to be formed between collagen and the a2b1 and GP VI receptors (11). The importance of the GP Ib-vWF interaction is illustrated by the pathologic conditions occurring in the absence of either receptor or ligand, respectively, the Bernard-Soulier syndrome and von Willebrands disease (12, 13). GP Ib-V-IX

The GP Ib-V-IX complex consists of four subunits: GP Iba, GP Ibb, GP IX and GP V, in a recently proposed 2:4:2:1 stoichiometry, where 2 GP Ibb (25 kDa) subunits are bound to each GP Iba subunit (135 kDa) by disulphide bonds. GP IX (22 kDa) and GP V (82 kDa) in turn are non-covalently associated with GP Ib (14). All subunits belong to the Leucine Rich Repeat protein superfamily, characterized by the presence of one or more stretches of approximately 24 amino acids containing multiple Leu residues, and flanked by conserved N- and C-terminal sequences containing disulphide bonds.

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Article in press - uncorrected proof S4 Thijs et al.: Normal platelet function

Figure 1 Schematic representation of the main biochemical pathways involved in the formation of a platelet-dependent hemostatic plug wbased on (10)x.

GP Iba is the major subunit and presents binding sites for most extracellular ligands, such as, vWF, thrombin, highmolecular weight kininogen, P-selectin and FXII. The crystal structure of GP Iba bound to the A1 domain of vWF has been resolved and suggests long-range electrostatic interactions as the major driving force, with two contact sites emerging in the final complex. One of these contact sites depends on conformational changes in the b-switch of GP Iba, thereby forming a b-hairpin and b-bimolecular sheet, with the other depending on changes in the vWF A1 domain to uncover the binding site (15, 16). Upon binding, clustering of GP Ib-V-IX receptors occurs, enabling multiple interactions between platelets and multimeric vWF molecules. The GP Ib-V-IX complex localizes to the lipid raft fraction of the cell membrane. This location is attractive for signalling molecules, allowing it to transmit weakly activating signals through a number of cytoplasmic proteins including immunoreceptor tyrosine-based activation motif (ITAM) containing proteins (17). 14-3-3z, another adaptor molecule, is also functionally associated with GP Ib-V-IX and participates in the inside-out activation of aIIbb3. This has been demonstrated in a transgenic mouse model expressing the human GP Iba receptor, but lacking the six C-terminal residues responsible for 14-3-3z binding (18, 19). A recent paper by Yuan et al. (20) suggests a double role for 14-33z, inhibiting association to the cytoskeleton, while promoting vWF binding. Other molecules associated with the cytoplasmic domain of the GP Ib-V-IX complex are proteins involved in Ca2q signalling, such as the regulatory p83 subunit of phosphoinositide-3-kinase, calmodulin and Src

kinases, as well as proteins playing part in GP Iba-induced inside-out aIIbb3 activation, such as the recently discovered Adhesion and Degranulation promoting Adaptor Protein (21–24). Finally, the GP Ib-V-IX complex is also linked to the cytoskeleton by its binding site for filamin, which in turn can bind to F-actin, facilitating GP Iba-mediated platelet adhesion to and translocation on a VWF matrix under high shear conditions (25). The best known inhibitor of the GP Iba-vWF interaction is the vWF A1 binding aptamer, ARC1779, which directs a dose-dependent inhibition of VWF activity and platelet function in human volunteers without causing any bleeding effects. In addition, it may serve as a new antithrombotic for the treatment of patients with acute coronary artery syndromes (26), and as a novel therapeutic for thrombotic thrombocytopenic purpura (27). Another drug candidate targeting the GP Iba binding site on vWF is the bivalent humanized Nanobody ALX-0081. This compound inhibits thrombus formation in human patients with stable angina undergoing percutaneous coronary intervention (28). Also, GP Ib-binding monoclonal antibody Fab fragments wreviewed by (29)x, such as 6B4 Fab, prevent platelet adhesion and thrombus formation in vivo without increasing bleeding time (30). Platelet collagen receptors

Although platelets possess a number of receptors capable of interacting with collagen, such as CD36 and GP V, almost

Article in press - uncorrected proof Thijs et al.: Normal platelet function S5

all attention has focused on GP VI and a2b1. GP VI (63 kDa) is part of the immunoglobulin superfamily and relies strictly on its association with the g subunit of the FcReceptor (FcR) in order to be successfully expressed on the platelet surface (31). In addition, the presence of FcRg is also important for triggering signalling events. Upon interaction with collagen, the cytoplasmic tail of GP VI is able to bind the SH3 domain of Fyn and Lyn, both members of the Src tyrosine kinase family. Fyn and Lyn subsequently phosphorylate Tyr residues present in the ITAM motif of FcRg. This phosphorylation allows binding of the tyrosine kinase Syk, which in turn induces a signalling cascade in a so-called signalosome, resulting in the activation of phosphoslipase Cg2 (PLCg2) and formation of second messengers and Ca2q release from internal stores (32–34). The a2b1 collagen receptor, part of the integrin family of proteins, consists of an a2 subunit (165 kDa) and b1 subunit (130 kDa). It has a dependency on Mg2q for collagen binding that increases with higher shear rates (35, 36). In resting platelets, a2b1 is present in a low-affinity state, which upon activation is changed to a high-affinity conformation, a characteristic shared by all integrins (37). Unlike most other integrins, a2b1 is not able to recognize the RGD sequence present in adhesive proteins, such as fibronectin and fibrinogen (35). By using a number of synthetic collagen-related peptides, the GFOGER (Oshydroxyproline) sequence in collagen type I was identified as the main a2b1 binding site. Other related sequences, generally designated GxOGER, that are present in collagen have also been implicated in a2b1 binding, although to a lesser extent (38–42). Signalling through a2b1 is associated with Src tyrosine kinases, Syk, and further downstream with PLCg2. Thus, although both collagen receptors belong to distinct protein families, they seem to share at least part of their outside-in signalling cascade. Recent research from our group suggests two other cytosolic proteins, RhoGDI-b and Slap-2, that interact with the a2 and b1 tail (43). Despite extensive research on both collagen receptors over the years, and the fact that they share at least part of their signalling molecules, the exact interplay between the two has long been a topic of discussion. This debate arises mainly because of conflicting results obtained by different groups, which can be ascribed partially to methodological differences and the difference between human and murine mechanisms. Based on results obtained in human platelets, the original 2site, 2-step model was proposed where a2b1 is important for establishing a high-affinity bond with collagen, followed by a lower-affinity bond with GP VI which then would initiate the intracellular signal transduction (11). This model was questioned when it was found that a2b1 can induce platelet activation via the Src kinase signalling cascade (44), and therefore, a new unifying model was proposed (45). In this model both GP VI and a2b1 are important for mediating stable adhesion, with the specific contribution of both depending on individual platelet responsiveness and the amount and nature of exposed collagen. A recent paper by Pugh et al. (46) might provide the answer regarding the exact role of the two collagen receptors

and the GP Ib-V-IX – VWF interaction. Using the synthetic peptides CRP-XL, GFOGER and VWF-III, specific for GP VI, a2b1 and GP Ib-V-IX, respectively, the authors managed to study individually the effects of the three main adhesionreceptors. They conclude that a2b1 and GP Ib-V-IX can both support substantial platelet activation and thrombus formation on collagenous substrates, whereby the relative importance of the GP Ib-VWF interaction increases at higher shear rates. However, even the combined action of a2b1 and GP Ib-V-IX is insufficient to promote full aIIbb3 activation and requires the strong activating signals generated by GP VI, which contributes little to platelet adhesion itself at all shear rates (Figure 1). Due to their shared signalling molecules and their importance in platelet adhesion, especially under low shear conditions, GP VI an a2b1 can both serve as suitable drug targets (47). Most of the work has been conducted on GP VI, which is not surprising given its important role in generating activation signals (46). A successful strategy has been the development of inhibitory monoclonal antibodies such as OM2 or the 9O12.2 Fab. These have antithrombotic effects in baboons, only slightly influencing the bleeding time (48, 49). In addition, it has been shown that the angiotensin II type I receptor-antagonist Losartan, a drug now prescribed to patients with high blood pressure, and its active metabolite EXP3179, reduced platelet adhesion in a mouse carotid artery injury model by directly interacting with the N-terminal Ig-like domain of GP VI (50, 51).

Amplification of the platelet activation Phospholipase C, Ca2H and granule release

Binding of platelets through its direct collagen receptors triggers tyrosine kinase activity as described above, and induces the platelet activation cascade. In this process, the tyrosine phosphorylated PLCg, and especially PLCg2, plays a central role. PLCg hydrolyses its substrate phosphatidylinositol 4,5bisphosphate (PtdIns4,5P2) yielding water soluble inositol 1,4,5 trisphosphate wIns(1,4,5)P3x and membrane associated diacylglycerol (DAG). Ins(1,4,5)P3 binds and opens intracellular Ca2q channels on calcium storage sites within the platelets, known as the dense tubular system. This allows an influx of free calcium into the cytoplasm. Depletion of Ca2q concentrations in the storage sites activates store-operated calcium entry in platelets by releasing the sensor STIM1 from the endoplasmic reticulum into the cytosol. This then further translocates to interact with plasma membrane calcium channels, the most prominent in platelets being Orai1 or CRAC channel moiety 1 (52, 53). This results in replenishment of Ca2q from the surrounding medium into the platelet through capacitative Ca2q entry. The increase in cytosolic Ca2q concentrations allows numerous Ca2q dependent processes to begin or to accelerate. Alternatively, membrane bound DAG functions together with Ca2q, bound to phosphatidylserine, as an internal recep-


tor for the serine/threonine protein kinase C (PKC). As a consequence, PKC translocates from the cytosol to the membrane to become activated. Activated PKC phosphorylates a variety of substrates, resulting primarily in platelet stimulatory activity, and in particular in platelet secretion of a- and dense-granules. This results in the release of proteins, such as fibrinogen, vWF, thrombospondin-1, factor V, GAS6, plasminogen activator inhibitor 1, and platelet derived growth factors from the a-granules, and ADP, ATP, serotonin (or 5-hydroxytryptamine), Ca2q and polyphosphates from the dense granules (54). Contradictory, PKC is also involved in some inhibitory responses, such as for example phosphorylation of the thromboxane A2 (TXA2) TP receptor that results in receptor desensitisation (55). Apart from its main substrate pleckstrin, PKC also phosphorylates myristoylated alanine-rich C kinase substrate. Upon phosphorylation, the latter loses its ability to crosslink actin filaments, thus facilitating secretion of the granular contents (56). In addition, phosphorylation of syntaxin 4 and platelet secI protein by PKC relieves their inhibitory action on the formation of Soluble N-ethylmaleimide-sensitive fusion protein Attachment protein Receptorcomplexes (SNARE) which are involved in membrane fusion and are necessary to mediate the exocytosis of a-granules (57). Taken together, PKC activation stimulates platelets to secrete agonists, such as ADP, ATP, serotonine and GAS6 through several mechanisms. Together with TXA2, this leads to amplification of platelet recruitment. Phospholipase A2 and amplification via TXA2

Phospholipase A2 (PLA2), a key enzyme in the formation of arachidonic acid (AA) and TXA2, is activated by increased concentrations of cytosolic Ca2q. Platelets contain secreted (s)PLA2, localized in a-granules, and cytosolic (c)PLA2-a. However, only (c)PLA2-a is activated upon agonist stimulation. Activated (c)PLA2-a cleaves platelet glycerolphosholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol at their sn-2 position to release AA. (c)PLA2-a contains a C2 domain at its N-terminus which is a Ca2q ligand binding domain and is involved in the transfer of (c)PLA2-a from the cytosol to the membrane in response to increased concentrations of cytosolic Ca2q. Ca2q and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) has been identified as a link between increased cytosolic Ca2q and activation of (c)PLA2-a (Figure 1). CalDAG-GEFI responds to increased concentrations of cytosolic Ca2q and activates Rap1, a small GTP binding protein of the Ras family. It has been suggested that Rap1 in turn activates extracellular signal-regulated kinases (ERK) which have been implicated in the phosphorylation of (c)PLA2-a (58). Phosphorylation of (c)PLA2-a is important for efficient AA release. However, high concentrations of Ca2q can overcome the need for phosphorylation (59). After its release from the membrane, AA is transformed into the unstable prostaglandin (PG)H2 by cyclooxygenase 1 (COX-1), and finally isomerized into TXA2 by thromboxane synthase. Once formed, TXA2 can diffuse across the plasma

membrane and activate other platelets by interacting with the TP receptor. The TP receptor is known to be a G-protein coupled receptor (GPCR), communicating with G12/G13 and Gq. Platelets lacking G13 and Gq are unresponsive to TXA2, indicating their role in platelet activation (60). Stimulation of Gq will activate PLCb, resulting in accumulation of Ins(1,4,5)P3 and DAG, which in turn activate Ca2q release and PKC. Changes in platelet shape are dependent on G12/ G13 stimulation, activating Rho-mediated signalling (61). The short half life of TXA2 in solution (30 s) limits the spread of platelet activation to the original site of injury. Cyclooxygenase is the target enzyme for aspirin or acetylsalicylic acid. Aspirin covalently acetylates Ser530 on COX (62), which in the anucleated platelet results in an irreversible inhibition of the enzyme for the life time of the platelet (8–10 days), in contrast to the endothelial cells where prostacyclin (PGI2) production recovers much more rapidly. In addition, low dose aspirin is also believed to spare PGI2 production, while inhibiting platelet thromboxane formation: a first passage through the liver results in deacetylation of the low concentrations of aspirin such that the endothelium in the systemic circulation will not be exposed to aspirin, in contrast to the circulating platelets that are inhibited during their passage through the portal vein. Whatever the role of PGI2 in the aspirin story may be, it is clear that daily intake of a low dose of aspirin is highly effective in preventing secondary thrombotic events resulting in an overall 25% reduction of the risk of thrombotic events in patients with confirmed disease of the coronary, cerebrovascular, or peripheral artery beds (63). Platelet activation via G-protein coupled receptors

Almost all soluble agonists induce platelet activation by interacting with GPCRs, such as the TP-receptor discussed above. The only exceptions known to date are ATP, that activates the Ca2q-channel P2X1 resulting in a direct Ca2qinflux into the platelet cytosol and GAS6 that interacts with its Tyr-kinase receptors Axl, Mer, and Sky (64, 65). All GPCRs, except for the protease activated receptor (PAR), reversibly bind their ligands and hence the effects induced depend on the dose of ligand present. Upon ligand or tethered ligand binding, the activated GPCR interacts with intracellular G-proteins, that will activate their effector molecule (66). Platelet GPCRs couple to a variety of G-proteins namely members of the Gq-family that activate PLCb2/b3, of the Gi-family that inhibit adenylyl cyclase and/or activate MAPkinases, or members of the G12/G13-family that signal to small GTPases controlling actin polymerisation. All these Gprotein signalling cascades can act as positive feedbackmechanisms thus ensuring further platelet (integrin) activation, shape change, granule secretion and recruitment of platelets in the growing thrombus (67). The soluble platelet agonist ADP binds to two GPCRs, P2Y1 and P2Y12, both required to ensure a full plateletresponse, but each coupled to its own G-protein. P2Y1 is coupled to Gq and involved in intracellular Ca2q release,


whereas P2Y12 is coupled to Gi-type proteins (68, 69) (Figure 1). Patients lacking this receptor are suffering from a mild form of hemorragia (70). A number of compounds that target the ADP-receptor P2Y12 are now in clinical practice with clopidogrel as currently most used. Clopidogrel is a prodrug that first needs to be converted to its active metabolite by liver cytochrome P450 (CYP)-dependent oxidation. The active metabolite forms a disulfide bond with the P2Y12 receptor, hence, like aspirin, irreversibly covalently modifying the receptor. However, due to the conversion that is needed, clopidogrel has a rather slow onset of action and is sensitive to genetic variations in CYP (so-called drug resistance), therefore, newer compounds with better characteristics are being developed, such as prasugrel (still a prodrug) and products like, ticagrelor and cangrelor (both reversible direct-acting P2Y12 inhibitors) (70). The platelet agonist thrombin binds to the PAR, PAR1 and PAR4 at respectively low and high thrombin concentrations (Figure 1) (71). These receptors couple to a number of Gproteins, most notably G12/G13 and Gq and in some cases also Gi. PAR receptors are activated when an extended Nterminal part of the receptor is being cleaved at a consensus sequence by their ligand. By this, a new N-terminus is exposed that folds back into the central part of the receptor and serves as a tethered ligand that induces receptor activation (72). As the cleaved PAR-receptor thus is expected to be continuously active, it is clear that in order to control PAR-dependent activation, turnoff signals are required, next to the classical ones, such as receptor desensitization, endocytosis and downregulation. Indeed evidence has been obtained that activated PAR-receptors, much like b-adrenergic receptors, become phosphorylated and inactivated by the b-adrenergic receptor kinase-2 (73). Finally, as the affinity of thrombin for the PARs clearly is lower than for the GP Ib-IX-V complex, there also is evidence that initial binding of thrombin to GP Ib might facilitate the consequent cleavage of PAR1 (74). SCH 530348 (vorapaxar) is an orally administered antiplatelet agent that attenuates thrombin induced platelet aggregation through inhibition of the PAR1 receptor without increasing bleeding risk. Phase II clinical trials indicate that this drug may be useful in addition to standard-of-care therapy in patients with coronary disease (75). Some other Gq-coupled receptors that are present on platelets are 5HT2A for serotonin, the PAF-1-receptor for PAFacether and the V-1 receptor for vasopressin (76). In platelets, both the a-subunit of Gq (Gaq) and bg-subunits efficiently activate PLCb3, whereas Gaq-subunits do not activate PLCb2 (77, 78). Activation of PLCbs again results in Ins(1,4,5)P3 and DAG-formation and joins in with the activation pathways already described as a consequence of PLCg phosphorylation-dependent activation (see Phos-

pholipase C, Ca2q and granule release) ultimately leading to integrin aIIbb3 activation (Figure 1). PAR and TP receptors can also couple to G12/G13 which activates a Rho/Rho-kinase-mediated pathway resulting in the phosphorylation of the myosin light chain and therefore, contributes to platelet shape change (79). However, it has also been shown that upon blocking of G12/G13, shape change can still occur by signalling through Gq when sufficiently high amounts of agonist are present. Gi coupled receptors, such as P2Y12 or the a2A-adrenergic receptor for epinephrine, can act in a different way. It has been shown that Gi2, which has the highest expression levels of any Gi family member in platelets, can bind to adenylyl cyclase thereby inhibiting its function. This leads to a reduced production of cAMP, the main inhibitory signalling molecule in platelets (see 4) and thus contributes to activation (Figure 1). To further support this activating role, it has also been shown that platelets lacking Gai2 show reduced aggregation in response to ADP, thrombin and epinephrine (80). Not all GPCR-induced signalling is activating however, PGI2 for instance, one of the major endothelium-derived inhibitors of platelet activation, has signals through a Gs protein upon binding to its IP receptor, leading to activation of adenylyl cyclase and an increase in cAMP (see Signal transduction to limit platelet activation) (Figure 1) (81). Platelet procoagulant formation

Platelet activation and thrombin generation, through the coagulation cascade, are intimately linked processes. Activated platelets can initiate and propagate coagulation on their surface, adequate thrombin formation is not possible without activated platelets. However, thrombin is a strong platelet activator, able to activate platelets at concentrations as low as 0.1 nM. The platelet response to thrombin is largely mediated by PAR1 and PAR4, but signaling through other receptors on the platelet surface, such as GP Ib, can play an accessory role. In platelet membranes, phospholipids are not evenly distributed between the inner and outer leaflet. In resting platelets, phosphatidylserine and phosphatidylethanolamine, negatively charged phospholipids, are found at the inside of the membrane. Upon activation, platelet membranes undergo changes by which more of these negatively charged phospholipids appear on the outside of the membrane (82). These negatively charged phospholipids then complex Ca2q, forming a bridge with coagulation factors, such as factor Xa and factor Va. Binding of coagulation factors on the platelet membrane increases their local concentration which facilitates interaction and accelerates thrombin formation. Finally, this results in further thrombin-induced platelet activation, fibrin formation and coagulation.

Platelet aggregation and integrin aIIbb3 Platelet adhesion and activation are steps leading to the end point of the signaling cascade, platelet aggregation. In this


final step, the integrin aIIbb3 shifts from a low affinity state to a high affinity state and efficiently binds the symmetrical fibrinogen. This step results in cross-linking of the platelets and formation of a firm platelet aggregate (Figure 1). Integrin aIIbb3, or GP IIb-IIIa, is the only integrin expressed uniquely on platelets. With 50,000–80,000 copies per platelet, it is the major platelet integrin receptor mediating both adhesion and bidirectional signaling. Fibrinogen is the main ligand of aIIbb3, other ligands include VWF, fibronectin and vitronectin. The absence or deficiency of aIIbb3 leads to Glanzmann thrombasthenia, characterized by a tendency for severe bleeding and a lack of platelet aggregation in response to all physiological agonists. The shift of integrin aIIbb3 from a low to a high affinity state is considered the ‘final common pathway’ of platelet activation. Therefore, the molecular machinery regulating this process represents a potential target for antithrombotic agents and has been studied intensively. Integrins are ab heterodimeric cell surface receptors consisting of a ligand binding extracellular domain sitting on two membrane-spanning legs. Ligand binding can trigger signal transduction into the cell (outside-in signaling). Integrin tail binding proteins can also induce conformational changes that alter the affinity for their ligands (inside-out signaling). Next to conformational changes that increase ligand affinity, activation can also occur by increased expression and/or clustering of integrins at the cell surface (83). The structural changes that occur when aIIbb3 is activated have been nicely reviewed by Arnaout and co-workers (84). On the basis of structural studies, a model has been proposed that integrins are in a low-affinity state when their extracellular domains are bent. Upon activation, the integrins switch to a high-affinity state with their extracellular domains extended upwards. The exact conformational changes that occur when integrins become activated remain unclear. The ‘switchblade model’ suggests that in response to an insideout signal, the integrin snaps from an inactive bent structure to an active straight conformation, analogous to the opening of a pocket knife. The ‘deadbolt model’, however, predicts that a defined region locks the integrin in an inactive state; upon activation the deadbolt slides away rendering the integrin ligand competent. In both models, these structural rearrangements finally result in a gain in ligand affinity. To date, it is still not clear how platelet agonists, acting through PLC-mediated increased Ca2q concentrations and/or PKC-activation, results in aIIbb3 activation (Figure 1). Although more than 20 proteins have been identified that interact with the cytoplasmic tails of aIIbb3, the functional significance is understood for only a few. The cytoplasmic tail of the integrin b-subunit contains two well-defined motifs which serve as recognition sequences for phosphotyrosine binding (PTB) proteins, including talin and the kindlins (85). The talin binding site on the b-tail was mapped to the membrane proximal NPxY PTB protein binding motif. However, kindlins bind to the membrane distal to the NxxP PTB binding motif (86, 87). Talin is known as a key integrin activator. Binding of talin to the cytoplasmic tail results in a disruption of the salt bridge between the two integrin tails,

with subsequent activation of aIIbb3 (88). How talin is activated is not clear, but it has been proposed that it binds to the lipid second messenger PtdIns4,5P2 (89). Also, Rap1 has been implicated in recruitment of talin to the integrin tails. Rap1 induces the formation of an integrin activation complex consisting of the Rap1-GTP-interacting adaptor molecule which binds directly with Rap1-GTP and talin (90). Although abundant evidence supports the essential role of talin in activation of integrin, recent work shows that talin alone is not sufficient. Kindlin-3 deletion prevents platelet integrins to bind to their ligands. In addition, platelet aggregation is defective despite normal expression of talin (91, 92). Platelets deficient in talin show the same phenotype, suggesting that both proteins are required for integrin activation (93). How kindlins and talin cooperate to regulate integrin activation remains unclear. It is possible that they bind simultaneously or sequentially to the b-tail causing integrin activation (94). The integrin aIIbb3 receptor recognizes its ligands through RGD motifs that are localized at the distal ends of dimeric fibrinogen (95). The binding of fibrinogen to aIIbb3 triggers a number of signaling events that promote cytoskeletal changes, which in turn lead to spreading and stabilization of platelet thrombi. During this outside-in signaling process kindlins again seem to play a role as they bind to integrin linked kinase and the filamin binding protein migfilin, which link kindlins indirectly to the cytoskeleton (96–98). Upon ligand binding, aIIbb3 activates non-receptor tyrosine kinases, such as Src and Syk which contribute to the stability of thrombi in vivo. Ephrin kinase A4 (EphA4), a transmembrane protein expressed on the platelet surface, is constitutively associated with aIIbb3. When platelets are activated the expression of this protein is increased. Binding of EphA4 to its ligand, ephrinB1, facilitates tyrosine phosphorylation of the b3-tail required for stable platelet aggregation (99). Since aIIbb3 is only expressed on platelets and binding of aIIbb3 with fibrinogen is the final major step in platelet aggregation, GP IIb-IIIa antagonists are expected to have a strong and specific effect. The GP IIb-IIIa antagonists that are available clinically include abciximab, tirofiban and eptifibatide. Abciximab is a chimeric Fab fragment of the murine anti-human aIIbb3 monoclonal antibody that blocks ligand binding to aIIbb3 through steric hindrance. Tirofiban is a specific non-peptide antagonist of GP IIbIIIa while eptifibatide is a cyclic heptapeptide derived from a protein found in the venom of the southeastern pygmy rattlesnake. The main mechanism of action of these drugs is the inhibition of fibrinogen binding to GP IIb-IIIa, thereby preventing platelet aggregation. Next to aspirin and clopidogrel, GP IIb-IIIa antagonists form an important component of the pharmacological management of patients undergoing percutaneous coronary intervention, such as patients with coronary artery disease (100). One practical difference between abciximab and the two small molecule agents, tirofiban and eptifibatide, is that abciximab has a longer duration of action (101). The


occurrence of minor bleeding complications and thrombocytopenia are side effects of GP IIb-IIIa antagonists. Drug induced thrombocytopenia often appears suddenly and can cause major bleeding and death. Severe thrombocytopenia is seen in 0.1%–2% of patients treated with GP IIb-IIIa antagonists within several hours after first exposure; up to 12% of patients show thrombocytopenia following a second exposure (102).

Signal transduction to limit platelet activation The signal amplification and platelet recruitment mechanisms require tight control in order to limit platelet aggregate formation to the place where it is needed. Therefore, endothelial cells produce PGI2 and nitric oxide (NO), which, respectively, increase platelet cAMP and cGMP concentrations, the main secondary messengers involved in platelet inhibition (Figure 1). Vascular endothelial cells produce PGI2, which upon diffusion, binds platelet PGI2 receptors (IP) which are coupled to adenylyl cyclase stimulating Gs-protein. Adenylyl cyclase catalyses the conversion of ATP into cAMP, a potent inhibitor of platelet activation (Figure 1). Mutations in the PGI2 receptor may lead to a deficiency in PGI2 signaling and contribute to atherothrombosis (103). Also, other PGs, such as PGD2 (via DP), PGE2 and PGE1 (via EP2 and EP4), and adenosine (via A2A) couple to Gs-adenylyl cyclase. Increased cAMP concentrations result in activation of the cAMP-dependent protein kinase, or PKA. However, Gambaryan et al. recently showed that cAMP-independent mechanisms may activate PKA (104). PKA-mediated phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) inhibits platelet aggregation. Aggregation and spreading of platelets is accompanied by changes in platelet morphology and in reorganization of the actin cytoskeleton, a process in which VASP is one of the key regulators. In addition, phosphorylation of VASP also inhibits binding of fibrinogen to GP IIb-IIIa (105). In 2006, Pula and co-workers, reported that PKCd also has a negative signaling role in regulating platelet aggregation through phosphorylation of VASP (106). PKA further phosphorylates the myosin light chain kinase (MLCK), impairing its binding to calmodulin and consequently reducing MLCK activity, resulting in reduced phosphorylation of myosin light chain which hampers cytoskeletal reorganisation (107, 108). Although no genetic disorders associated with VASP or MLCK have been described, defects in key proteins of the cytoskeleton organization, as found in Wiskott-Aldrich Syndrome, result in severely impaired platelet function. Synergistically to cGMP, cAMP inhibits Ca2q mobilization from intracellular stores, without any major effects on the ADP-regulated cation channel (109). Nitric oxide is produced primarily in endothelial cells from L-arginine by NO synthase. Recently, however, the role of platelet produced NO in regulating platelet activation has also drawn attention wreviewed in (110)x. After diffusion of

endothelial-produced NO through the platelet membrane, NO stimulates soluble guanylyl cyclase causing an increase in cGMP and concomitant activation of cGMP-dependent protein kinase (PKG). PKG inhibits Ins(1,4,5)P3-stimulated Ca2q release from the sarcoplasmic reticulum and promotes ATPase-dependent refilling of intraplatelet Ca2q stores, thereby decreasing the amount of cytosolic Ca2q (111, 112). In addition, PKG inhibits TXA2 receptor function through phosphorylation, thus blocking platelet activation and aggregation (113). The cGMP that is produced, in turn, might prevent platelet activation by indirectly increasing intracellular cAMP through inhibition of phosphodiesterase type 3 and inhibiting the activation of phospatidylinositol 3-kinase (114, 115). In addition, VASP also becomes phosphorylated following increases in cGMP by activation of guanylyl cyclase (116). Reduced responsiveness of platelets to NO, called ‘platelet NO resistance’, is involved in symptomatic ischemia, chronic heart failure and other risk factors for cardiovascular disease, and might be due to decreased bio-availability of NO or to disturbances in the integrity of the NO/ cGMP signaling pathway. Pharmacological strategies that may enhance platelet responsiveness to NO have been reviewed by Rajendran and Chirkov (117).

Perspectives Recent comparative transcriptome analyses of in vitro differentiated megakaryocytes and erythroblasts have led to the identification of new platelet proteins with structural features that suggest a role in platelet function (6). In order to functionally characterize these newly identified proteins in platelet signaling pathways, there is a need for functional genomic approaches in new, previously unexplored model organisms. O’Connor et al. (7) recently demonstrated the suitability of the zebrafish, Danio rerio, for functional analysis of novel platelet genes in vivo by reverse genetics in a laser-induced thrombosis model. In this model, the relatively easy knockdown in gene function following the injection of antisense morpholino oligonucleotides allows for screening of large populations of fish lacking one or more platelet receptors or signaling molecules. In a parallel approach, xenotransplantation models have been optimized allowing the in vivo investigation of human platelet function and thrombopoiesis in NOD/SCID mice (9). This model can be further explored to functionally study genetically modified platelets or new pharmacologic strategies. All the aforementioned findings discussed in this review have resulted in a significant increase in our knowledge of platelet function over the past decade. However, there still is no unifying model for the prediction or treatment of cardiovascular diseases available at the present time. However, thanks to recent large scale platelet transcriptome studies, soon we will be able to associate many of the gene polymorphisms in platelet surface receptors or signaling proteins with changes in the risk for atherothrombotic events. Fur-


thermore, it is anticipated that the development of low-cost high-throughput genotyping techniques will allow clinical application and individualization of therapies, although there is still ongoing discussions regarding the benefits and limitations of this approach (118). Platelet pharmacogenomics has nevertheless already started to link this genetic information to inter-individual variability in drug response, and as reviewed by Zuern and co-workers (119), several genetic polymorphisms can now be associated with ‘aspirin resistance’ or explain the variability in treatment response to clopidogrel or abciximab.

Conflict of interest statement

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Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: Research funded by the Fund for Scientific Research (FWO G.0564.08). TT and BPN are funded by a fellowship from the Agency for Innovation by Science and Technology in Flanders (IWT). Employment or leadership: None declared. Honorarium: None declared.

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Review

Hypercoagulable state, pathophysiology, classification and epidemiology

Zrinka Alfirevic´1,* and Igor Alfirevic´2 1

Department of Internal Medicine, Medical School, University Hospital ‘‘Sestre Milosrdnice’’, Zagreb, Croatia 2 Special Hospital for Cardiovascular Surgery and Cardiology ‘‘Magdalena’’ Krapinske Toplice, Croatia

Abstract Hypercoagulable state is not a uniform disease. It is a complex condition with an abnormal propensity for thrombosis that may or may not lead to thrombosis, depending on complex gene-gene and gene-environment interactions. The prevalence of the hypercoagulable state depends on the ethnicity and clinical history of the population being studied. The consequences of a hypercoagulable state due to thrombosis of veins and arteries are the most important cause of sickness and death in developed countries at present. Primary hypercoagulable state is an inherited condition caused by the reduced level of natural anticoagulants due to a qualitative defect or quantitative deficiency of an antithrombotic protein, or increased concentrations or function of coagulation factors. Most of the inherited abnormalities recognized to date have little or no effect on arterial thrombosis and are associated primarily with venous thromboembolism. Arterial thrombosis usually develops as a complication of atherosclerosis and patients usually have more than one traditional risk factor. Secondary hypercoagulable states generally occur as a result of a large number of transient or permanent acquired conditions that increase the tendency for formation of blood clots. New epidemiological data and clinical trials suggest that many acquired risk factors in the pathophysiology of arterial and venous thrombosis overlap and coexist for both disorders. Clin Chem Lab Med 2010;48:S15–26. Keywords: acquired conditions; arterial and venous thrombosis; hypercoagulable state; inherited thrombophilia.

Introduction The hypercoagulable states are a group of inherited or acquired conditions that cause a pathological thrombotic ten*Corresponding author: Zrinka Alfirevic´, Department of Internal Medicine, Medical School University Hospital ‘‘Sestre Milosrdnice’’, Vinogradska 29, 10,000 Zagreb, Croatia Phone: q385 98 417 307, E-mail: [email protected] Received June 16, 2010; accepted October 16, 2010; previously published online November 16, 2010

dency due to an abnormality in the blood itself or changes in vasculature. There are no specific signs or symptoms associated with hypercoagulable states. The most common clinical manifestation is venous thromboembolic disease, although hypercoagulable states increase the risk for both arterial and venous thrombosis. Some conditions are associated to a greater extent with venous, and others with arterial thrombosis, but there are also conditions that are associated with the development of both arterial and venous thrombosis (1). The term thrombosis refers to the formation, from constituents in the blood, of a mass within the venous or arterial vasculature of a living being (2). The presence of hypercoagulability is not associated uniquely with the development of thrombosis, and there is no data to suggest that hypercoagulable states reduce survival in patients who carry an inherited predisposition to hypercoagulability. A person with a well-defined hypercoagulable state does not necessarily develop thrombosis, and not all people with thrombosis have an identifiable hypercoagulable state. Many of these patients with inherited defects are asymptomatic until they experience some acquired risk factor. There is no specific therapy and we do not cure the cause, but the consequences. Antiplatelets, fibrinolytics and anticoagulants are utilized to treat arterial thromboembolism (ATE), and anticoagulants and fibrinolytics may be utilized to treat venous thromboembolism (VTE). Patients are considered to have hypercoagulable states if they have laboratory abnormalities or clinical conditions that are associated with an increased risk of thrombosis (prethrombotic states), or if they have recurrent thrombosis without recognizable predisposing factors (i.e., they are thrombosis-prone). Among the factors that may indicate a primary hypercoagulable state are a family history of thrombosis, recurrent thrombosis without apparent precipitating factors, thrombosis at unusual anatomic sites, thrombosis at an early age, and resistance to conventional antithrombotic therapy. In everyday practice, accurate determination of causes that have led to a hypercoagulable state is useful in deciding on the length of therapy. Testing should also be directed at identifying any underlying systemic disorder since treatment may improve the thrombotic tendency (1, 3). Today, we know that the development and location of thrombosis depends on a wide range of diseases, clinical circumstances or molecular variants associated with an increased risk of thrombosis. However, the cause remains unknown in many patients. Thrombus formation depends on complex interactions between heritage and the gene-environment relationship (4).

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Arterial thrombosis usually develops as a complication of atherosclerosis resulting from hypertension, hyperlipidaemia, diabetes mellitus, metabolic syndrome and homocystinuria. These patients have hypercholesterolaemia and other lipoprotein abnormalities, high fibrinogen concentrations and increased von Willebrand factor (vWF), tissue plasminogen activator antigen and factor VIII activity (5). Coronary arterial thrombosis and cerebrovascular thrombotic occlusion are the leading causes of morbidity and mortality in developed countries. Myocardial infarction and thrombotic stroke cause more than one third of all deaths in developed countries (6). In contrast, venous blood stasis initiates local activation of procoagulant events to form venous thrombus by accumulation of fibrin and trapped erythrocytes. Hypercoagulability due to inherited genetic abnormalities plays a crucial role in the development of venous thrombosis. Thrombophilia is generally associated with venous thrombosis and may be detected in a significant number of patients who present with their first episode of venous thromboembolism. The term thrombophilia, as opposed to haemophilia, refers to inherited or acquired conditions in the coagulation system, with thrombosis as their primary manifestation and is often not evident without specific laboratory testing. These patients are at higher risk for thrombosis as compared with the normal population, and the risk increases according to the number of risk factors (6). Venous thromboembolic disease is the third leading cause of cardiovascular death in developed countries. It causes 50,000–100,000 deaths in the US annually (7). The disease can manifest itself through thrombotic obstruction of any venous system and usually occurs as deep vein thrombosis of the leg. An unusual venous location occurs in fewer than 5% of all cases of thrombosis, and is frequently associated with some genetic defect. Foetal complications in the form of massive pulmonary embolism occurs in 20% of cases (8, 9). From the first review article registered in 1964 in the Medline database, the view on hypercoagulable states has been updated continuously (10). Interest in these disorders has increased in the last 50 years due to the discovery of several genetic factors that contribute to the incidence of thrombosis. The initial report was published in 1965 by Egeberg who described a Norwegian family with an identified hereditary tendency to thrombosis, caused by antithrombin deficiency (11). From 1965 to 1993, a heritable cause of thrombosis was detected in 5%–15% of patients with thromboembolic disease. Today, inherited thrombophilia can be found in over 60% of cases following the first clinical episode of VTE (12). This article will focus on the pathophysiological mechanisms of arterial and venous thrombosis, and underlying inherited and acquired conditions in epidemiological situations.

Haemostasis The human haemostatic system maintains blood fluidity on the basis of a well-controlled interaction between platelets, the vascular endothelium and protein (procoagulant, natural anticoagulant, fibrinolytic and antifibrinolytic) components.

Haemostatic thrombosis in a normal healthy organism is a self-limited, well-controlled condition that prevents leakage of blood from the blood vessel. It is subdivided into four well-known conditions: vascular constriction, primary and secondary haemostasis, and dissolution of the clot. Immediately after vascular injury, vascular vasoconstriction limits the flow of blood to the area of injury. At the same time damage to the blood vessel walls exposes underlying subendothelium proteins (vWF) to blood proteins, initiating primary haemostasis (13). The aim of primary haemostasis is formation of a platelet plug. Circulating platelets adhere to damaged endothelium with the help of von Willebrand factor, by binding to collagen where they enlarge and flatten. Adhesion, activation and aggregation of platelets is a process mediated by several factors secreted by surrounding cells and by platelets themselves. These mediators activate other platelets to link by fibrinogen through the surface glycoprotein IIb/IIIa receptors to form the platelet plug, and further activate vasoconstriction and the clotting cascade to form an organized clot (14, 15). The aim of secondary haemostasis is to continue and complete the process of haemostasis through a series of wellknown reactions known as the coagulation cascade. Secondary haemostasis can take place through two common pathways initiated by distinct mechanisms which lead to formation of a thrombus: the tissue factor pathway (formerly known as the extrinsic pathway) and the contact activation pathway (formerly known as the intrinsic pathway). In both pathways, inactive enzyme precursor of a serine protease and its glycoprotein co-factor are activated to become active components that then catalyse the next reaction in the cascade. This results ultimately in cross-linked fibrin which stabilizes a platelet plug and forms a durable thrombus. The intrinsic pathway has low significance under normal physiological conditions; its activation begins when negatively charged collagen inside the damaged endothelium, or negatively charged phospholipids on the surface of activated platelets, activate formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor). The main pathway of in vivo coagulation takes place through tissue factor. Tissue factor is the only protein of coagulation which does not circulate in the blood. As non-functional precursors, it is fully functional only in cases of cell damage when it is expressed on the surface of endothelial cells, platelets or leukocytes. It is present on almost all extra-vascular cells, with the exception of joint coatings. Also, it may be expressed during physiological processes, such as normal parturition. The coagulation cascade occurs during thrombus formation in response to tissue injury. Activation begins with the release of tissue factor from injured cells where activated factor VII, expressed on tissue-factor-bearing cells, forms an activated complex (TF-FVIIa). This complex further activates factor X and leads to formation of a small amount of thrombin. Factor VIIa can also form a tenase complex through activation of factor XI, which then further initiates the activation of factor IX. Factor IX, together with activated factor VIII,

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activates factor X. Both pathways convert to activation of thrombin, the key enzyme of haemostasis. The common point is factor X. Factor Xa activates prothrombin (factor II) which is converted to thrombin (factor IIa). The activation of thrombin occurs on the surface of activated platelets and requires formation of a prothrombinase complex. This complex is composed of platelet phospholipids, phosphatidylinositol and phosphatidylserine, Ca2q, factors Va and Xa, and prothrombin. Thrombin converts fibrinogen to fibrin, and also activates factors VIII and V, and their inhibitor, protein C (in the presence of thrombomodulin). Factor XIII acts on fibrin polymers to form activated monomers which form bridges between aggregated and adjacent stimulated platelets and the stabilized platelet plug (16–18). The procoagulation system is very powerful, and potentially dangerous. Therefore, the production of thrombin, its generation from prothrombin, and its activity in plasma are carefully regulated by thromboregulatory anticoagulants at each step of the cascade. Natural anticoagulants, such as protein C, protein S, antithrombin III, the tissue factor pathway inhibitor (TFPI) and protein Z, inhibit activated enzymes and associated cofactors to prevent pathological thrombosis, limiting the clotting process to the actual site of injury. However, despite all the mechanisms of control, if thrombus formation occurs, conter regulatory balancing mechanisms from the fibrinolytic system attempt to reduce and eliminate the thrombi. Plasmin is the most important enzyme of physiological fibrinolysis and limits thrombus size and dissolves a thrombus by degrading a cross-linked fibrin once the vascular injury has been repaired (19).

Pathophysiology Abnormalities in blood flow due to turbulence (aneurysm, hypertension), blood stasis (immobilization, tumour compression) or hyperviscosity (myeloproliferative disorder, thrombocytosis) may contribute to thrombogenesis due to local hypoxia which stimulates endothelial cells to release an activator of factor X (20). Disturbances of the vessel wall (vascular endothelial damage) may occur as a result of direct physical trauma during surgical procedures, or as a result of local exposure to proinflammatory or procoagulant mediators (21). Normal endothelium is physiologically non-thrombogenic and responsible for normal blood fluidity. Endothelial cells defend themselves and control thromboregulation through continuous communication with platelets and with several of their own mechanisms, such as the thrombomodulin protein C system, the tissue factor pathway inhibitor system, the plasmin generating system and antithrombin system. Endothelial prostacyclins, ADP and nitric oxide inhibit platelet aggregation and support vascular relaxation, endogenous heparin, and block thrombin production. At the same time, prostacyclin released locally from platelets inhibit platelet adhesion and aggregation. In situations where damage occurs, endothelial cells release endothelin-1 and platelet activating factor, increase production of vWF, PAI-1 and factor V, and also

produce one of the strongest drivers of haemostasis, a cellular membrane protein known as tissue factor. Coagulation can also be activated by factor X, activated platelets, monocytes or factor XII (22). Hypercoagulability is the propensity to develop thrombosis due to an abnormality of any haemostatic cellular and protein component caused by a hyperactive procoagulant or hypoactive anticoagulant and/or defect in fibrinolysis. Hypercoagulability is due to disruption of the highly regulated coagulation mechanism and occur as a result of hereditary defects in one or more of clotting factors, or some situational or acquired condition. The level of lifelong, baseline hypercoagulability in any individual may be determined by the type and number of defects that are inherited. The inheritance of more than one genetic polymorphism increases the risk (23). Environmental risk factors, such as pregnancy, use of oral contraceptives and heparin, induce thrombocytopenia and represent transient circumstances, whereas increased risk lasts as long as the disorder that has caused it to occur. Acquired risk factors are, for the most part, the result of a non-reversible medical disorder, such as cancer, the nephrotic syndrome, myeloproliferative syndrome and paroxysmal nocturnal haemoglobinuria (24).

Arterial thrombosis Arterial thrombus formation usually begins with endothelial injury (atherosclerotic plaque rupture, aneurysm formation, vessel dissection, hyperhomocysteinaemia) in a situation where there is high velocity blood flow and increased platelet activity. Hyperviscosity due to myeloproliferative syndromes, cryoglobulinemia and plasma cell dyscrasias, may also precipitate arterial thrombosis. The core of an atherosclerotic plaque is rich in inflammatory cells, lipids, cholesterol crystals, and tissue factors generated by activated macrophages. In cases of plaque ulceration, the arterial endothelium becomes damaged and highly thrombogenic lipid components are exposed to the bloodstream. These thrombogenic lipid components activate adherent neutrophils and platelets to cause release of inflammatory and procoagulant mediators that further amplify thrombosis (5). In this case, platelet granules release adenosine diphosphate, adenosine triphosphate, calcium and serotonin and induce additional platelets in the surrounding milieu to activate and aggregate. Platelet activation by ADP leads to a conformational change in platelet glycoprotein IIb/IIIa receptors that induces them to bind to fibrinogen (25). Stimulated platelets bind related adhesive proteins of vWF, fibronectin, and vitronectin. Next, the GpIIb/IIIa-bound protein cross-links these platelets into aggregates that bind to the layer of adherent platelets, forming a haemostatic plug or an occlusive thrombus (26). An arterial (white) thrombus is formed primarily from platelets. Its formation resembles a normal haemostatic plaque except that it is located on the external surface of a ruptured atherosclerotic plaque. Thrombus growth and fibrin deposition may produce a red thrombus and totally occlude the flow of


blood. This can result in transition from stable or subclinical atherosclerotic disease to acute myocardial infarction, stroke, or peripheral arterial occlusion (27). Most arterial thromboses develop on the basis of endothelial injuries in association with traditional cardiovascular risk factors which themself are potent procoagulant factors (28). Genetic abnormalities in platelet function due to polymorphisms in platelet glycoprotein IIIa polypeptide, high fibrinogen concentrations, increased plasma vWF concentrations, and increased concentrations of factors VII, VIII, IX, XI, XII are also responsible for hypercoagulability state in the pathophysiology of arterial thrombosis. Any qualitative or quantitative changes in platelet count or function due, for example, to a polymorphism in the b-subunit of G protein increases prothrombotic risk (29). A strong association between the (gp)IIIa, (PlA2), (gp)Ia (8807T allele), and (gp)Iba polymorphism of the glycoprotein IIIa gene due to altered adhesion, spreading and actin cytoskeletal rearrangement and acute coronary thrombosis has been described in patients who had coronary events before 60 years of age. The odds ratio for coronary thrombosis is six-fold higher in patients younger than 60 years who have a glycoprotein IIb/ IIIa allele (30). Sticky platelet syndrome is an example of a congenital, autosomal dominant disorder of platelets, characterized by hyperaggregable platelets in response to ADP and epinephrine, and associated with arterial and venous thromboembolic event (31). An increased fibrinogen concentration is a strong and independent predictor of cardiovascular risk, both in healthy individuals and in those with manifest coronary artery disease (32–34). Many studies have found a connection between increased plasma factor VII activity and arterial thrombosis (35). Other disorders of the fibrinolytic system due to defective synthesis of tissue plasminogen activator or deficiency or a functional defect in the plasminogen molecule can also be a possible cause of a hypercoagulable state in the pathogenesis of arterial thrombosis. The plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G insertion/deletion polymorphism has been associated with 25% higher plasma PAI-1 activity. The prevalence of the 4G allele is significantly higher in patients with myocardial infarction before the age of 45 years than in population-based controls, (allele frequencies of 0.63 vs. 0.53). Both alleles bind a transcriptional activator. Increased gene transcription is associated with four guanine bases (the 4G allele), and results in the binding of a transcriptional activator alone, whereas the 5G allele also binds a repressor protein that decreases the binding of the activator (36).

Venous thrombosis In contrast, venous blood stasis is known to initiate the formation of a venous thrombus by the local activation of procoagulant zymogens. A red venous thrombus is composed of large amounts of fibrin that contains entrapped erythrocytes, and very often occurs in intact endothelium. Exceptions include direct venous trauma, extrinsic venous compression, and biochemical vascular endothelial cell injury resulting

from the toxic effect of many prothrombotic mediators (37). Venous stasis due to immobilization, hospitalization, limb paralysis, pregnancy, varicose vein, or venous insufficiency increases venous retrograde pressure and potentiates local hypoxia. Damaged endothelial cells release prothrombotic and proinflammatory mediators. The process begins with tissue factor and leads to the sequential activation of coagulation factor X (FXa), cleavage of prothrombin by FXa to liberate thrombin, and cleavage of fibrinogen by thrombin to produce fibrin monomers. Fibrin monomers spontaneously polymerise into fibrin strands that are then irreversibly crosslinked by thrombin-activated factor XIII to produce an insoluble clot. Thrombosis can occur either when the production of FXa and thrombin is enhanced or when the activity of control proteins is diminished. Endothelial cell injury increases the surface expression of cell adhesion molecules, promoting leukocyte and platelet adhesion and activation. This initiates and amplifies inflammation and thrombosis (38, 39). Hypercoagulability, due to inherited genetic abnormalities, plays a crucial role in the development of venous thrombosis (40). These conditions are characterized by disruption in the normally highly regulated coagulation mechanism, resulting in increased thrombin generation and an increased risk of clinical thrombosis. In normal conditions, clotting factors are deceptive and wait for a signal for activation (41, 21). Activated protein C (APC) resistance due to a single point mutation in the factor V gene is the most common relatively mild inherited disorder that causes hypercoagulability. It is responsible for 20%–60% of cases of thrombosis in Caucasians. The risk in carriers of one polymorphic allele of Factor V Leiden (FV Leiden) is 4–8/ 1000, and for homozygotes the risk is 80/1000 per year (42). APC resistance is a laboratory clotting parameter, but it is also a clinical name for familial thrombotic disorder, a condition where the patient’s plasma does not produce an appropriate anticoagulant response to APC. In 90% of cases, patients have the factor V allele that is resistant to the proteolytic effect of protein C. FV Leiden is a factor V variant which is APC inactivated 10 times more slowly (43). The second most common cause of inherited thrombophilia is a mutation in prothrombin. People with the prothrombin G20210A mutation have higher amounts of prothrombin than normal due to a decreased rate of degradation of mRNA, with a three to seven-fold increased risk of thrombosis in heterozygotes. Compound heterozygosity with FV Leiden increases the risk of VTE 20-fold (44, 45). A deficiency in natural anticoagulants is a strong, but rare, prothrombotic risk factor. Severe AT deficiency may increase the risk of VTE up to 50-fold (46). The patients heterozygous for protein C deficiency have a 10-fold increased risk for thrombosis, develop thrombosis between the age of 15 and 30 years, and have 60% less than normal antigenic protein C (47, 48). Increased factor VIII (FVIII) concentrations have also been associated with increased prothrombotic risk (49). Epidemiological data have shown that high concentrations of factors IX and XI are also associated with an increased risk of venous thrombosis, but an adequate association has not been well-established (50, 51).


Deficiency of any other fibrinolytic com-ponent may be a potential cause of venous thrombosis, but arterial events may also occur. There are five forms of dysfibrinolysis: congenital plasminogen deficiency, congenital deficiency of tissue plasminogen activator or congenital increases in plasminogen activator inhibitor, congenital dysfibrinogenaemia and factor XII deficiency. Plasminogen deficiency is an autosomal dominant disorder caused by mutations of the PLG gene; thrombotic events occur when plasminogen values are -40% of normal (52). Deficiency of heparin cofactor II, deficiency of tissue factor pathway inhibitor, and thrombomodulin mutations are extremely rare, but are potential prothrombotic risk factors.

Classification According to the mechanism of action and time of occurrence, the hypercoagulable state can be divided into an inherited or acquired condition, primary or secondary, severe or rare and permanent or transient. Some states are associated more with arterial thrombosis, while others are associated with venous thrombosis (Table 1) (1, 53). Hypercoagulable states are usually divided into primary and secondary states. Until 1993, the primary hypercoagu-

lable states included antithrombin III, protein C and protein S deficiencies, dysfibrinogenemias, plasminogen deficiency, and decreased plasminogen activator activity I (53). Since then, the number of specific genetic polymorphisms that play a role in the pathogenesis of arterial and venous thrombosis have been rising. It was recognized that most, if not all, patients with venous thromboembolism have a genetic basis for the disorder, i.e., ‘‘thrombophilia’’ (54). Thrombophilia is an inherited or acquired clinical phenotype manifesting in selected individuals as a greater risk for the development of recurrent thrombosis at a younger age compared with the general population, with considerable differences in the magnitude of risk among individuals in the same family and with the same thrombophilic gene defect. Hereditary thrombophilia is a condition with thrombosis as its primary manifestation, and is often not evident without specific laboratory testing to reveal its mechanism of action. After 1993, the term thrombophilia has often been identified as the primary hypercoagulable state (55, 56). There are 21,000 published articles in the Medline electronic data base associated with the hypercoagulable state. Most of the articles are focused on a single association of each factor with venous thrombosis, and/or the role of thrombophilia in the pathogenesis of venous thrombosis. Only a few of the articles generally address hypercoagulable states (57, 58) and mentioned con-

Table 1 Genetic disorders associated with hypercoagulable states. Hypercoagulable states Disorders

Arterial

Both arterial and venous

Venous

Inherited

Increased fibrinogen concentrations (a, b chain polymorphism) Platelet glycoprotein polymorphism GPIIIa Leu 33Pro GP1BA 5T/C GP&1325T/C Thrombomodulin deficiency Lipoprotein(a) Factor VII excess

PAI-1 excess Dysfibrinogenaemia Homocystinuria

Definite AT Protein C Protein S Factor V G1691A (APC) PT G20210A Elevated factor VIII Possible Factors XI, IX, TAFI, von Willebrand, heparin cofactor II

Mixed Acquired

Hypertension Smoking Diabetes melitus Hypercholesterolaemia

Hyperhomocysteinaemia Antiphospholipid antibody Cancer Age Obesity Metabolic syndrome HITTS Malignancy Oral contraceptives Hormone replacement therapy Infection Myeloproliferative disorders (essential thrombocytosis polycythaemia rubra vera, chronic myeloid leukaemia, myelofibrosis) Paroxysmal nocturnal hemoglobinuria

Previous thrombosis Immobilization Major surgery Orthopedic surgery Pregnancy and puerperium Nephrotic syndrome


ditions that are associated with both arterial and venous thrombosis. Thus, it may be concluded that classification differs slightly from author to author.

Primary hypercoagulable states All authors agree that the primary hypercoagulable states include inherited conditions caused by decreased amounts of natural anticoagulants due to a qualitative defect or quantitative deficiency of an antithrombotic protein, or by increased amounts or function of coagulation factors. Most of the inherited abnormalities recognized to date have little or no effect on arterial thrombosis, and are associated primarily with venous thromboembolism (55). Reduced concentrations of the inhibitors of the coagulation cascade involve a rare but strong risk of inherited mutations (deficiency of antithrombin III, protein C and protein S) commonly found in Southeast Asia, and are rare in the black population. Deficiency is divided into subgroups according to antigen and activity levels. Most patients will have an episode of thrombosis by 60 years of age. Homozygous deficiencies are rare and can result in a severe phenotype with neonatal purpura fulminans (deficiency of PC and PS), or are incompatible with normal life (antithrombin deficiency) (48, 55). At present, the prothrombin G20210A mutations or APC resistance due to FV Leiden are the most prevalent causes of thrombosis in Caucasians (59). The risk is mild and increases with age and in proportion to the number of risk factors. Most of these individuals will not have had an episode of venous thrombosis by the age of 60 years, or the disorder will be expressed by exposure to another risk factor (43, 44). Not as a result of an acute phase reaction, increased factor VIII concentrations are also a risk factor for venous thrombosis, particularly in the black population (60, 61). Some studies suggest that a weak association exists between FV Leiden and prothrombin G20210A arterial thrombosis (62). A number of less frequent hereditary abnormalities (increased concentrations of factors IX and XI, VII, von Willebrand, TAFI, deficiency of heparin cofactor II) have also been associated with an increased tendency for venous thrombosis. However, such associations have not been wellestablished, even though the relevance varies from author to author. For example, Schafer mentioned, among other factors, the increased levels of factors VII, XI, IX, and von Willebrand in the primary hypercoagulable state. Nachman, in the primary condition, included MTHFR mutation that is rarely mentioned by other authors, but dysfibrinogenemia, mentioned by many other authors, is not listed (57). The pathophysiological mechanism of the primary hypercoagulable state is a result of impaired regulation of endothelial function (AT III, PC, PS) or alterations in the kinetics of coagulation zymogens (FV Leiden, prothrombin G20210A) that are associated with surface expression of tissue factor that lead to venous thrombosis. Hereditary thrombophilia factors are clearly associated with venous

thrombosis; the exception is dysfibrinogenaemia which also plays a role in the development of arterial thrombosis (57). When discussing the primary hypercoagulable state, we should also mention the rare genetic disorders that play a role in the pathophysiology of arterial thrombosis (platelet glycoprotein polymorphism, PAI-1 polymorphism, fibrinogen polymorphism, thrombomodulin polymorphisms, plasminogen deficiency). Hyperhomocysteinaemia is an example of inherited or acquired conditions that increase thrombotic risk due to direct toxic effects on endothelial cells as a result of increased tissue factor activity, increased platelet activation, suppression of thrombomodulin expression, and impaired fibrinolysis (58).

Secondary hypercoagulable state Secondary hypercoagulable states generally occur as a result of a large number of transient or permanent acquired conditions that increase the tendency for formation of bloods because they convert constitutive non-thrombogenic surface of endothelial cells to the active proinflammatory thrombogenic phenotype. The conversion is a result of biochemical damage to endothelial cells mediated by several proinflammatory and prothrombotic mediators. This leads to increased expression of leukocyte adhesion molecules, increased expression of macrophages and tumour cell tissue factor, and inhibition of the protein C system. Endothelial conversion to the proinflammatory thrombogenic phenotype may be result of many transient and permanent disorders (57, 63, 64). Some of these such as the presence of antiphospholipd antibody, hyperhomocysteinaemia, polycythemia rubra vera, cancer and infection increase the risk of both arterial and venous thrombosis. Pregnancy, puerperium, the early postoperative state, sepsis, and oral contraceptives are examples of transient conditions where increased thrombotic risk is present as long as these transient disorders are present. Permanent disorders are the result of irreversible processes that produce alterations in basic homeostasis. Examples of these disorders are the antiphospholipid antibodies syndrome, carcinoma and other malignancies, nephrotic syndrome, previous thrombosis, myeloproliferative disorders and homocysteinaemia. The most common cause of an acquired hypercoagulable state is the antiphospholipid syndrome. Thrombotic events associated with the antiphospholipid syndrome (APS) more often affect the venous rather than arterial system. A heterogeneous family of antiphospholipid antibodies (anticardiolipin antibodies or lupus anticoagulans) are associated with many collagen vascular disorders, infections and some medications. The prevalence in the general population is 2%–5%, and it has also been described as an isolated abnormality. However, the incidence of APLA is in patients with SLE as high as 50%–60%. Although b2 glycoprotein 1 is the most common target of APLAB, they have an affinity for the phospholipid surface of thrombin, high molecular weight kininogens, activated protein C and S and factor XII. Many


mechanisms have been proposed for the manner of how antiphospholid antibodies induce thrombosis. These range from endothelial defects to platelet and monocyte activation and alteration in plasma proteins. Many studies have confirmed the association between antiphospholipid antibodies and thrombosis (65–67). Cancer is the second most common acquired condition associated with the hypercoagulability state. It can lead to thrombus formation by local compression and invasion of the vessel wall, or indirectly with inflammatory and proangiogenic cytokines and tissue factor rich microparticles released from tumour cells. It is a well-recognized (AmE) risk factor for arterial and venous thrombosis (68). Use of some medications, such as aspariginase thalidomide, prothrombin complex/intermediate purity factor IX concentrate, oral contraceptives, and tamoxifen have shown a strong association with thrombosis (69). Oral contraceptives are an example of a well-defined condition associated with thrombosis (arterial and venous) as a result of endothelial dysfunction, changes in the concentrations of several coagulation factors and lipoprotein components. Oral contraceptives increase the risk of arterial thrombosis by approximately three-fold, and the risk for venous thrombosis by about four-fold (70). Many studies performed over the years have confirmed that acquired risk factors for venous thrombosis differ from those that cause arterial vascular disease. The latest study was a large LITE study in 2002 that included 19,293 people which corroborated the view that the aetiology of VTE differs from that of atherosclerotic cardiovascular disease (71). Hypertension, smoking, metabolic syndrome, and hyperlipidemia have been considered as risk factors for arterial thrombosis, whereas surgery, immobilisation, pregnancy and oestrogen use are the risk factors for venous thrombosis (71, 72). In the last few years, studies have shown that the pathophysiology of the hypercoagulable state has a complex mechanism and that risk factors for arterial and venous thrombosis that are traditionally divided, do overlap (73). In 2003, Prandoni was the first to report a higher prevalence of asymptomatic atherosclerosis lesions in patients with idiopathic deep vein thrombosis compared with patients with secondary DVT and controls (OR 2.3 95% CI 1.4–3.7) (74). Additional comparative studies have shown that patients with arterial thrombosis (myocardial infarction and stroke) also had an increased relative risk of venous thrombosis in the first 3 months following the events mentioned above (75). In some patients, VTE might occur as the first symptomatic cardiovascular event (76, 77). Recently, a number of studies have found many common links between these two different clinical conditions that supported the finding that patients with venous thromboembolism and arterial thrombosis share common risk factors (78, 79). In addition to all these positive studies, it should also be mentioned that there are those who have not found any relationship (80, 81). The incidence of arterial and venous thrombosis increases exponentially with age due to increased activation of blood coagulation and fibrinolysis (82). Results of a meta-analysis

by Ageno and colleagues from 2008 confirmed the hypothezis that cardiovascular risk factors for obesity (OR 2.33 95% CI 1.68–3.24), hypertension (OR 1.51 95% CI 1.23–1.85), diabetes mellitus (OR 1.42 95% CI 1.12–1.77), smoking (OR 1.18 95% CI 0.95–1.46) and hypercholesterolaemia (OR 1.16 95% CI 0.67–2.02) are associated significantly with an increased risk of VTE (83). Patients with idiopathic VTE are at a higher-risk for complications of arterial thrombosis compared with those with VTE caused by well-known factors. Obesity is a well-known acquired risk for venous thrombosis due to immobility and venous stasis. In addition, abdominal obesity is a well-known component of the metabolic syndrome which is also an established risk factor for atheromatosis. In 2006, Ageno et al. first described the metabolic syndrome as an independent predictor of idiopathic DVT (OR 1.93 95% CI 1.05–3.56) (84). In this study, patients with unexplained DVT had a significantly higher prevalence of the metabolic syndrome compared with controls. The patients with metabolic syndrome had increased platelet activity, increased plasma concentrations of PAI-1 and coagulation factors VIII, VII, XIII, and decreased production of nitric oxide and prostacyclin due to endothelial cell dysfunction. Procoagulant activity due to metabolic syndrome may act as a link between VTE and atherosclerosis. It is a result of the increased lipoprotein concentrations, increased amounts of circulating microparticles and increased proatherosclerotic mediators released from adipose cells (85). New results from the Copenhagen City Heart Study (2010) confirmed that obesity and smoking, two well-known cardiovascular risk factors, were both important risk factors for VTE body mass index whazard ratio (HR) for G35 vs. -20s2.10 (95% CI 1.39–3.16)x; smoking wHR for G25 g tobacco per day vs. never smokers1.52 (95% CI 1.15–2.01)x; gender wHR for men vs. womens1.24 (95% CI 1.08–1.42)x (86). This new perception suggested a closer link between the two different clinical conditions. However, the strength of this association is still controversial (87). A positive association between diabetes mellitus, dyslipidaemia with VTE and atherothrombosis can be explained by endothelial dysfunction, increased procoagulant concentrations and platelet activation and dysfibrinolysis. An improved understanding of inflammation, the role of lipoproteins, local hypercoagulability and endothelial injury integrates these processes, suggesting that venous and arterial thrombosis are two aspects of the same disease (73). The data indicate that inflammation of the vessel wall initiates thrombus formation, and inflammation and coagulation systems are coupled by a common activation pathway. According to current understanding, inflammation appears to be a common pathogenic determinant. Leukocytes are observed approximately 2 min after any type of endothelial cell injury and its proinflammatory mediators can further enhance thrombosis. It has been hypothesized that selections, expressed after a thrombogenic stimulus, facilitate these proposed interactions between leukocytes and endothelial cells, leukocytes and leukocytes, and leukocytes and platelets. Neutrophils and platelets then become activated, generating other mediators that amplify thrombosis (73, 88–90). Structural (increased col-


lagen and calcium content) and functional (degeneration of elastic fibrils) changes in the vessel wall during aging, or increased concentrations of plasma coagulation factors increase the prothrombotic risk (63). The formation of venous as well as arterial thrombosis is a result of complex gene-gene and gene-environment interactions involving many risk factors (72). Which clinical manifestations of thrombosis develop in genetically predisposed individuals is dependent on environmental and acquired risk factors. The most recent results from the Iowa Women’s Health Study has confirmed this hypothesis. The risk for VTE was increased in elderly women who were smokers, physically inactive, overweight and diabetic, indicating that lifestyle contributes to risk of VTE (91).

Epidemiology The prevalence of the hypercoagulable state depends on the ethnicity and clinical history of the population studied (63, 92). Inherited conditions are associated with venous thrombosis rather than arterial thrombosis (93). The incidence of venous thrombosis increases with age and number of inherited risk factors. In contrast, the influence of inherited risk factors in patients with arterial thrombosis declines with age. These individuals usually have more than one acquired factor rather than rare thrombophilic disorders (7, 63, 94). Testing for an inherited hypercoagulable state is successful in more than 60% of patients presenting with idiopathic venous thromboembolic disease, and informative in a very restricted population with arterial events (non-arteriosclerotic thromboembolic event in a young person or in population with premature occlusion after revascularization procedures) (92,

95). The association between inherited thrombophila and arterial thrombosis is weak (96). Mutations in factor II and FV Leiden are the two most common inherited causes of a hypercoagulable state in Caucasians. In Europe, the prevalence of FV Leiden and factor II varies across different geographical regions. The frequency of FV Leiden in Croatian healthy volunteers was 2.9%, in Greeks 7%, and Scandinavians 15% (59, 97–99). The prevalence is lowest in the general population, greater in individuals with a single thrombosis, and the highest in those with recurrent thrombosis or in family member who are thrombophilic (59). These mutations are rare in Asians and Africans and ethnic groups of Asian decent (Inuit Eskimos, Native Americans, Australian aborigines) (97, 98, 100). The prevalence of mutations in two natural anticoagulants, protein C and protein S, is higher in South Asians compared with Caucasians. These mutations were observed in up to 3% of Caucasians and in 6% of Southeast Asians with venous thromboembolism (100, 101). Asians also had the highest PAI-1 activity (102). Factor VIII is an important prothrombotic risk factor in the UK black population (levels )150 U/L; more than a 10-fold increased risk of VTE in blacks and is found in 35% of patients with VTE (102). The phenotypic frequency and genotypic frequency of PlA2 was estimated to be 26.5% and 15%, respectively, in individuals from Northern and central Europe (103). The antiphospholipid syndrome is the most common acquired condition associated with the hypercoagulable state. The risk for thrombosis increases with increased amounts antibody and lupus anticoagulants (104). Patients with more than one inherited thrombophilia defect are often at significantly greater risk of thrombosis than those

Table 2 Prevalence of genetic disorders associated with the hypercoagulable state and relative risk for venous thrombosis. Hypercoagulable state Genetic disorders

Factor V Leiden (G1691A) qProthrombin G20210A qOral contraceptives Factor V Leiden (A1691A) Oral contraceptives Prothrombin G20210A Protein C x Protein S x Antithrombin x Dysfibrinogenaemia Hyperhomocysteinaemia ≠ Factor VIII ≠ Factor IX ≠ Factor XI ≠ Lipoprotein (a) ≠ Thrombin-activatable fibrinolysis inhibitor (TAFI) Antiphospholipid antibody Lupus anticoagulant Anticardiolipin antibody

Frequency, %

Relative risk

Normals

VTE patients

Venous thrombosis

0.05–4.8

18.8

0.02

1.5

0.06–2.7 0.2–0.4 0.16–0.21 0.02 -0.01 5–7 11 10 10 7 9

7.1 3.7 2.3 1.9 0.8 10 25 20 19 20 14

4 20 30 80 2 2.8 6.5 5.0 20 Unknown 2.95 4.8 2.8 2.2 3.2 1.7

0–7

5–15

5.5 11 3.1


with only a single genetic risk factor. The risk of thrombosis is usually expressed as relative risk and depends on the underlying cause of hypercoagulability. The prevalence of genetic disorders associated with the hypercoagulable state and relative risk for venous thrombosis is shown in Table 2 (105–107). According to data from the literature, the relative risk of thrombosis is two times higher in patients with the prothrombin gene mutation, three times higher in patients with the mutation for FV Leiden, and six times higher in patients who are carriers of both mutations when compared to patients with the wild type genotype. The risk increases dramatically following exposure to some acquired conditions, such as oral contraceptives. FV Leiden and oral contraceptives raise the risk by 30-fold (108).

Conclusions The hypercoagulable state is not a uniform disease, it is a complex condition that may or may not lead to thrombosis as a result of complex gene-gene and gene-environment interactions. Disorders of the hypercoagulable state due to thrombosis of veins and arteries are the most important cause of sickness and death in developed countries throughout the world today. It is well-known that tests for defects of inherited thrombophilia are not helpful in discovering the aetiology of arterial thrombosis, but are very useful in detecting venous thromboembolic disease. Inherited thrombophilia can be detected in more than 60% of patients with VTE. Screening tests for a primary hypercoagulable defect (inherited thrombophilia) are useful in individuals with thrombosis at an early age, a family history of thromboembolic disease, unusual sites of thrombosis, or recurrent thrombosis. There are no specific prophylactic therapies for any inherited conditions associated with the hypercoagulable state. Thrombosis associated with inherited thrombophilia is very often precipitated with some acquired conditions. Adequate identification of risk factors is essential in making decisions about the duration of anticoagulant therapy and prevention of recurrence (53, 108). Until recently risk factors for arterial and venous thrombosis have been considered to be strictly distinct. New epidemiological data and clinical trials have suggested that many acquired risk factors overlap and coexist for both disorders. Lifestyle changes, or timely recognition of acquired risk factors can reduce morbidity and mortality from these conditions.

Conflict of interest statement Authors’conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Diagnostic algorithm for thrombophilia screening

Sandra Margetic* Department of Laboratory Coagulation, University Department of Chemistry, Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia

Abstract Thrombophilia screening is aimed at detecting the most frequent and well-defined causes of venous thrombosis, such as activated protein C resistance/factor V Leiden mutation, prothrombin G20210A gene mutation, deficiencies of natural anticoagulants, such as antithrombin, protein C and protein S, the presence of antiphospholipid antibodies, hyperhomocysteinemia and increased factor VIII activity. At this time, thrombophilia screening is not recommended for those possible congenital or acquired risk factors, whose association with increased risk of thrombosis has not been proven sufficiently. Laboratory investigations should include a stepwise approach to the diagnosis of thrombotic disorders with respect to the assays and methods of analysis that are used. The assays recommended for the first diagnostic step of screening should establish, whether the subject has one of the common causes of thrombophilia. If one or more abnormal results are obtained, the second diagnostic step includes the assays recommended for confirmation and/or characterization of the defect. When performing the investigation of thrombophilia, it is important to consider all pre-analytical and other variables that may affect the results of thrombophilia testing, including time of testing, age, gender, liver function, hormonal status, pregnancy or the acute phase response to inflammatory diseases. This is necessary, in order to avoid, any misinterpretation of the results. This review summarizes the current knowledge concerning thrombophilia investigations, with special focus on the diagnostic algorithm regarding patient selection, the assays and methods of analysis used and all the variables that should be considered when employing tests for the diagnosis of thrombophilia. Clin Chem Lab Med 2010;48:S27–39. Keywords: diagnostic algorithm; laboratory investigation; risk factors; thrombophilia; venous thromboembolism. *Corresponding author: Sandra Margetic, M.Sc., Department of Laboratory Coagulation, University Department of Chemistry, Medical School University Hospital Sestre Milosrdnice, Vinogradska 29, 10000 Zagreb, Croatia Phone: q385 1 3787 115, Fax: q385 1 3768 280, E-mail: [email protected] Received June 8, 2010; accepted September 5, 2010; previously published online November 6, 2010

Introduction The term thrombophilia is defined as the tendency to develop thrombosis due to pre-disposing factors that may be genetically determined, acquired, or both. Thrombosis may occur in both venous and arterial vessels, but thrombophilia is usually considered within the context of venous thromboembolism (VTE) (1–3). Although hereditary thrombophilia is associated primarily with VTE, it can also manifest with an arterial event specifically in the setting of a paradoxical embolism (4). Furthermore, some of the well-defined acquired risk factors for VTE, such as the presence of antiphospholipid antibodies (aPLAs) and hyperhomocysteinemia (HHC), can also present with arterial thrombosis. However, since at present there is no established causal relationship between hereditary thrombophilic risk factors and arterial thrombosis, testing for heritable thrombophilia is not indicated in patients with arterial thrombosis (5), but is directed primarily towards patients with VTE. Additionally, another indication for thrombophilia testing occurs in women with a history of adverse outcomes during pregnancy including severe pre-eclampsia, placental abruption, intrauterine growth restriction and unexplained consecutive first trimester abortions and second and third trimester unexplained fetal loss (6, 7). During the past few decades, the clinical importance of thromboembolic events has progressively increased. Today, VTE is a severe condition in all areas of medicine due to its endemic nature (8, 9). Each year, VTE affects approximately 1–2 individuals per 1000 in the general population of Western countries (10). Over the last two decades, knowledge on the etiology of VTE has increased considerably, and various hereditary and acquired risk factors have been discovered. As a consequence, the investigation of thrombophilia has contributed considerably to pressure on clinical laboratories, with enormously increased ordering of tests over the last 10 years. To date, well-defined risk factors for VTE include resistance to activated protein C (APCR)/factor V Leiden (FVL) mutation, prothrombin G20210A gene mutation (FIIG20210A), deficiencies in the natural anticoagulants antithrombin (AT), protein C (PC) and protein S (PS), the presence of aPLAs and HHC. In addition, since recent studies have clearly shown persistently increased concentrations of factor VIII (FVIII) to be an independent risk factor for VTE, it should be included in thrombophilia screening. In addition to these inherited and acquired risk factors for VTE, several environmental or transitional conditions, such as advancing age, malignant diseases, trauma, surgery, pregnancy and puerperium, immobilization and estrogen therapy, are asso-

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ciated with an increased risk for VTE (11–14). These conditions not only pre-dispose apparently healthy individuals to thrombosis but may also trigger thrombosis in persons with thrombophilic risk factors. Each individual risk factor constitutes an element of increased risk for VTE, which is compounded when several different conditions are present. Hence, individuals with multiple defects have considerably increased risk for VTE (15, 16). Furthermore, interactions between hereditary and transitional risk factors significantly increase the risk of VTE, suggesting the multifactorial nature of VTE (17). Thus, VTE often occurs in susceptible patients having one or more thrombophilic abnormalities when they are exposed to exogenous prothrombotic stimuli (18). However, single genetic or acquired risk factors do not necessarily lead to thrombosis without interaction with other genetic or environmental risk factors. When a thrombotic episode occurs, it is important to establish whether it represents an isolated event caused by a transitional condition, or whether the patient has an underlying genetic and/or acquired predisposition to life-long risk for thrombosis. At this time, the laboratory investigation of thrombophilia permits recognition of underlying inherited thrombotic risk factors in approximately 50%–60% of patients being evaluated (19). The aim of this review is to summarize the current knowledge concerning the laboratory diagnosis of thrombophilia. The review focuses on the most important aspects of laboratory investigation, with special interest on the diagnostic algorithm regarding careful patient selection, the assays and methods of analysis used, and all the pre-analytical variables and other conditions that should be considered when employing tests for the diagnosis of thrombophilia.

General guidelines for laboratory investigation of thrombophilia Thrombophilia screening of an unselected population is not indicated. In general, the prevalence of any known risk factor for VTE is not sufficient to justify indiscriminate screening of the general population (20–23). Furthermore, the diagnostic investigation of thrombophilia is not indicated either in unselected patients presenting with a first episode of VTE, but is medically and economically justifiable in patients who have a history of unexplained thromboembolism. Therefore, all subjects with a confirmed episode of VTE should be considered for thrombophilia investigation if at least one of the following is present: thrombosis prior to the age of 50 years, recurrent spontaneous thrombosis, family history of VTE or thrombosis in unusual sites, such as portal, mesenteric, splenic, hepatic, renal or cerebral veins (24, 25), as shown in Figure 1. The initial investigation should begin with a complete personal and family medical history. Although a family history of thrombosis is a significant selection factor, it is important to note that a negative family history does not exclude a thrombophilic abnormality because the defects may have low penetrance and new mutations can occur (26). Because of the beneficial effect that thrombophilia screening may have on asymptomatic individuals, laboratory inves-

tigation is also justified for first degree family members of the symptomatic patient with previously identified inherited thrombophilic risk factors (27, 28). Thrombophilia screening in certain populations with a well-known increased risk for VTE, such as pregnant women and those on oral contraceptives is continually debated in the literature (29). Although indiscriminate testing of all pregnant women or those prior to prescription of oral contraceptives is not supported by the of majority investigations to date (5, 30), thrombophilia screening is indicated in selected patients with a previous history of VTE or who have a positive family history (5, 31). Furthermore, the results of some studies have shown that relatively weak inherited risk factors, such as heterozygosity for FVL and FIIG20210A mutations can be associated with spontaneous VTE in subjects )50 years of age (32, 33). This suggests that increased age acts as an additional risk factor (34). These results indicate that age at the time of VTE should not be taken as strict criteria to determine laboratory testing. The laboratory investigation of thrombophilia is aimed at detecting the common and well-established causes of thrombophilia in carefully selected patients with VTE. Thrombofilia testing should begin with global coagulation tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen concentrations in order to rule out anticoagulant therapy and other coagulation disorders. The PT test indicates the effect of any anticoagulant treatment and functional status of the liver. The APTT and TT can reveal heparin therapy or contamination that interferes with some functional assays. Laboratory investigations should include a step-wise approach to the diagnosis of thrombotic disorders regarding the assays and methods of analysis used. As shown in Figure 1, the first diagnostic step should be restricted to the high priority tests for the most frequent and well-established causes of thrombophilia, including APCR/FVL, FIIG20210A mutation, deficiencies of natural anticoagulants AT, PC and PS, the presence of aPLAs, homocysteine (HC) and FVIII concentrations (35, 36). The general strategy of thrombophilia evaluation is to investigate individually each of these welldefined risk factors know to be associated with increased risk of thromboembolism. Since combined defects are common and their detection is clinically relevant because of the highly increased risk for thrombosis, a complete investigation should always be performed, even if one defect has already been identified. If none of the high priority test results are abnormal in patients with a family history of VTE or recurrent thrombosis, it is reasonable to perform low priority tests, such as the laboratory evaluation of dysfibrinogenemia (5, 37). Moreover, in the case of patients with unexplained intraabdominal thrombosis (portal, splenic, mesenteric or hepatic vein thrombosis), there is a need to consider molecular testing for Janus-activating kinase-2 (JAK-2) mutation to exclude an underlying myeloproliferative disorder and to evaluate CD55/CD59 expression by flow cytometry of peripheral blood to exclude paroxysmal nocturnal hemoglobinuria (PNH) (27, 38–40).

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Figure 1 Diagnostic algorithm in the thrombophilia screening in patients presenting with VTE.

At this time, the laboratory evaluation of thrombophilia is not recommended for other possible congenital or acquired risk factors, such as abnormalities of the fibrinolytic system (increased concentrations of plasminogen activator inhibitor-1, deficiencies of plasminogen or tissue plasminogen activator), deficiency of heparin cofactor II (HCII), increased concentrations of fibrinogen, FVII, FIX, FXI, or deficiency of FXII, whose association with an increased thrombotic risk has not been sufficiently proven at present (36, 41).

The recommended assays in the first diagnostic step for screening should establish, whether the subject has one of the common causes of thrombophilia. If one or more abnormal results are obtained, a second diagnostic step should include recommended assays for the confirmation and/or characterization of the defect (Figure 1). The laboratory tests chosen in the investigation of thrombophilia should be specific and the results should be clinically relevant. Methodology issues in thrombophilia testing


are important because some methods for certain test are better than others. Hence, careful selection of specific recommended assays should be undertaken in order to ensure both test sensitivity and specificity (21, 42). Laboratory experts are in the best position to recognize the limitations of the tests they use, and they have an obligation to provide clinicians with both pre-test education and the limitations of the assays used. Laboratories should identify and report the most common pre-analytical causes of potential false-positive and false-negative results for all tests employed (2, 42). Also, quality assurance should be maintained through use of internal quality control as well as external quality assessment programmes in order to minimize errors (43–45). When performing the investigation of thrombophilia, it is particularly important to consider all of the pre-analytical and other variables that may affect the results of thrombophilia testing, including time of testing, age, gender, liver function, hormonal status, pregnancy or an acute phase response to inflammatory diseases in order to avoid misinterpretation of the results (21, 46). The appropriate time of testing is of critical importance because the acute phase of thrombosis and anticoagulant therapy considerably affect the results of many phenotypic assays, making interpretation of the results difficult (30, 46). Since the clinical management of an acute thrombotic event is not influenced by the immediate demonstration of a specific defect, the laboratory investigation should be delayed for 6 months after the acute phase of thrombosis, and at least 2 weeks after anticoagulant therapy has been discontinued. However, it is important to note that in some exceptional cases, immediate certain thrombophilic tests are indicated, such as testing for aPLAs in patients with catastrophic antiphospholipid syndrome (APLS) which may require more aggressive and/or prolonged therapy. Also, as discussed later in the section on natural anticoagulant deficiencies, in the case of patients with possible hereditary AT deficiency where the use of higher doses of heparin or AT concentrates may be life saving, determination of AT is indicated even in the acute phase of thrombosis and anticoagulant therapy. Furthermore, when evaluating pediatric patients for inherited thrombophilic risk factors, such as AT, PC and PS deficiencies, it is important to note that pediatric reference ranges are necessary because age-related changes occur in the concentrations of these proteins. It is recommended that appropriate age- and gender-dependent reference ranges for each parameter being evaluated be employed (2). Ideally, reference ranges should be determined for the method and test system being used locally, with the normal donors recruited from the local population. All pre-analytical and other variables that affect the results of thrombophilia testing will be discussed in the next section that describes the laboratory aspects in the evaluation of individual risk factors. In order to confirm any positive result in the investigation of thrombophilia that is obtained with non-genotyping tests, it is recommended that a second analysis be performed using a newly collected blood sample for any case with questionable or positive results (2, 21). Moreover, genetic defects can

be definitely demonstrated through the investigation of first degree relatives.

Laboratory aspects in the evaluation of individual risk factors in the thrombophilia screening Activated protein C resistance (APCR) and factor V Leiden (FVL)

In 1993, Dahlback et al. first described a hereditary defect in the anticoagulant response to activated protein C (APC), known as APCR, that was associated with an increased risk for VTE (47). One year later, Bertina and colleagues at the University of Leiden identified the molecular basis of this defect (48). In over 90% of cases, inherited APCR is caused by a single point mutation in the factor V gene, known as FVL. The mutation results in inefficient inactivation of activated factor V (FVa) by APC, because the mutant FV is much more resistant to proteolytic degradation by APC. This mutation is the most common inherited risk factor for VTE in whites, with different prevalence in different populations and geographical regions. In Europe, the frequency of the FVL mutation is lowest in North Europe and highest in Southern Europe (49, 50). The FVL mutation is rarely found in African, Australian and South Asian populations (49). However, FVL accounts for most, but not all, cases of APCR. In the absence of FVL, an acquired APCR phenotype may be present during pregnancy, use of oral contraceptives, in the presence of lupus anticoagulants (LA) or with increased concentrations of FVIII, and in patients with myeloma who develop thrombosis (51–53). Recent studies have shown that acquired APCR is a risk factor for VTE, independent of FVL (51). The laboratory evaluation of APCR/FVL includes functional coagulation assays and genotyping for FVL by DNA analysis (54). Functional assays identify individuals who have resistance to APC, while DNA analysis identifies FVL mutation as the cause of the APCR. The most commonly used phenotypic screening test for APCR is based on prolongation of the APTT by the addition of APC, as originally described by Dahlback. Results are expressed as an APC ratio; the ratio between the APTT measured in the presence and then the absence of added APC. Although simple and inexpensive, this method is not sufficiently sensitive and specific for FVL. In the modified second generation of APTTbased methods for APCR, patient plasma is pre-diluted (1q4) with factor V-deficient plasma. This modification of the APCR assay is highly sensitive and specific for FVL mutation and can even be used in patients taking vitamin K antagonists (VKA) (27, 55). Standardization of the APTTbased APCR assay may be further improved by determination of a normalized ratio where the assay ratio is divided by the ratio of pooled normal plasma or standard human plasma analyzed in the same test run (42, 56). A general strategy to evaluate APCR/FVL is first-line screening by the APCR functional phenotypic test using the second generation APTT-based assay with FV-deficient plas-


ma. This test is automated, cost-effective and detects causes of APCR other than FVL. Borderline and positive results should undergo genotyping to confirm heterozygosity or homozygosity for FVL. A search for FVL by DNA analysis alone would not identify cases of APCR caused by acquired conditions. The variables that affect test results of phenotypic assays for APCR/FVL are shown in Table 1. Prothrombin G20210A mutation

In 1996, Poort et al. described the mutation of the factor II (FII) gene which results in increased concentrations of functionally normal prothrombin in plasma due to increased synthesis (57). FIIG20210A mutation is the second most prevalent inherited risk factor for VTE, associated with a three-fold increase in risk. The mutation is present in approximately 2% of the general population and in 6%–7% of unselected patients with VTE (58). Although prothrombin activity is often raised mildly in carriers of the mutation, there is an overlap of values in subjects with and without mutation using the available functional test for FII activity. Therefore, the measurement of prothrombin activity in plasma is not an adequate test to screen thrombophilic patients for this mutation because it is unable to clearly distinguish carriers from non-carriers of the mutation (59). Thrombophilia investigations should include DNA analysis of the FIIG20210A mutation only (60), as shown in Figure 1. The acute phase of thrombosis and anticoagulant therapy do not affect DNA based assays. Deficiencies of natural anticoagulants

Deficiencies of natural anticoagulants AT, PC and PS are less common genetic risk factors for thrombosis compared with APCR and FIIG20210A mutation, being present in -1% of the general population. Together they account for only 1%–5% of genetic defects found in patients with VTE (61, 62). AT deficiency was the first recognized inherited risk factor for VTE, described by Egeberg in 1965 (63). Later in the 1980s, deficiencies of PC and PS were identified as causes of inherited thrombophilia (64, 65). Since congenital deficiencies of natural anticoagulants are caused by a large number of different mutations, DNA testing is generally not available for thrombophilia investigations, outside of specialized research laboratories. Instead, the laboratory evaluation of natural anticoagulants includes functional and immunochemical assays. When considering a diagnosis of hereditary deficiencies of AT, PC and PS, it is of great importance to exclude the

causes of acquired deficiencies since a large number of conditions are associated with a reduction in plasma concentrations of natural anticoagulants (Table 2). Also, there are numerous other variables that should be taken into consideration when employing thrombophilia assays for the natural anticoagulants, as shown in Table 2. However, in spite of a general recommendation to avoid thrombophilia testing for natural anticoagulants in patients on anticoagulant therapy and during the acute phase of thrombosis, in case of suspected hereditary AT deficiency, for example in patient with VTE from a family with known AT deficiency, measurement of AT is justified because it can modify therapy with higher doses of heparin, or even with AT concentrates. Further, in cases of suspected heparin resistance due to markedly decreased AT values, determination of AT is also justified because these patients may require very high doses of heparin in order to obtain an adequate therapeutic effect. However, in order to diagnose congenital AT deficiency, a decreased value of AT obtained during the acute thrombotic event or during heparin therapy must definitely be verified after the acute phase of thrombosis and anticoagulant therapy have been terminated. There are two major types of AT and PC deficiencies. Type I deficiency (quantitative) is caused by decreased synthesis of a biologically normal molecule, resulting in reductions in both the activity and concentration. Type II is a qualitative defect, resulting in decreased functional activity, but normal concentrations. In the first diagnostic step of laboratory investigation, the tests aimed at detecting AT and PC deficiencies should include functional assays capable of identifying both type I and type II abnormalities. Immunoassays are useful in combination with functional assays for the classification of the type of deficiency. If the result of functional assay is decreased, the immunochemical (antigenic) assay allows differentiation between type I and type II deficiencies (Figure 1). The immunochemical assay should not be performed without the functional assay because type II deficiencies in AT and PC will not be detected. Commercially available functional assays for AT are chromogenic methods in which AT activity can be measured according to its ability to inhibit thrombin or activated factor X (FXa). (66). Chromogenic methods with FXa are considered more appropriate compared with those based on thrombin as the target enzyme because they are not affected by the presence of other inhibitors of thrombin in plasma, i.e., HCII. On the basis of the nature of the functional defect, type II AT deficiency is subdivided into three subtypes depending

Table 1 Variables that can affect the results of APCR/FVL phenotypic assays. Variable

Impact on result

Recommendation

Contamination of plasma sample with platelets, particularly in frozen and thawed samples Anticoagulant therapy with heparin, hirudin, argatroban Heparin contamination

False-positive result

Double centrifugation of plasma sample is advised if frozen samples are used Do not perform testing in patients on therapy with these anticoagulants TT test can be used for excluding heparin contamination

False-positive result False-positive result

Decreased AT and PC because of increased consumption Lower concentrations of free PS because of increased C4B-BP as acute phase reactant At birth levels of AT, PC and PS are decreased until the age of 6 months for AT and PS and until adolescence for PC PS in newborn is largely or entirely in the free form, because C4B-BP is low or undetectable Healthy women have slightly lower PS levels than men, and total PS concentrations increase with age in women Decreased AT and PS

Acute phase of thrombosis

Reduced level of free PS may be obtained because of increased binding to C4B-BP Falsely low PS result

Chromogenic functional assays are recommended as test of choice for PC in order to avoid interferences Abnormal result of functional assay for PS should always be evaluated with an immunoassay for free PS In case of low PS activity it is advised to determine FVIII activity and a routine marker of acute phase (CRP, fibrinogen) Double centrifugation of plasma sample is advised if frozen samples are used

Avoid testing: – in pregnant women until 6 weeks postpartum – in women taking oral contraceptives or HRT for three months with the exception of AT measurement in the cases listed above concerning to the anticoagulant therapy Acquired states of natural anticoagulants deficiencies should be considered and excluded prior to thrombophilia investigation

Avoid testing in patients on anticoagulant therapy. There are some exceptional cases in which AT measurement is needed in spite of acute phase of thrombosis or heparin therapy: – patients with suspected congenital AT deficiency, as in case of patient with VTE from a family with known AT deficiency – patients with suspected heparin resistance because of markedly reduced levels of AT In order to diagnose congenital AT deficiency in these cases, AT measurement must definitely be verified after the acute phase of thrombosis and anticoagulant therapy. Avoid testing in the acute phase of thrombosis with the exception of AT measurement in the cases listed above concerning to the anticoagulant therapy For pediatric patients, separated reference ranges should be constructed due to different levels of AT, PC and PS. It is recommended to determine separate male and female reference ranges of PS for adult population.

Recommendation

VKA, vitamin K antagonists; DIC, disseminated intravascular coagulation; C4B-BP, C4B-binding protein; CRP, C-reactive protein.

Increased levels of C4B-BP in inflammatory states Contamination of plasma sample with platelets, particularly in frozen and thawed samples

Clot-based functional assays for PC and PS

Pregnancy and puerperium Estrogen therapy oral contraceptives (OC) or hormone replacement therapy (HRT) Acquired states of PC, PS and AT deficiencies

AT: liver disease, sepsis, pre-eclampsia, nephrotic syndrome, pregnancy and puerperium, surgery, post-traumatic state, therapy with L-asparaginase PC: DIC, acute thrombosis, sepsis, vitamin K deficiency, liver disease, postoperative state PS: DIC, acute thrombosis, vitamin K deficiency, liver disease,pregnancy and puerperium, nephrotic syndrome, inflammatory illness of any cause Falsely low values of PC and PS may be obtained due to APCR/FVL or FVIII)150%, and falsely increased values in the presence of LA or therapy with heparin or new anticoagulant drugs (hirudin, argatroban)

AT activity decreases up to 30% during therapy with heparin VKA cause notably reduced levels of PC and PS since both are vitamin K-dependent proteins

Anticoagulant therapy: heparin and vitamin K antagonists (VKA)

Age and gender

Impact on result

Variables

Table 2 Variables that affect the test results for natural anticoagulants AT, PC and PS.

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S32 Margetic: Thrombophilia – diagnostic algorithm


on whether the mutation affects the heparin binding site (HBS), reactive site (RS) or has pleiotropic (multiple) effects (PE) (56). Among these defects, HBS mutations are associated with a low-risk of thrombosis. This increases the clinical relevance of distinguishing the subtypes of AT deficiencies (67). Crossed immunoelectrophoresis, with and without heparin, or a variant of functional chromogenic assay that measures progressive inhibitory activity may be used to identify subtypes of type II AT deficiencies (56, 67). Commercially available functional assays for PC are either clot-based or chromogenic methods (42, 56). Chromogenic methods are generally recommended because clot-based assays are affected by several interferences, such as the presence of LA, increased FVIII concentrations ()150%) and APCR (Table 2). Although at present, there is no evidence that distinguishing between type I and type II PC deficiency is clinical relevant, immunochemical PC assays are often routinely employed in cases of reduced functional activity, since it can serve as a useful additional quality control step by comparison with functional PC levels (42). Immunochemical assays for AT and PC measurements include enzyme-linked immunosorbent assays (ELISAs) and newer automated immunoturbidimetric assays. The laboratory investigation of PS deficiency is more complex than that for AT and PC because of the lack of well-standardized functional assays. There are three types of congenital PS deficiencies on the basis of PS activity and free and total PS antigen concentrations (Table 3). Type I and type III PS deficiency are quantitative defects with both low free PS antigen concentrations and low PS activity, and account for 95% of cases (68). However, type I deficiency is associated with low total PS antigen, whereas type III deficiency is associated with normal PS antigen. PS type II deficiency accounts for approximately 5% of cases, representing a qualitative defect where concentrations of both total and free PS antigen are normal, but PS activity is diminished (68). The laboratory evaluation of PS deficiency should include a functional assay and immunological assay for PS antigen (69). A functional assay can be used as the initial screening assay because it detects all three types of deficiency. If only antigenic assays are performed in the first diagnostic step, type II deficiencies will not be detected. Functional assays for PS are clot-based methods that are widely used for initial screening of thrombophilia. These methods measure the ability of PS to serve as a cofactor to APC, enhancing its degradation of activated FV and FVIII, thereby prolonging clotting time. However, the limitations of these methods regarding interferences of LA, increased FVIII concentraTable 3 Classification of PS deficiencies on the basis of PS activity and free and total PS antigen concentrations.

Type I Type II Type III

PS activity

Free PS

Total PS

Decreased Decreased Decreased

Decreased Normal Decreased

Decreased Normal Normal

tions and APCR should be taken into consideration (42, 70), as shown in Table 2. Therefore, abnormal (low) result for PS obtained using functional assay should be further investigated using immunochemical assays (5, 71). Immunochemical methods, including ELISA and newer automated immunoturbidimetric assays, can measure total and the free PS concentrations, depending on the assay design. Free PS antigen assays measure only unbound or free PS, while total antigen assays measure bound (inactive) and unbound (free and active) PS. Namely, in plasma PS is present in two forms: 60% of entire PS is in a complex with the C4B-binding protein (C4B-BP), while 40% is present in an free form, which is the active component of PS. In order to determine the free antigen, in older methods, bound PS is precipitated using polyethylene glycol (PEG), followed by measurement of the remaining PS portion in the supernatant. Newer methods use specific monoclonal antibodies for free PS, which makes the precipitation step unnecessary. It is generally considered that free PS antigen, being closely related to the functional form of PS, shows better discrimination between subjects with and without PS deficiency compared with total PS concentrations (69, 72). If free PS antigen is decreased only, a total antigen assay can be performed to further determine the deficiency subtype (type I and type III), although it is not necessary to routinely measure total PS antigen (72). In addition to the functional and immunochemical assays for PC and PS natural anticoagulants described above, a commercial global screening test for the PC pathway was developed as an attempt for the global testing of the entire potential of the PC anticoagulant pathway (35, 73). Defects that affect the PC pathway are PC or PS deficiencies and APCR. However, these global tests for PC anticoagulant system have shown acceptable diagnostic efficacy only for APCR and PC deficiency, but not for PS deficiency where variable proportions of patients with this deficiency were not identified using the global test (74, 75). Therefore, at this time, the available PC pathway functional assays cannot be proposed as a global test for the PC anticoagulant system in thrombophilia screening because of insufficient sensitivity (76). Antiphospholipid antibodies (aPLAs)

APLS, characterized by the presence of circulating aPLAs, represents an important cause of acquired thrombophilia associated with both VTE and arterial thromboembolism, recurrent fetal loss and thrombocytopenia (77). Diagnosis of APLS requires demonstration of persistently increased concentrations of aPLAs in the setting of thrombosis or certain complications of pregnancy (78). The aPLAs are a heterogenous group of autoantibodies directed against the phospholipid-protein complexes. Currently, no single test is sufficient for the diagnosis of aPLAs because of the lack of specific tests, and the heterogeneity of the antibodies (79, 80). Based primarily on the method of detection, there are three major subgroups of aPLAs: LA, anticardiolipin anti-


bodies (ACL) and anti-b2-glycoprotein-1 (anti-b2GP1) antibodies. LA antibodies are identified by functional coagulation assays, where they prolong clotting times. In contrast, ACL and anti-b2GP1 antibodies are detected by immunoassays that measure the immunological reactivity to a phospholipid or a phospholipid-binding protein. In accordance with recommended guidelines, the laboratory investigation of aPLAs should always include determination of all three groups of antibodies (78, 81). Lupus anticoagulants In vitro, LA prolong various clot-

ting times because of binding to phospholipids where they interfere with the ability of phospholipids to serve its essential cofactor function in the coagulation cascade. According to the International Society of Thrombosis and Haemostasis guidelines, laboratory detection of LA consists of a panel of coagulation-based assays that include 1. prolongation of at least one phospholipid-dependent coagulation test, 2. evidence of inhibitor activity in the test plasma determined by mixing tests with pooled normal plasma, and 3. confirmation that the inhibitory effect is due to blocking of phospholipiddependent coagulation by the addition of excess phospholipids (82). Therefore, patients may be considered to be LA positive if one or more phospholipid-dependent tests are prolonged, if the prolonged test is not corrected when plasma from the patient and a healthy control are mixed and if prolongation of the test is corrected by increasing the concentration of phospholipids in the test system. There is no definite recommendation on the assays of choice for LA testing. Because no one single screening test is 100% sensitive for LA, it is advised that two or more screening tests with different assay principles be used (83, 84). The most common used combination of LA screening tests includes the APTT, the mixing test and dilute Russell’s viper venom time (dRVVT) test; the later of which is considered to be the most sensitive. The test is performed with Russell viper venom that directly activates coagulation factor X leading to the formation of a fibrin clot. LA prolongs the dRVVT clotting time by interfering with phospholipids. The most common used confirmatory LA test is dRVVT which contains an excess of phospholipids that neutralize the effect of LA resulting in correction of the dRVVT screening test (85). Variables that affect the results of coagulation assays for LA are presented in Table 4.

Anticardiolipin antibodies and anti-b2-glycoprotein-1 antibodies The laboratory evaluation of ACL and anti-

b2GP1 antibodies is performed using ELISA methods (86). Both isotypes IgG and IgM of ACL and anti-b2GP1 antibodies should be measured. Generally, ACL antibodies are sensitive, but not particularly specific, while anti-b2GP1 antibodies have greater specificity for APLS (87–89). Testing for the presence of anti-b2GP1 has been shown to be very useful in patients that have persistently low titers of aPLAs. Patients who are anti-b2GP1 positive should be considered as having aPLAs. The laboratory criteria for the diagnosis of aPLAs should be positive on two or more separate occasions performed at least 12 weeks apart. This is of particular importance because the transient occurrence of aPLAs, which is sometimes observed in conjunction with microbial infection or drugs, is not associated with an increased risk of thrombosis. Among the variables affecting the results of ACL, it is important to note that false-positive IgM ACL result can be associated with rheumatoid factor and cryoglobulins (90). Hyperhomocysteinemia (HHC)

HHC, or increased plasma HC concentrations have been shown to be associated with an increased risk for VTE, as well as for arterial thrombosis (91–94). HHC may be caused by a congenital deficiency of enzymes, such as cystathionine-b-synthetase (CBS) and methylenetetrahydrofolate reductase (MTHFR) involved in its metabolism, or may be attributable to poor dietary intake of vitamins B6, B12 or folate that act as cofactors in HC metabolism (36). Homozygous deficiency of CBS, a rare hereditary defect with a prevalence in the general population only of 0.3%, is characterized by increased plasma concentrations of HC, the presence of HC in the urine, atherosclerosis, and arterial and venous thrombosis occurring at a young age. In contrast, mutation of the MTHFR gene appears in homozygous form in 10%–13%, and in heterozygous form in 30%–40%, of the general population (95). A common variant in the gene for MTHFR that results in a thermolabile variant of the enzyme has been shown to be associated with mildly increased HC concentrations. However, recent studies have indicated that MTHFR polymorphism does not predispose to HHC when

Table 4 Variables that affect the results of coagulation assays for the detection of lupus anticoagulants. Variables

Impact on result

Recommendation

Plasma sample with platelet count )10=109/L

False-negative LA result because of neutralization of LA

Acute phase of thrombosis

False-negative LA result because of possible consumption False-positive result for LA screening tests (APTT, mixing tests)

Double centrifugation of plasma sample is advised in order to avoid the interference of platelets Do not perform testing in the acute phase of thrombosis Do not perform testing in patients on any anticoagulant therapy

False-positive LA result False-negative results for LA screening tests (APTT, mixing tests)

FVIII activity may be measured in order to exclude acute phase reaction

Anticoagulant therapy: heparin and new anticoagulant drugs (hirudin, argatroban, danaparoid, bivalirudin) Vitamin K antagonists (VKAs) FVIII activity)150%


folate status is adequate. Only when combined with vitamin deficiencies, heterozygous subjects often have mildly increased HC concentrations. Most heterozygotes do not experience HHC or increased risk for thrombosis, unless they have other thrombotic risk factors. The recent MEGA Study, a large population-based case-control study by Bezemer and collaborators, has clearly shown that there is no association between the MTHFR 677CT polymorphism and risk for VTE (96). Therefore, genetic testing of the MTHFR polymorphism is not indicated in thrombophilia screening, as it has not been shown to be risk factor for thrombosis if HC concentrations are normal (97, 98). The laboratory diagnosis of HHC as a part of the investigation of thrombophilia should be performed exclusively by determining a fasting HC concentration in plasma, without genotyping for genetic defects. Until recently, HC concentrations have been measured primarily using high-pressure liquid chromatography (HPLC). Now, newer and simpler immunoassays are commercially available. These methods are more suited for use in the general clinical laboratory because they require simpler instrumentation and less technical expertise compared with HPLC (99). The acute phase of thrombosis and anticoagulant therapy do not affect the results of HHC investigations. EDTA or citrated plasma may be used, but plasma should be separated from cells within an hour of collection to avoid contamination with HC from red cells. Controlled blood collection and proper sample handling for HC measurement is essential to obtain valid results. Testing for HC concentrations should be done following an overnight fast of 10 h before collection of blood. The diet should be normal and not supplemented with vitamins in the few weeks preceding testing. Immediately after collection, the blood sample should be placed on ice or refrigerated at 2–88C until analysis. Elevated factor VIII

Increased FVIII concentrations were first associated with increased thrombotic risk by Koster et al. in 1995, as part of the Leiden Thrombophilia Study (100). This study has demonstrated that FVIII values )150% are an independent risk factor for VTE, with a relative risk of three-fold and a highrisk of recurrence. These findings were confirmed by O’Donnell et al. who found increased FVIII to be the common abnormality in patients referred for thrombophilia screening because of unexplained VTE (101). To date, no genetic variation in the FVIII gene has been identified that might account for this variation in phenotype. However, in 1998, Kamphuisen observed a positive correlation of FVIII concentrations within families (102, 103). Although the molecular basis of an increased FVIII is not presently known, the high reported prevalence of increased FVIII concentrations suggest that a genetic component may be at least partly responsible for factor increases. Among patients with VTE, the prevalence of increased plasma FVIII concentrations is approximately 20%–25%, and high FVIII persists over time

and appears to be independent of the acute phase response (104). The significant number of patients with isolated persistent increased FVIII concentrations as their only risk factor, as well as the increased risk of recurrent VTE in these patients suggests that routine screening for thrombophilia should also include measurement of this coagulation factor (105, 106). For thrombophilia screening purposes, assays that measure both the activity and antigen of FVIII are suitable. The activity of FVIII can be measured using APTT-based methods with FVIII-deficient plasma or by chromogenic methods. The antigen can be measured using ELISA-based methods. The most widely used methods are clot-based methods. Measurements of FVIII concentrations should be delayed for at least 6 months after an acute thrombotic event, since FVIII is an acute phase reactant. Furthermore, since FVIII concentrations are increased physiologically during pregnancy and the puerperium period, thrombophilia testing should be delayed for at least 6 weeks postpartum. In cases with increased FVIII concentrations as a result of thrombophilia screening, it is advised to repeat the testing 3–6 months after initial testing in order to confirm FVIII increases. Dysfibrinogenemia

Dysfibrinogenemias are a very rare, but heterogenous group of congenital disorders resulting in a structurally altered fibrinogen molecule that may cause altered fibrinogen function (107). These disorders may be clinically asymptomatic or may cause bleeding and venous or arterial thrombosis (108). The prevalence of the disorder in patients with VTE is 0.8% (108). Laboratory investigation of dysfibrinogenemias should include simple screening assays, such as TT and reptilase time (RT). These are typically prolonged in dysfibrinogenemias because of a dysfunctional fibrinogen molecule. Positive cases identified using TT and RT assays should be evaluated by parallel analysis of functional and immunoreactive fibrinogen. Functional fibrinogen assays show considerably lower concentrations compared with immunological assays that measure fibrinogen quantity. This is because fibrinogen function is impaired, but the quantity of fibrinogen is not. A decreased fibrinogen activity/antigen ratio is considered the confirmatory test for the diagnosis of dysfibrinogenemia. The most commonly used functional activity assay for fibrinogen is the Clauss method, while immunoreactive fibrinogen may be determined by ELISA methods or by newer automated immunoturbidimetric methods. As dysfibrinogenemia is very uncommon risk factor for VTE, laboratory evaluation of this disorder is not performed as a part of routine thrombophilia screening, but only if an initial panel of high priority tests excludes more common causes of VTE (5, 109), as shown in Figure 1. Since fibrinogen is an acute phase reactant, laboratory evaluation of dysfibrinogenemia should be delayed 6 months after acute thrombotic event.


Conclusions and perspectives The laboratory investigation of thrombophilia should be performed in accordance with the recommended evidence-based guidelines on testing, regarding who and when to evaluate and what tests and test methods to use. Following these guidelines is necessary in order to ensure that an accurate diagnosis is made. Excessive or inappropriate thrombophilia testing outside the recommended guidelines is likely to be more harmful than beneficial for the patient due to the possibility of misinterpretation of the test results. In addition, because of the lack of global tests for screening thrombophilic patients, the laboratory investigation of thrombophilia requires an expensive approach by performing a panel of different assays for each patient being evaluated. Future efforts should be aimed at developing a diagnostic strategy that makes testing more cost-effective by developing new screening tests able to assess globally the increased tendency for VTE. Since a substantial proportion of patients have no identifiable cause of thrombosis, future efforts should focus attention on the identification of additional risk factors for VTE. Also, studies in progress should investigate the relevance of interactions between different genetic risk factors, or interactions between genetic and acquired risk factors in the development of VTE. More knowledge of risk factors and particularly of their inter-relationships would help to improve our understanding of the mechanisms of thrombus formation in a variety of conditions, as well as enable us to individualize risk and guidelines for thrombosis prevention.

Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Genetic basis of thrombosis

Valeria Bafunno1 and Maurizio Margaglione1,2,* 1

Genetica Medica, Dipartimento di Scienze Biomediche, Universita` degli Studi di Foggia, Foggia, Italy 2 Unita` di Emostasi e Trombosi, I.R.C.C.S. ‘‘Casa Sollievo della Sofferenza’’, S. Giovanni Rotondo, Italy

Abstract Venous thrombosis (VT) represents a common and serious disorder that occurs as the result of clotting of the blood in the venous system and venous obstruction. Environmental risk factors and genetic predisposition play an important role in the development of thrombosis. It is therefore seen as a classic example of a complex common disease. We have focused on the role of genetic risk factors, primarily related to the hemostatic system, in triggering thrombotic events. Since the identification of antithrombin deficiency in 1965, major efforts have been made during the past 15 years to identify other genetic entities that lead to increased thrombotic risk. Results of early genetic studies demonstrated that two types of genetic defects cause VT: loss of function mutations in the natural anticoagulants antithrombin, protein C and protein S and gain of function mutations in procoagulant factors V (FV Leiden) and II (prothrombin G20210A). The high incidence of these mutations in Caucasians induced a shift from family studies to case-control association studies. Several investigations have been performed on the role of other candidate genetic risk factors predisposing to VT, including such variants in FXIII, FIX and fibrinogen genes. Moreover, the contribution of genetic variation in genes encoding less-well studied proteins that are part of the anticoagulant pathways has been evaluated. Recently, different genome-wide association studies have been performed in which several single nucleotide polymorphisms were investigated and related to the risk of VT. However, further studies are needed to identify additional genetic causes of thrombosis and to assess functional molecular mechanisms. Clin Chem Lab Med 2010;48:S41–51. *Corresponding author: Maurizio Margaglione, MD, Istituto di Genetica Medica, Dipartimento di Scienze Biomediche, Universita` degli Studi di Foggia, Viale Pinto, Foggia 71100, Italy Phone: q39 0881 733842, Fax: q39 0881 736082, E-mail: [email protected] Received July 6, 2010; accepted September 5, 2010; previously published online October 30, 2010

Keywords: case-control studies; family studies; genetic risks factors; venous thrombosis.

Introduction Thrombosis has been described as hemostasis in the wrong place (1). Genetic studies in thrombosis started with coining of the term thrombophilia by Jordan and Nandorff in 1956. Since then, researchers have attempted to elucidate the role of genetic factors in venous thrombosis (VT) (2). VT, whose main clinical presentations include deep vein thrombosis (DVT), usually in the leg, and pulmonary embolism (PE), represents a common and serious disorder with an age dependent incidence of 1–3 per 1000 individuals per year (3). It is still the major cause of morbidity and mortality during pregnancy and childbirth in developed countries, and is a frequent cause of disease in young women using oral contraceptives. Both sexes are equally afflicted by a firsttime VT, but the risk of recurrent thrombosis is higher in men than in women (3, 4). One of the first organizations founded to bring together basic scientists and specialists, clinicians interested in different field related to thrombosis was the Mediterranean League against Thromboembolic Diseases. The goal of this organization was promoting research into the pathogenesis of thromboembolic diseases, prevention, diagnosis and treatment, and thus of providing overall better care of patients (5). There is no doubt that the concept of common complex disease is pertinent in this clinical condition: VT may be seen as a consequence of a heterogeneous collection of genetic and environmental factors. Although the role of environmental factors (such as trauma and immobilization, surgery, pregnancy, use of oral contraceptives) in triggering thrombotic events have captured the public’s interest, major advances have been made over the past 15 years in understanding the genetics of VT. This review focuses on the role of genetic risk factors, mainly related to the haemostatic system, in triggering thrombotic events. Data on each of the well-established genetic conditions predisposing to VT are summarized, and the contribution of other candidate genetic risk factors is discussed. Data from family based studies of the genetics of thrombosis in Spanish individuals suggest that more than 60% of the variation in susceptibility to common thrombosis is attributable to genetic components (6). Reports describing families with a marked predisposition to VT were published as early as in 1905 (7). Subsequently, hundreds of mutations

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in different genes have been identified, enabling an understanding of the etiology and genetic complexity of VT. Genetic risks factors for VT may be classified as strong, moderate and weak. Deficiencies in the natural anticoagulants, antithrombin (AT), protein C (PC) and protein S (PS) are strong risk factors (risks 5–10-fold increased). Moderate risk factors are Factor V Leiden and prothrombin 20210A (risk increase 2–5-fold). Weak genetic risks factors include variants that increase risk no more than 1.5-fold (8). A large population-based case-control study showed that a positive family history increased the risk of VT more than 2-fold wOR, 2.2 (95% CI, 1.9–2.6)x and up to four-fold w3.9 (95% CI, 2.7–5.7)x when more than one relative was affected. Therefore, a positive family history increases susceptibility to VT. For those with a genetic and environmental risk factor, and a positive family history, the risk was about 64fold higher than for those with no known risk factors and a negative family history (9).

Deficiencies of natural anticoagulation inhibitors Deficiencies in the group of proteins usually referred to as natural coagulation inhibitors, i.e., AT, PC and PS are considered rare disorders and are found in -1% of the general population; prevalence rates for AT deficiency of 1 in 500 to 1 in 5000 in the overall population have been reported (10). AT is a serine protease inhibitor that physiologically inactivates thrombin and factor Xa and, to a lesser extent, factors IXa, XIa, XIIa. AT deficiency was first reported in 1965 by Egeberg, who described a Scandinavian family in which several subjects with decreased plasma AT concentrations presented with thrombotic events. Since then, numerous studies have described similar clinical conditions in additional families, establishing the concept of AT deficiency as a risk factor for thrombosis (11). AT deficiency is inherited as an autosomal dominant trait. Most cases are heterozygous; homozygosity for AT deficiency is rare and is almost always fatal in utero. Heterozygosity for AT deficiency can be found in 4% of families with inherited thrombophilia, in 1% of consecutive patients with a first DVT, and in 0.02% of healthy individuals (12). AT deficiency is divided into type I (low plasma concentrations of AT) and type II (dysfunctional protein in plasma). Type I AT deficiency is much more prevalent, often representing up to 80% of total cases. Type II is further subdivided into three types depending on the location of the mutation: IIa (defective reactive site), IIb (defective heparin-binding site) and IIc (pleiotropic group of mutations) (13). The gene AT3 coding for AT is located on chromosome 1q23–25, spanning 13.4 kb with 7 exons that encode for a mature secreted peptide of 432 amino acids (14). The molecular basis of AT deficiency is highly heterogeneous. Following the identification of the first mutation in 1983, the analysis of the AT3 gene has revealed a myriad of mutations (11, 15). The most frequent genetic defects are

missense mutations, but other types of gene lesions, such as nonsense mutations, splice site mutations, deletions and insertions, have also been reported (13). The current online AT3 gene database contains 256 entries (13) and a total of 127 different mutations have been reported. Type I AT deficiencies are most commonly caused by short deletions and insertions, and less commonly by single base pair substitutions, especially nonsense mutations. Three preferred regions for these deletions have been identified, codons 244/245, codon 81 and codons 106/107. The type II AT deficiencies are commonly secondary to single base pair substitutions. Most mutations involve the reactive domain occurring at residues Ala382 and Ala384 in the hinge region of AT, and near the reactive domain at residues 392, 393 and 394. Most mutations altering the heparin binding capacity of AT are missense mutations, often involving residues 41, 47, 99 and 129. The type IIc deficiencies include a host of different mutations, especially involving residues 402, 404–407 and 429. These mutations are located in strand Ic of AT, impairing the function of the reactive domain and also giving rise to a reduction in AT concentrations. This decrease is because strand Ic is necessary for the structural and functional integrity of AT (16). The estimates of thrombotic risk linked to AT deficiency and its prevalence in thrombotic patients also vary between different investigations, probably reflecting differences in study design and patient selection. In general, data obtained from family studies indicate increased risks (17, 18). PC and PS deficiencies result in defects in the activated PC anticoagulant system. PC, a vitamin K-dependent plasma glycoprotein, is cleaved to its activated form, ‘activated protein C’ (APC), and then acts as a serine protease to inactivate factors Va and VIIIa. In the 1980s, genetic defects leading to PC and PS deficiencies were for the first time reported as causes of inherited VT (19, 20). PC deficiency is inherited as an autosomal dominant disorder, and heterozygosity for PC deficiency is a significant risk factor for VT. However, autosomal recessive inheritance has been observed in families with severe thrombosis resulting from homozygous or compound heterozygous PC deficiency (21). Heterozygosity for PC deficiency is found in 6% of families with inherited thrombophilia, in 3% of patients with a first DVT, and in 0.3% of healthy individuals (12). The PROC gene encoding for PC has been localized to chromosome 2q13–14 where it spans approximately 10 kb and contains 9 exons. Similar to AT, PC deficiency is classified into type I (low plasma concentration of both functional and immunological PC) and type II (low plasma concentrations of functional protein with normal antigen concentrations). Mutations in the PC gene resulting in deficiency of PC are highly heterogeneous (22, 23). The mutation database for PC deficiency in its 1995 edition contained 331 entries (160 different) for 315 apparently unrelated probands from 16 different European and American countries (24). The majority of these entries are missense, nonsense and splicing mutations. PS is a potent anticoagulant protein that down regulates thrombin formation by stimulating the proteolytic inactiva-

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tion of factors Va and FVIIIa by APC. The PROS1 gene encoding for PS has been mapped to 3p11.1, spanning 80 kb and containing 15 exons. The inheritance pattern of familial PS deficiency is usually autosomal dominant. PS deficiency is classified as type I (quantitative deficiency of both total and free PS), type II (qualitative deficiency characterized by decreased activity and normal total and free PS antigen concentrations) and type III (normal concentrations of total PS and low concentrations of free PS). At present, more than 200 mutations have been described in the PROS1 gene, and large deletions/duplications have been identified as being relatively common causes of PS deficiency (25–27). Because AT, PC and PS deficiencies are so rare, most reports on the prevalence and thrombotic risk of heterozygotes come from family studies (17, 21, 28, 29). Heterozygous carriers of AT, PC and PS deficiencies show a highly penetrant phenotype, and similar thrombotic risks 10-fold higher than seen in non-carriers. However, the risks conferred by these deficiencies appear lower in unselected consecutive patients with thrombosis than in the families with penetrant thrombophilia (28, 30). This is probably because these thrombophilic families present with additional risks for thrombosis, such as resistance to APC or other unknown genetic defect (31, 32). Bucciarelli et al. performed a retrospective cohort family study to assess the risk for venous thromboembolism (VTE) in individuals with AT, PC, or PS deficiency, or activated PC resistance (APCR). The lifetime risk for VTE was 4.4 for AT vs. APCR, 2.6 for AT vs. PS, 2.2 for AT vs. PC, 1.9 for PC vs. APCR, and 1.6 for PS vs. APCR. AT deficiency seems to have a higher risk for VTE compared with the other genetic defects (18). In 2008, another retrospective family cohort study was performed in order to assess whether hereditary PS, PC, or AT deficiency was associated with arterial thromboembolism (ATE). The authors found that deficient subjects had a 4.7fold (95% CI, 1.5–14.2; ps0.007) higher risk for ATE before the age of 55 years vs. 1.1 (95% CI, 0.5–2.6) thereafter, compared with non-deficient family members. For separate deficiencies, the risks were 4.6- (95% CI, 1.1–18.3), 6.9- (95% CI, 2.1–22.2), and 1.1- (95% CI, 0.1–10.9) fold higher in PS-, PC-, and AT-deficient subjects, respectively, before 55 years of age (33).

Factor V Leiden In 1993, Dahlba¨ck and colleagues described an inherited abnormality that was highly prevalent in patients with VT. This condition was referred to as ‘‘APCR’’ defined as a poor anticoagulant response of plasma to the addition of APC (34). The phenotype of APC resistance is, in most cases, the result of a gain-of-function point mutation in the coagulation factor V gene: a G™A transition at nucleotide position 1691 leading to substitution of Arg 506 by Gln in the FV molecule (FV Leiden), one of the three cleavage sites for APC. FV Leiden is the most common genetic defect involved in the etiology of VT. In Europeans, this mutation is found in

up to 20% of familial thromboses. The transmission is autosomal dominant (1q21) (35). The extent of the increased thrombotic risk linked to FV Leiden is probably less than that associated with AT, PC and PS deficiencies. Martinelli et al. made a direct comparison of the thrombotic risk associated with deficiency of anticoagulant proteins and FV Leiden in 150 Italian families with different thrombophilic defects. They found higher risks for thrombosis in subjects with AT wrisk ratio 8.1 (95% CI, 3.4–19.6)x, PC w7.3 (95% CI, 2.9–18.4)x, PS deficiency w8.5 (95% CI, 3.5–20.8)x, and FV Leiden w2.2 (95% CI, 1.1–4.7)x, compared with individuals with normal coagulation. The risk of thrombosis for subjects with FV Leiden was lower than that for those with all three other coagulation defects, even when the analysis was restricted to DVT w0.3 (95% CI, 0.2–0.5)x (36). However, data obtained in case-control and cohort studies show that the relative risk for VT is about 3–10 for heterozygotes and 50–100 for homozygotes (35, 37). The prevalence of FV Leiden carriers in the healthy population varies between different geographical regions and is clearly population dependent. The FV:Q506 allele has a high frequency in Caucasians (the prevalence of heterozygotes varies from 2% to 13%), and is extremely rare in indigenous populations of Asia, Africa, America and Australia. This population-based distribution may explain the higher incidence of thromboembolism in Caucasians compared with other groups (38). Haplotype analysis of individuals homozygous for the FV:Q506 allele shows strong evidence for a founder effect, with the single mutational event occurring 21,000– 34,000 years ago (39, 40). Lindqvist et al. demonstrated that women carrying the FV:Q506 allele have a significantly lower risk of intrapartum bleeding complications compared with non-carriers, providing evidence that the mutation has conferred selective advantages in Caucasian populations (41). Alfirevic et al. performed a prospective study to assess the prevalence of FV Leiden, FII, MTHFR and plasminogen activator inhibitor (PAI)-1 polymorphisms in Croatian patients with thromboembolic disease. Of the polymorphisms tested, only the FV Leiden polymorphism showed to be significantly associated with VTE, with an odds ratio OR (95% CI)s6.41 (1.81–22.76); ps0.004 (42). Whether FV Leiden is also associated with an increased risk of recurrent VTE remains controversial (43–45). In 1997, Bernardi et al. identified a FV haplotype, denoted HR2, as a group of six polymorphisms in the FV gene associated with a decreased APC response (46). This was probably related either to the amino acid substitutions predicted by the HR2 haplotype, or to linkage disequilibrium with another mutation in the FV gene. However, conflicting results about the relationships between the HR2 haplotype and the risk of thrombosis have been obtained in different studies. Other mutations affecting other APC cleavage sites in FVa have been described including FV Cambridge, FV:R306T and FV Hong Kong, FV:R306G that could potentially give rise to APC resistance, hypercoagulability and an increased risk of VT (47, 48). These gene variations are rare in the general population, and it remains to be demonstrated


whether FV Hong Kong and FV Cambridge are a risk factor for thrombosis.

Prothrombin 20210A A G-to-A transition at nucleotide 20210 of the prothrombin gene has been identified as the second most common independent risk factor for VT (49). The mutation is located in the 39UTR of the gene and is associated with increased plasma concentrations of prothrombin (approx. 30% in heterozygotes and 70% in homozygotes) which may be the molecular mechanism behind the increased risk of thrombosis. The transmission is also autosomal dominant (11p11). Data obtained from the Leiden Thrombophilia Study (LETS) have established that the prothrombin 20210 A allele was associated with an increased risk of a first DVT wOR 2.8, (95% CI, 1.4–5.6)x. Its distribution is elective in Caucasians, with a North to South gradient in Europe (prevalence of 0.017 and 0.03, respectively) (50). This gain of function mutation was found in 1%–3% of subjects in the general population, approximately in 6% of unselected patients with VT, and in 10% of probands of thrombophilic families. As previously reported, it remains uncertain if individuals homozygous or double heterozygous for FV Leiden and/or prothrombin G20210A have an increased risk of recurrent VT. A recent case-control study calculated the risk of recurrent VT in individuals with homozygosity or double heterozygosity of FV Leiden and/or prothrombin G20210A. The OR for recurrence was 1.2 (95% CI, 0.9–1.6) for heterozygous carriers of FV Leiden, 0.7 (95% CI, 0.4–1.2) for prothrombin G20210A, 1.2 (95% CI, 0.5–2.6) for homozygous carriers of FV Leiden and/or prothrombin G20210A, and 1.0 (95% CI, 0.6–1.9) for double heterozygotes of both mutations. Adjustments for age, gender, family status, first event type, and concomitance of natural anticoagulant deficiencies did not alter the risk estimates. Therefore, individuals homozygous or doubly heterozygous for FV Leiden did not have a high-risk of recurrent VT (51).

Several mutations in MTHFR and CBS have been identified in patients with homocystinuria (54–56). However, this review we will focus on two mutations in MTHFR (C677T and A1298C) and one mutation in CBS (844ins68). Individuals with compound heterozygous or homozygous MTHFR polymorphisms may have slight increases in homocysteine concentrations, especially when folate deficiency is present (57). The most common polymorphism is C677T which causes a change from alanine to valine, rendering the enzyme thermolabile. Conflicting results have been reported regarding the prothrombotic role of the 677T variant (58). A meta-analysis suggested a weak effect (relative risk 1.2), but a recent population-based case-control study (the MEGA study) showed no association with VT (59). The prevalence varies widely according to populations, and is related to food content of folic acid. In Europe, there is a North to South gradient with a very high prevalence among Mediterranean countries. In addition, a certain level of microheterogeneity is seen in some populations, e.g., between northern and southern parts of Italy with allele frequencies of 0.448 and 0.556, respectively (60). However, in Africans from the SubSaharan zone, this frequency is among the lowest in the world (38). The A1298C polymorphism occurs in the heterozygous state in approximately 9%–20% of most ethnic groups (61). By itself, MTHFR A1298C does not seem to be associated with hyperhomoysteinemia, but compound heterozygosity with MTHFR C677T results in decreased enzyme activity and increased homocysteine concentrations (62). MTHFR polymorphisms have also been associated with an increased risk of neural tube defects (63). It has been described that defects in the CBS gene may result in enzyme deficiency and hyperhomocysteinemia. A 68-bp insertion into the CBS gene (844ins68) has been described as a frequent mutation in various populations (64). Although this variant does not influence homocysteine concentrations or the risk of DVT (65); in combination with MTHFR C677T it may increase thrombotic risk (66).

Other candidate genetic risk factors for VT Hyperhomocysteinemia

O-blood group

Hyperhomocysteinemia is an abnormal increase in plasma concentrations of homocysteine, and is associated with a mild (approx. 2- to 4-fold) increase in thrombotic risk (52). Genetic and acquired factors interact to determine plasma homocysteine concentrations. Genetic causes include gene defects in methylenetetrahydrofolate reductase (MTHFR) and cystathionine b-synthase (CBS); two enzymes involved in homocysteine intracellular metabolism that may result in enzyme deficiency and hyperhomocysteinemia. Most of the mutations in MTHFR and CBS genes are rare and only have clinical consequence in homozygotes or compound heterozygotes. This condition, known as homocystinuria, is an autosomal recessive disorder characterized by multiple neurological deficits, psychomotor retardation, seizures, skeletal abnormalities, lens dislocation, premature arterial disease and VTE (53).

The moderate thrombotic risk associated with non-OO blood group (OR: 1.8–2.5), and the high prevalence in the general population of prothrombotic non-OO genotypes ()50%), make the ABO blood group of general interest in thrombosis (67). The O blood group is associated with decreased concentrations of von Willebrand factor and factor VIII, whose increased plasma concentration represent an established risk factor for VTE (68). Minãno et al. studied the effect of ABO blood group on the risk and severity of VTs in carriers of FV Leiden and prothrombin 20210A polymorphisms. Their results confirmed the significant contribution of the non-OO blood group to the expression of VTE associated with FV Leiden wOR: 1.76; (95% CI: 1.06–2.91)x. They suggested that the non-OO blood group also increases significantly the risk of VTE in carriers of prothrombin 20210A wOR: 2.17; (95% CI: 1.33–3.53)x, in contrast with previous studies (69).


Fibrinogen

Abnormalities in fibrinogen have been reported to affect the risk for DVT. Qualitative deficiencies of fibrinogen (dysfibrinogenemias) have been found in patients with thrombosis and a prolonged thrombin time (70). Most of these patients have a mutation in the fibrinogen a-chain gene (FGA) or the g-chain gene (FGG). In the LETS, de Willige et al. investigated the association between haplotypes of FGA, FGB, and FGG genes, total fibrinogen concentrations, and the risk of DVT. The authors showed that the haplotype H2-tagging single nucleotide polymorphism (SNP) 10034C/T in the FGG gene reduced the fraction of the variant g9 chain in plasma, increasing the thrombotic risk (OR: 2.4; 95% CI, 1.7–3.5) (71). Recently, the same authors evaluated whether FGG polymorphisms 10034C)T and 9340T)C were associated with risk of VTE in African Americans and Caucasians. In African Americans, 10034C)T and 9340T)C marginally influenced risk of VTE, with a 20% increase in risk for 10034TT carriers, and a 20% reduction in risk for 9340CC carriers. In Caucasians, 10034TT was associated with a 1.7-fold increase in risk, which increased to 2.1-fold for idiopathic VTE patients. 9340CC significantly reduced risk of VTE by approximately 2-fold (72). Although predisposition to thrombosis is a well-known feature of dysfibrinogenemia, relatively frequent thrombotic manifestations were seen in congenital afibrinogenemia. Simsek et al. reported a mutation analysis of a young afibrinogenemic man with multiple thrombo-embolic events involving both arteries and veins. Genetic analysis found a homozygous G to A transition in exon 5 of the Aa chain gene that predicted a homozygous W315X in the Aa chain. The patient was free of known risk factors as well as diseases associated with thrombosis. Therefore, it seems probable that afibrinogenemia itself might have contributed to both arterial and VT (73). FXIII

The physiological role of FXIII in normal hemostasis is to improve the mechanical strength of the fibrin clot and to protect its elimination by the fibrinolytic system. In addition, it has also been implicated in the pathology of arterial and VT. Most recently, the polymorphisms in the FXIII subunit genes and their influence on the risk of thrombotic diseases have stirred much interest (74). Several studies have investigated the effect of FXIII Val34Leu polymorphism on the risk of DVT. There is wide variation in the prevalence of the Val34Leu mutation in different populations. In one study, the Val34Leu polymorphism was present in 2.5% of Asians, 28.9% of Blacks, 44.3% of Caucasians and 51.2% of American Indians. The 34Leu allele is virtually absent in Japanese, and present in 11%–27% of Australian Caucasians (75). Catto et al. determined the Val34Leu genotype in patients with DVT and PE, demonstrating a significant protection by the Leu34 allele wOR: 0.63, (95% CI: 0.38–0.82)x (76). However, additional studies have not found a significant association. In 2006, Wells et al. published a comprehensive

meta-analysis of 12 studies that showed a slight (OR: 0.89), but statistically significant protection afforded by the Leu34 allele (77). The Val34Leu polymorphism has been associated consistently with increased activation by thrombin in vitro. The clots formed in the presence of the FXIII 34Leu polymorphism are thinner and less porous. Thinner fibers are more resistant to fibrinolysis in vitro, and should theoretically increase the risk of a clinical thrombotic event. The putative protective effect of this SNP against VT is paradoxical. One study has suggested that the FXIII 34Leu polymorphism may protect against thrombosis, specifically in patients with high fibrinogen concentrations, who are known to be at increased risk for thrombotic complications (78). An interesting study was performed concerned the effect of a His95Arg substitution in the FXIII subunit B on the risk of DVT (74). This relationship was investigated in patients and controls from Leeds. The His/ArgqArg/Arg genotypes were found to be more frequent in patients than in controls (22.4% vs. 15.1%). FXIII-B His95Arg was also investigated in the LETS, in which a similar difference was observed between patients and controls (18.5% vs. 14.0%), with a pooled OR of 1.5 (CI: 1.1–2.0) (74). Tissue factor pathway inhibitor (TFPI)

TFPI is a circulating protease inhibitor that plays a major role in the inhibition of TF-factor VIIa proteolytic activity in vivo. Data obtained from the subjects enrolled in the LETS suggest that low concentrations of TFPI constitute a risk factor for DVT (79). The TFPI gene contains 9 exons that and have been mapped on 2q31-q32.1. To date, different polymorphisms have been described in the TFPI gene, but it is uncertain whether these polymorphisms influence the risk of thrombosis. A –399C)T transition in the 59 UTR of the gene was described, but this polymorphism neither alters the expression level of the TFPI protein, nor its association with the risk for VT (80). Moatti et al. performed an association study of –287 C)T in 59UTR, –33C)T transition in intron 7 with coronary artery disease and plasma TFPI concentrations. No association was found between these polymorphisms and coronary syndromes (81). No association between the Val264Met polymorphism and DVT has been described (82). However, a study of total TFPI antigen concentrations in 122 patients with DVT and 126 controls demonstrated an association between TFPI concentrations and VT (ps0.0001). However, these results do not support a link between DVT and Val264Met mutation. Kleesiek et al. described a Pro151Leu polymorphism in patients with a history of VT (83). They reported a statistically significant association between the C536T transition in the TFPI gene and the risk for VT (84). However, this result has not been confirmed in other populations (85). Thrombomodulin (TM)

TM is a transmembrane protein expressed on the luminal surface of vascular endothelial cells. TM catalyzes thrombin activation of PC to APC, binds and alters thrombin substrate


specificity, and catalyzes the inhibition of thrombin by antithrombin. Based on the important antithrombotic role of TM, it has hypothesized that polymorphisms within the TM gene that alter TM expression and/or anticoagulant function could predispose to VTE. Heit et al. screened a large Caucasian population containing unrelated patients with idiopathic DVT or PE to identify mutations within the TM gene and to assess the association with VTE. Four mutations, Ala25Thr, Ala455Val, 2470C deletion (39-UTR) and 4363A)G (39-flanking region) were more common in the study cohort, but were not associated with VTE by genotype or haplotype (86). However, Sugiyama et al. found that genetic variations in the TM gene, especially those with a haplotype consisting of 2729A)C SNP and A455V missense mutation, affect soluble TM concentrations and may be associated with DVT in the Japanese (87). Endothelial protein C receptor (EPCR)

EPCR is a type I transmembrane protein expressed on the endothelium of large vessels that binds PC with high affinity. EPCR enhances the rate of PC activation by increasing the affinity of PC for the thrombin-thrombomodulin complex. In the last few years, several reports have suggested an association between mutations/polymorphisms in the EPCR gene and venous and arterial thrombosis (88). The human EPCR gene consists of 4 exons, spans approximately 6 kb and is located on chromosome 20q11.2. Several variations have been reported in the EPCR gene, some of which are associated with the risk of thrombosis. The first EPCR gene mutation to be described was a 23 bp insertion in exon 3 resulting in a stop codon and truncated protein. Given its low frequency in the population (-1%), it will be difficult to assess the effect of this mutation on the risk of venous and arterial thrombosis (89). Moreover, different point mutations have been reported in the EPCR gene promoter, but they were rare in patients with VTE or myocardial infarction. A substitution of Arg for Cys at position 98 was identified with a prevalence of 0.44% in patients and 0.83% in controls, suggesting no role for this mutation in VTE. Four haplotypes have been reported in the EPCR gene; H1, H2, H3 and H4. Three haplotypes contain one or more SNPs that are haplotype-specific. The H3 haplotype, tagged by the rare allele of 4600A/G, is associated with increased plasma concentrations of sEPCR, but its association with risk of VTE is controversial. The H1 haplotype, tagged by the rare allele of 4678G/C, was reported to be associated with a decreased risk of VTE wORs0.59, (95% CI, 0.41–0.84)x as well as in carriers of FV Leiden. Moreover, APC concentrations were found to be significantly higher in carriers of the H1H1 genotype than in those not having the H1 haplotype, both in patients and in controls and resulting in a protective effect against VTE. It has been observed that individuals carrying the H3 haplotype present with increased sEPCR concentrations that could increase the risk of VT. Finally, the H4 haplotype was reported to be associated with a slight increase in the risk of VTE (90).

Thrombin activatable fibrinolysis inhibitor (TAFI)

TAFI is an important inhibitor of fibrinolysis which acts by inhibiting the assembly of fibrinolytic factors on the fibrin surface. It has been shown that increased TAFI antigen concentrations are associated with a mild (1.7-fold) but significantly increased risk for DVT (91). The human TAFI gene has been mapped to chromosome 13q14.11, spans approximately 48 kb and is organised in 11 exons (92). TAFI concentrations are also determined genetically, and the –438 G/A SNP in the promoter region and the 505 G/A SNP and 1040 C/T SNP in the coding region are associated with TAFI plasma antigen concentrations. The –438 G/A polymorphism was reported to be associated with an increased risk for development of VT (93). Martini et al. reported that the 505 G-allele is associated with low TAFI antigen concentrations and carriers of the 505G allele showed a mildly increased risk of VT (94). P-selectin

P-selectin, a member of the cell adhesion molecules, is primarily stored in the a-granules of platelets and the WeibelPalade bodies of endothelial cells. The interaction of P-selectin and its main-counter receptor leads to several mechanisms that induce a procoagulant state by triggering the generation of procoagulant microparticles from leukocytes, upregulating the expression of tissue factor on monocytes and inducing an increased surface-dependent thrombin generation on monocytes. High plasma concentrations of soluble P-selectin (sP-selectin) are strongly associated with VTE (95). Moreover, sP-selectin concentrations were found to be higher in cancer patients with VTE compared to those without VTE, allowing prediction of the occurrence of cancer-associated VTE, with a hazard ratio of 2.6 (95% CI: 1.4–4.9) (96). A number of P-selectin gene variants that affect the protein sequence have been described. One such variant, Thr715Pro, has consistently been associated with lower sPselectin concentrations in plasma. Ay et al. investigated sPselectin and the Thr715Pro variant in a high-risk population of patients with a history of recurrent VTE. This study demonstrated that sP-selectin concentrations are lower in individuals carrying the Pro715 variant than those without. Therefore, the Pro715 variant may be seen as a protective allele, providing defense against increases in sP-selectin concentration that are commonly seen in Thr715 homozygotes (97).

Genome wide association studies During the last years, several investigations have been performed where a great number of SNPs are tested in association studies in order to fully understand the genetic causes of thrombosis. Bezemer and colleagues investigated almost 20,000 potentially functional SNPs for association with DVT. They used three case-control studies derived from the LETS and the


MEGA study. The evidence for an association with DVT was strongest for the three SNPs (p-0.05): rs13146272 in CYP4V2 (risk allele frequency, 0.64), rs2227589 in SERPINC1 (risk allele frequency, 0.10), and rs1613662 in GP6 (risk allele frequency, 0.84). The OR for DVT per risk allele was 1.24 (95% CI, 1.11–1.37) for rs13146272, 1.29 (95% CI, 1.10–1.49) for rs2227589, and 1.15 (95% CI, 1.01–1.30) for rs1613662. It is interesting to note that these SNPs are in or near genes that have a clear role in blood coagulation. In the region of CYP4V2, there have been identified four additional SNPs in the CYP4V2/KLKB1/F11 locus that were also associated with both DVT (highest OR per risk allele, 1.39; 95% CI, 1.11–1.74) and coagulation factor XI concentrations (highest increase per risk allele, 8%; 95% CI, 5%–11%) (98). One of the SNPs identified was an A)G sequence variant in the gene encoding factor IX (rs6048, also known as F9 Malmo¨). The G allele was associated with a 15%–43% decrease in risk of DVT compared with the A allele. This common variant (minor allele frequencys0.32) leads to the substitution of alanine for threonine at amino acid 148 in the portion that is cleaved to activate factor IX. The OR for the G allele of F9 Malmo¨, compared with the A allele, was 0.80 (95% CI, 0.69–0.93). The SNP rs422187 in FIX was strongly linked to F9 Malmo¨, and was similarly associated with DVT. No other SNP or haplotype tested was more strongly associated. However, the biological mechanism by which F9 Malmo¨ influences the risk of DVT remains unclear (99). It has been suggested that the overall effect of the major proinflammatory cytokine interleukin-1 (IL-1) on coagulation and fibrinolysis is prothrombotic. Van Minkelen et al. have investigated whether common variations in the genes coding for IL-1b, IL-1Ra, IL-1R1, and IL-1R2 (IL1B, IL1RN, IL1R1, and IL1R2) influence the risk of VT by modulating the IL-1 pathway. The authors genotyped 18 SNPs in these genes, together with the Tag 25 haplotype groups, in all patients and control subjects in the LETS. Global testing using a recessive model showed a difference in haplotype frequency between patients and controls for IL1RN (ps0.031). Subsequently, the risk of VT was calculated for each haplotype of IL1RN. Increased thrombotic risk was found for homozygous carriers of haplotype 5 (H5, tagged by SNP 13888T/G, rs2232354) of IL1RN (ORs3.9; 95% CI: 1.6–9.7; ps0.002) (100). Recently, Reiner et al. screened 290 common SNPs in 51 coagulation-fibrinolysis and inflammation candidate genes for association with VTE in the Cardiovascular Health Study (CHS), a large, well-characterized prospective populationbased study of older adults. TagSNPs within four genes encoding factor XIII subunit A (F13A), factor VII activating protease (HABP2), protease activated receptor-1 (F2R), and the urokinase receptor (PLAUR) showed the strongest evidence for association with VTE. The rs3024409 variant allele of F13A1 was associated with a 1.66-fold increased risk of VTE, while the minor alleles of HABP2 rs6585234 and rs3862019, F2R rs253061 and rs153311, and PLAUR rs344782 were each associated with lower risk of VTE (hazard ratios in the range of 0.49–0.66). Consistent with the

observed protective association on VTE risk, the HABP2 rs3862019 variant allele was also associated with lower activity levels of coagulation factors VIII, IX, X, and plasminogen (101).

Conclusions VT represents a significant public health problem in Western countries resulting from the interaction of genetic predisposition and environmental risk factors. In this review we have focused on the role of genetic risk factors, mainly related to the haemostatic system, in influencing thrombotic risk. The most significant evidence emerging from genetic studies over the past few decades has been the confirmation that inherited hypercoagulable conditions are present in a large proportion of patients with venous thromboembolic disease. These include mutations in the genes that encode AT, PC and PS, that may be viewed as strong risk factors, and the FV Leiden and factor II G20210 A mutations commonly referred to as milder risk factors. The gain-of-function mutations in procoagulant factors were more prevalent than abnormalities in anticoagulant proteins in the typical Caucasian population. Thus, the focus shifted from family studies to populationbased association studies in a case-control format. In fact, common genetic variations in coagulation proteins were recently tested in association studies. In addition to these well-established conditions predisposing to VT, different studies have pointed to the role of other candidate genetic risk factors, such as variants in FXIII, FIX and fibrinogen genes. Moreover, less-well studied proteins that are part of anticoagulant pathways, such as TFPI, TM and EPCR, have also been investigated as risk factors for VT. Recently, different genome-wide association studies have been performed where several SNPs are tested in association studies and linked to thrombotic risk. This extensive list of genetic factors illustrates that VT can be understood by assuming an interaction between different mutations in candidate susceptible genes. By itself, the risk associated with each genetic defect may be relatively low, but the simultaneous presence of several mutations may dramatically increase disease susceptibility. Moreover, environmental factors may interact with one or more genetic variations to add further to the risk. Even considering the occurrence of gene-gene interaction, it is interesting to note that the known thrombophilic abnormalities do not explain all cases of variable penetrance of VT in families with a certain identified defect. This evidence points to the existence of additional unidentified prothrombotic defects that remain to be discovered.

Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared.


Employment or leadership: None declared. Honorarium: None declared. 19.

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Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game Zsuzsanna Bereczky*, Kitti B. Kovaćs and La´szlo´ Muszbek Clinical Research Center and Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary

Abstract Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins that play an important role in the regulation of blood coagulation as natural anticoagulants. PC is activated by thrombin and the resulting activated PC (APC) inactivates membrane-bound activated factor VIII and factor V. The free form of PS is an important cofactor of APC. Deficiencies in these proteins lead to an increased risk of venous thromboembolism; a few reports have also associated these deficiencies with arterial diseases. The degree of risk and the prevalence of PC and PS deficiency among patients with thrombosis and in those in the general population have been examined by several population studies with conflicting results, primarily due to methodological variability. The molecular genetic background of PC and PS deficiencies is heterogeneous. Most of the mutations cause type I deficiency (quantitative disorder). Type II deficiency (dysfunctional molecule) is diagnosed in approximately 5%–15% of cases. The diagnosis of PC and PS deficiencies is challenging; functional tests are influenced by several pre-analytical and analytical factors, and the diagnosis using molecular genetics also has special difficulties. Large gene segment deletions often remain undetected by DNA sequencing methods. The presence of the PS pseudogene makes genetic diagnosis even more complicated. Clin Chem Lab Med 2010;48:S53–66. Keywords: protein C deficiency; protein S deficiency; review; thrombophilia.

Introduction Venous thromboembolism (deep vein thrombosis and/or pulmonary embolism, VTE) and its consequences occur with high frequency Western societies. VTE is still a major cause *Corresponding author: Zsuzsanna Bereczky, MD, PhD, Clinical Research Center, University of Debrecen, Medical and Health Science Center, 98, Nagyerdei krt., Debrecen, 4032, Hungary Phone: q36 52 431 956, Fax: q36 52 340 011, E-mail: [email protected] Received July 4, 2010; accepted September 26, 2010; previously published online November 6, 2010

of morbidity and mortality during pregnancy and stillbirth, and occurs with a relatively high frequency in young women using oral contraceptives. VTE can be recurrent and may also lead to post-thrombotic syndrome, a chronic disease with disabling pain and ulceration. VTE is a typical example of common complex diseases; both acquired (environmental) and genetic causes play an important role in the development of the disease (1). Over the last decades, several genetic risk factors for VTE have been identified. Loss of function mutations in different components of the natural anticoagulant system lead to antithrombin III (ATIII), protein C (PC) and protein S (PS) deficiencies, while gain of function mutations in the genes of coagulation factors, such as Factor V Leiden (FVL) and prothrombin 20210A allele are responsible for the majority of inherited thrombophilia. The aim of this review is to give an overview of the physiology of PC and PS, on the molecular basis of their deficiencies and on the laboratory diagnosis of these disorders including the difficulties and challenges in this field.

The role of protein C-protein S system in the regulation of coagulation PC and PS play important roles in the regulation of blood coagulation as natural anticoagulants (2, 3). PC is activated by thrombin in the presence of thrombomodulin (TM). TM is an endothelial cell surface protein, and upon binding, thrombin becomes a potent activator of PC. Endothelial protein C receptor (EPCR) also is highly important in the activation process; EPCR binds PC through its Gla-domain and presents it to the thrombin-TM complex. Thrombin cleaves the activation peptide domain of PC at Arg169 resulting in a 12-amino acid long activation peptide being released from the N-terminal end of its heavy chain. Activated PC (APC) inactivates membrane-bound active factor VIII (FVIIIa) and factor V (FVa) by cleaving these factors specific arginine residues. FVa is cleaved at Arg506, which is the preferred cleavage site. However, full inactivation of FVa also requires cleavage at Arg306. Cleavage at Arg679 seems unimportant for inactivation of FVa. FVIIIa is cleaved at Arg336 and Arg562. Non-activated forms of FVIII and FV are poor substrates for APC. Esmon and co-workers showed that the membrane phospholipid requirement for the anticoagulant APC complex differs from that of the procoagulant complexes (4). Phosphatidyl ethanolamine, instead of phosphatidyl serine, is required for the binding of the APC complex to the membrane of endothelial cells. APC can also cleave intact FV at Arg506, which makes FV a cofactor of APC in inactivating FVIIIa. The main inhibitor of APC is protein C

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inhibitor, a single chain glycoprotein serine protease inhibitor synthesized in the liver. The inhibitor forms a 1:1 complex with APC and is cleaved at the reactive site (Arg354). APC is also inhibited by a-1 antitrypsin. In addition to its anticoagulant function, PC plays an important role in cytoprotection, which has been partially clarified in the last decade. The multiple cytoprotective effects of APC is not the subject of this review, it has been reviewed by Mosnier et al. (5). The free form of PS (described later) is an important cofactor of APC, enhancing its affinity to negative charged phospholipid surfaces. PS is able to displace FXa from its complex with FVa, allowing APC to cleave FVa at Arg506. In the process of FVIIIa inactivation APC activity is synergistically stimulated by PS and the non-activated form of FV, while in the process of FVa inactivation, free PS is the only cofactor of APC. PS also forms a complex with the complement 4b binding protein (C4bBP); this complex lacks APC cofactor activity. PS also has direct, APC-independent effects (6, 7). PS binds to FVa present on phospholipid vesicles, and inhibits prothrombinase activity by competing with prothrombin for binding to FVa. Binding of PS to FXa has also been demonstrated. APC-independent PS function does not seem to be restricted to the free form; C4bBP-complexed PS has the same activity. A direct interaction of PS with tissue factor pathway inhibitor (TFPI) in the inhibition of FXa was recently reported (8).

Protein and gene structure of protein C PC is a vitamin K-dependent glycoprotein synthesized by the liver as a single chain protein. It exists in the plasma as a precursor of a serine protease at a concentration of 3–5 mg/L (9). Its half-life is short, approximately 8 h in the circulation. The mature 62 kDa protein is composed of a heavy (41 kDa) and a light (21 kDa) chain, these chains are held together by a single disulfide bond between Cys141 and Cys265. The domain structure of PC shows high similarity to other vitamin K-dependent proteins. It has a pre-pro leader sequence (numbered as –42 to –1 by the traditional numbering system, where the first methionine corresponds to –42), which is required for g-carboxylation of glutamic acid residues in the Gla-domain and for secretion. The mature protein contains a Gla-domain (amino acids 1–37) with the nine glutamic acid residues that are carboxylated during posttranslational maturation. A short amphipathic helix (amino acids 38–45) connects the Gla-domain to the first epidermal growth factor (EGF) domain (amino acids 46–91). The second EGF domain (amino acids 92–136) is followed by the activation peptide domain (amino acids 137–184). This region contains the Lys156–Arg157 dipeptide that is released upon maturation, and the cleavage site for thrombin activation (Arg169). The heavy chain of PC contains the activation peptide and the catalytic domain (amino acids 185–419). The gene for human PC (PROC) is located at the 2q13–q14 position and contains nine exons encoding for a

1.7-kb messenger RNA (mRNA) and eight introns (10, 11). All the exon/intron boundaries follow the GT-AG rule. Exon 1 is a non-coding exon and a long intron separates it from the initiator ATG codon; this phenomenon is unique among the vitamin K dependent factors. The PROC contains two Alu repeats in intron 5. The major transcriptional start site is located 1515 base pairs upstream from the initiator ATG codon. Two minor transcription start sites were also recognized at –7 and q13 bp relative to the major start site. The regulation of transcription was extensively studied. Cis-elements within PROC are the HNF-1, HNF-3 and Sp1 binding sites. A strong silencer region and two liver specific enhancer regions have also been described.

Protein and gene structure of protein S PS is a vitamin K-dependent single-chain 71 kDa glycoprotein. It is synthesized primarily in the liver. However, significant amount of PS are also produced by endothelial cells and megakaryocytes (12). Its plasma concentration is 20–25 mg/L and PS circulates with a half-life of 42 h. Its domain structure is different from other vitamin K dependent proteins. The pre-pro leader sequence (numbered as –41 to –1), the Gla-domain (amino acids 1–36) with 11 glutamic acid residues and a short amphipathic helix (amino acids 37–46) also exists in other vitamin K dependent hemostatic proteins. In addition PS contains unique domains, a thrombin cleavage sensitive loop formed from 24 residues, four EGF domains, and a huge sex hormone domain with the binding site for the complement regulator C4bBP. About two third of PS circulates in complex with C4bBP. In this complex, PS is not a cofactor of APC (13). The gene for human PS (PROS1) is located at position 3q11.2 and contains 15 exons producing a 3.5-kb messenger RNA (mRNA) and 14 introns (10). All exon/intron boundaries follow the AG/GT rule. Exon 1 encodes for the first part of the pre-pro leader sequence, exon 2 codes for the second part of the leader sequence and the Gla-domain. Exon 3 encodes for the short hydrophobic region and exon 4 codes for the thrombin sensitive region. Exons 5–8 encode for the four EGF domains. Exons 9–15 code for the very large C4bBP-binding (sex homone binding) domain. Six Alu repeats are located within the PROS1 gene. In addition to the active gene, a pseudogene of PS (PROS2) has also been discovered. This inactive gene shows 97% homology to the active gene, but lacks exon 1 and contains multiple base changes. The presence of PROS2 makes the molecular genetic diagnosis of hereditary PS deficiency a difficult task (see later).

Protein C and S deficiencies: epidemiological aspects and clinical symptoms The first patient with PC deficiency was described by Griffin et al. in 1981 (14). PS deficiency was first reported in 1984 by Comp and Esmon (15). Both cases were associated with

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recurrent venous thromboembolism. Since then, a large number of deficient patients have been identified and the underlying genetic defects have been clarified in a number of cases (see later). Soon afterwards it was revealed that PC and PS deficiencies are associated with increased thrombotic risk. The degree of the risk and the prevalence of PC and PS deficiencies among patients with thrombosis and in the general population have been examined in several population studies, with conflicting results (16–29). The estimated prevalence of PC deficiency in the general population is surprisingly high (0.4%) (18, 19). The prevalence of PS deficiency seems to be less, but data are uncertain, which is due, at least in part, to difficulties in the laboratory diagnosis. In a Scottish study, it was within a range of 0.03%–0.13% (20), while PS deficiency was shown to be more prevalent in a general Japanese population (1%–2%) (21). Among 163 randomly selected patients with lower extremity venous thrombosis (LEVT), 2%–4% of the patients suffered from PC or PS deficiency (22). Interestingly in the same study, when patients with confirmed cerebral venous sinus thrombosis were examined (ns163), no PC deficiency was found, while the prevalence of PS deficiency did not differ significantly from that of the LEVT group. In an Italian cohort, the prevalence of PC and PS deficiency among patients with first time VTE was found to be 4.7% and 3.7%, respectively (23). PC or PS deficiency was diagnosed in 3% and 2% of patients with proximal deep vein thrombosis, respectively (ns920) (24). According to the results of the Leiden Thrombophilia Study, one of the first case-control studies on this topic, the risk of first VTE in PC deficiency was 3-fold, while in PS deficiency, no significant risk was demonstrated in unselected patients (25). In other studies, a 3–11-fold risk of VTE was demonstrated in PC or PS deficiency. The results depended on the selection of patients, including ethnicity, study design, and the methods for determining PC and PS activity and concentration. The annual incidence of recurrent VTE were 6.0% for PC deficiency and 8.4% for PS deficiency (23, 26). In the large prospective EPCOT study (European Prospective Cohort on Thrombophilia), the risk of first VTE in asymptomatic relatives of patients with confirmed thrombophilia was investigated (ns575). During the 5.7 years of follow-up, 4.5% of individuals developed VTE. The annual incidence of VTE in PC (0.7%) and PS (0.8%) deficiency was higher than in cases of FVL, and thrombosis developed at a mean age of 40 (27). Putting the results of all epidemiological studies together, one can conclude that the risk of developing thrombosis among individuals with genetic defect in PROC or PROS1 varies considerably in the various studies that have been preformed. In addition to methodological variability, this could be due to gene-gene or gene-environment interactions, many of which have not yet been discovered (30, 31). Symptoms of PC or PS deficiencies are deep venous thrombosis and/or pulmonary embolism in early adulthood, which is often recurrent. Thrombosis might also develop at unusual sites, such as the proximal extremities and in mesen-

terial and cerebral veins. Intracardial thrombus was reported in a 2-year-old child having inherited PC deficiency (32), and intracardial multichamber thrombi were identified in a middle aged patient with combined PC and PS deficiency (33). Pregnancy associated thrombosis has also been reported and PS deficiency was also found in the background of late fetal loss (34). In severe PC and PS deficiency when plasma PC or PS concentrations are extremely low, severe thrombosis develops in newborns, frequently in disseminated form, named purpura fulminans (35). Warfarin-induced skin necrosis is a severe complication of PC or PS deficient patients receiving vitamin K antagonist treatment. In addition to venous thrombosis, patients with PC or PS deficiency can also suffer from thrombotic complications of arterial origin (36). In addition to isolated case reports (37–39), a large cohort of relatives of VTE patients with PC, PS or ATIII deficiency (ns468) had a higher incidence of arterial thrombosis compared to subjects who were not deficient. The risk of arterial thrombosis was especially high in individuals -55 years of age; adjusted hazard ratios for PC and PS deficiencies were 6.9 (95% CI, 2.1–22.2) and 4.6 (95% CI, 1.1–18.3), respectively (40). In the ARIC cohort which enrolled more than 13,000 patients with coronary events or ischemic stroke, and had a follow-up time of almost 17 years, low PC concentrations were associated with the development of stroke (41). A Japanese study demonstrated that patients with PC deficiency were 10 years younger at the onset of myocardial or cerebral infarction compared with those without deficiency (16). A meta-analysis of studies involving children with arterial ischemic stroke calculated an odds ratios of 8.46 for PC and 3.20 for PS deficiency (42). These findings suggest the importance of screening for inherited thrombophilia, primarily PC and PS deficiencies in young patients with arterial thrombotic events, especially in those without any other obvious risk factor.

Molecular genetic background of protein C and S deficiency, genotype-phenotype correlations Protein C deficiency

PC deficiency is classified as type I (quantitative) and type II (qualitative) deficiency. In type I deficiency, PC activity and the antigen concentration are decreased equally, suggesting defective synthesis or secretion of the protein, while in type II deficiency, the activity is decreased without a significant decrease in antigen concentrations. The latter type could be due to abnormalities in substrate, calcium-ion or receptor binding. The inheritance of PC deficiency is not as clear as was first thought. It may show an autosomal recessive or dominant inheritance, often with incomplete penetrance. The majority of PC deficient patients are heterozygous for the defect, with typical PC activity values between 30% and 65%. Homozygous or compound heterozygous patients often have undetectable PC concentration and/or activity and exhibit life-threatening thrombosis very early in life. The molecular genetic background of PC deficiency is hetero-


geneous. Most of the mutations cause type I deficiency, type II deficiency is diagnosed in approximately 10%–15% of cases. Summary reports of mutations leading to decreased PC concentrations were first described in 1995 (43, 44). To date, approximately 250 causative mutations have been published, which are collected in different databases (http:// www.hgmd.cf.ac.uk and http://www.isth.org) (Figure 1). A recently developed mutation database, ProCMD, is an interactive tool that contains phenotypic descriptions with functional and structural data obtained by molecular modeling (47). Most of the mutations causing type I deficiency are single nucleotide substitutions within the coding region of PROC, leading to amino acid changes (approx. 70%). If performed, molecular modeling studies, in almost all cases, suggested misfolding and instability of the mutant proteins as a consequence. In vitro expression studies were performed in approximately one third of the cases only. Point mutations introducing a stop codon were also reported (approx. 5%). A smaller number of the point mutations (approx. 9% of all mutations) were found at the exon/intron boundaries leading to splicing defects. Most of the small deletions (approx. 8%) or insertions (approx. 4%) introduced frameshifts, resulting in a premature stop codon and truncated protein. Gross deletions were identified in only 1% of cases. Although almost all missense mutations result in an absolute block in secretion, a few mutations allow the protein to be secreted. However, the rate of secretion is much lower when compared to the wild type protein (48). In homozygous patients having such mutations, PC concentrations are low, but higher than 1%. Diagnosis of type II deficiency is based on the discrepancy between the results of functional testing and antigen measurements (see later for details). Missense mutations are the most frequently reported types; the resulting amino acid change involving the Gla-domain or the pro-peptide result in defective calcium and phospholipid binding (49–57). Mutations in the serine protease domain result in defective protease activity or decreased substrate binding (58–60). Of interest there are mutations that are enriched in certain populations, suggesting the presence of a founder effect. For example, all Finnish type II protein C deficient cases show a single mutation (p.W380G) (61). The p.R147W mutation within PROC is common in Taiwanese Chinese patients with VTE (62). Five frequent mutations account for almost 50% of all PC deficiencies in patients from Japan (c.1268delG, p.F139V, p.R211W, p.V339M and p.M406I) (63). A common ancestor was identified for probands with the p.R306X mutation in the Dutch population (64). The c.3363insC mutation was introduced by French settlers to North America (65). No mutation in the PROC is detectable in 10%–30% of families with PC deficiency (66). This does not necessarily mean that PROC lacks causative mutations in these cases. The larger gene deletions may remain undetectable when using the classical Sanger (i.e., chain termination) sequencing method for detection of mutations. There are known polymorphisms within the PROC gene which may affect meas-

ured PC activity or antigen concentrations (see later for details). This may be regulated by loci other than PROC. As part of the GAIT project, a genome-wide linkage study was performed to localize genes influencing variations in PC plasma concentrations. A region flanked by microsatellite markers D16S3106 and D16S516 on chromosome 16 (16q22–23) with one candidate gene was identified as a major quantitative trait locus influencing variation in PC concentrations. This gene encodes a quinone reductase, NADPH:menadione oxidoreductase 1 (NQO1), involved in vitamin K metabolism. The association of this locus with other vitamin K dependent factor concentrations was also demonstrated, however, the linkage was not as strong as in the case of PC (67). Protein S deficiency

Initially three types of PS deficiency were distinguished according to the results of the functional test, and free and total PS antigen concentration measurements. In type I deficiency, a low amount of activity, total and free PS antigen concentrations can be found. In type II deficiency, only the result of the functional test is abnormal, while in type III deficiency, low PS activity is associated with low free PS but normal total PS antigen concentrations. Based on extensive family studies, it was suggested that type I and type III deficiencies are phenotypic variants of the same genetic alteration (68). The molecular background of this finding was that C4BbP and PS could bind to each other with very high affinity. Therefore, in the case of mild PS deficiency, the complexed form of PS is not decreased (69). Later, genetic differences between type I and type III PS deficiency was demonstrated, and no linkage to the PROS1 locus was found in most of the patients having type III deficiency. In families having the PROS1 mutation, the phenotype more often shows type I rather than type III deficiency. These findings led to the conclusion that type I PS deficiency is a monogenic disease caused by PROS1 mutations, while type III PS deficiency is more complex or heterogeneous disorder (70, 71). The majority of protein S-deficient patients are heterozygous for an inherited defect, and homozygous or compound heterozygous deficiency can cause the same symptoms as in the case of PC deficiency. The molecular genetic background of PS deficiency is also heterogeneous (72). Most of the mutations cause type I deficiency, type II deficiency is diagnosed in approximately 5% of cases. The mutations are listed in the HGMD and in the ISTH databases (http://www. hgmd.cf.ac.uk and http://www.isth.org) (Figure 2). Among the 200 different mutations found to date, missense (approx. 53%) mutations are the most frequent. Approximately 20% of the mutations are small deletions or insertions; non-sense and splice-site mutations are present in approximately 14% and 10% of the cases, respectively. Type II PS deficiency is caused by missense mutations affecting the Gla-domain or EGF4 domain (73). Gross deletions are more frequent than in the case of PC deficiency, they comprise approximately 3% of all cases and are associated with quantitative PS deficiency. However, it is very likely that the number of large


Figure 1 Distribution of causative mutations published to date within the PROC gene according to the HGMD (www.hgmd.cf.ac.uk) database. Nucleotide numbering in exon 1 and nearby is given relative to the first nucleotide of the non-coding exon 1. Nucleotide numbering in the coding region is given relative to the first nucleic acid of the initiator ATG codon. Amino acids are numbered according to the mature protein, where the first methionine is numbered as –42. The literature also mentions two gross deletions: one includes the entire gene, the other includes exons 1–9 (45, 46) (not shown in the Figure.).


Figure 2 Distribution of causative mutations published to date within the PROS1 gene according to the HGMD (www.hgmd.cf.ac.uk) database. Nucleotide numbering is given relative to the first nucleic acid of the initiator ATG codon. Amino acids are numbered according to the mature protein, where the first methionine is numbered –41.


deletions in PROS1 is even higher, but often remains undetected. In the PROSIT study, mutations in PROS1 were found only in 70% of probands with PS deficiency (17). However, using DNA sequencing methods, large deletions or gene segment duplications may remain undetected (74, 75). Moreover, there are mutations affecting the transcription regulatory sequences at the 5’ of the gene (76, 77). Most recently, the first case of PS deficiency due to chromosome translocation has been reported. The diagnosis was established by painting fluorescence in situ hybridization (78). Although a founder effect is not confirmed, it is to be noted that PS Tokushima (p.K155E) shows a high prevalence in the Japanese population.

Polymorphisms in the protein C and S gene It has been reported that individuals with the homozygous C/G/T haplotype at the nucleotide positions -1654 (rs1799808), -1641 (rs1799809), and -1476 (rs1799810) in the promoter region of the PROC had lower plasma PC concentrations compared to individuals with the T/A/A homozygous haplotype (79). The -1654/-1641 CC/GG genotype was associated with a slightly increased risk of thrombosis (OR, 1.39, 95% CI, 1.04–1.87) (80). In a large populationbased case-control study, individuals having this genotype had the lowest plasma PC concentration, and the highest risk for venous thrombosis (OR, 1.27, 95% CI, 1.09–1.48) compared with individuals having the TT/AA genotype (81). In contrast to these findings, variation at the PROC structural locus did not influence plasma PC concentrations in the GAIT project, but the chromosome region 16q22-23 was found to be a major determinant (67). Polymorphisms in genes involved in the vitamin K dependent g-carboxylation of PC and PS may also be responsible for the inter-individual variation in the plasma concentrations of these proteins in the general population (82). PS Heerlen (p.S460P) results in the loss of N-glycosylation at Asn458. The concentration of free PS in the plasma of carriers was slightly lower than that of non-carriers and was considered to be a type III PS deficiency. However, the risk of thrombosis conferred by this mutation is a matter of debate. PS Heerlen displayed reduced anticoagulant activity as cofactor to APC in plasma based assays, as well as when using a FVIIIa degradation system. In a purified system using recombinant proteins, PS Heerlen was a good cofactor of APC in the degradation of normal FVa, but became a poor cofactor in the degradation of FVa carrying the Leiden mutation (83). This suggested a synergistic contribution between FV Leiden and PS Heerlen that increases the risk of thrombosis. However, this hypothesis was not confirmed by another study. The cause of the decrease in free PS concentrations associated with PS Heerlen has not been clarified, but most likely is a consequence of increased clearance (84). A transition of adenine to guanine transition at nt 2148 (p.P626P, silent) and an A to C substitution at nt 2698 have been suggested to decrease PS concentration in healthy individuals (85). However, this finding was not confirmed in

another study (86). No decrease in the secretion of p.P626P variant was demonstrated in an in vitro expression system; this variant was not a risk factor for VTE and did not modify the risk of patients with causative mutations (87).

Laboratory tests of protein C deficiency Two different types of assays are available for the diagnosis and classification of PC deficiency, functional tests and antigen assays. For screening, a functional test should be performed, and if the results are abnormal, the antigen assay can distinguish between type I and type II deficiencies, with concentrations of antigen being normal in the latter. There are two different methods for determination of PC activity (Figure 3A). In both assays, PC present in patient plasma is activated by the venom of Agkistrodon contortrix, now commercially available under the trade name Protac. The advantage of Protac is its insensitivity to plasma protease inhibitors. Also, it can be added directly to plasma. Protac activated PC can be measured either using a chromogenic assay or a clotting assay. In chromogenic assays, paranitroaniline (pNA) is cleaved-off from a small synthetic peptide by APC. Peptide bound pNA does not absorb light at 405 nm, while the liberated chromogenic compound has an intense color at this wavelength. The spectrophotometric measurement can be either end-point or kinetic, with the latter being preferred. The higher the APC activity, the more intense the increase in absorbance during the test. The rationale of the clotting time based assays is the fact that if APC degrades its natural substrates FVa and FVIIIa, it leads to clotting time prolongation. Determination of clotting time can be based on the prothrombin time, activated thromboplastin time (APTT) or Russell viper venom time (RVV), and there is a linear relationship between PC activity and clotting time. There are numerous advantages and disadvantages of both functional assays (88) (Table 1). Clotting tests are influenced by several pre-analytical or analytical variables. Despite predilution of the sample with PC deficient plasma, in the presence of lupus anticoagulant, heparin or direct thrombin (or factor Xa) inhibitor which prolongs the clotting time, falsely increased PC activity can be measured (89). Interference by heparin (up to 1–2 U/mL) is eliminated by adding a heparinneutralizing substance, e.g., hexadimethrine bromide (polybrene) to the reagent. The interference caused by lupus anticoagulant is more pronounced in tests using APTT as activator (90). In the case of increased Factor VIII, the opposite effect might be seen; shortening the clotting time (APTT) will lead to falsely decreased PC activity (91). The most important problem with the clotting method is the influence of the FV Leiden mutation. In patients having this mutation, falsely low PC activity could be detected despite pre-dilution of patient plasma with PC deficient plasma containing wild type FV. If PC antigen concentrations are normal, antigen measurements are not influenced by the mutation; such patients are easily misdiagnosed as having type II PC deficiency (92, 93).


Figure 3 Schematic presentations of protein C and protein S functional assays. PC, protein C; PS, protein S; APC, activated protein C; R-pNA, chromogenic substrate containing oligopeptide (R) and p-nitroaniline (pNA); DA, difference in absorbance.

Chromogenic assays are not sensitive to high FVIII, lupus anticoagulant or FV Leiden. They show lower inter-laboratory and intra-laboratory variation (94). There are two major limitations in the chromogenic assays; the chromogenic peptide substrates do not have exclusive specificity for APC and

may overestimate PC in the presence of other proteolytic enzymes, such as plasmin, kallicrein, and thrombin which also cleave the chromogenic substrate(s). The second problem is that chromogenic assays are insensitive to a certain type of qualitative PC deficiency. When the mutation affects

Table 1 The most important difficulties in the diagnosis of protein C and protein S deficiency. Analytical/methodical problems Functional clotting assays Lupus anticoagulant High heparin concentration Direct thrombin (or FXa) inhibitor High FVIII level (usually )250%) FV Leiden mutation Functional amidolytic assay Presence of enzymes cleaving the chromogenic substrate Mutations result in altered g-carboxylation, or phospholipid binding Problems with molecular genetic diagnosis

Physiological conditions which influence PC-PS levels Pregnancy Oral anticoncipients, hormonal replacement therapy Infants Age Gender Acquired deficiency Vitamin K antagonist therapy Hepatic disease Consumption (DIC, VTE) Presence of autoantibodies (SLE, varicella, malignancies, sepsis, HIV)

Protein C

Protein S Overestimation Overestimation Overestimation Underestimation Underestimation

Overestimation

NA

Normal result despite genetic defect

NA

Large gene segment alterations are not diagnosed by the DNA sequencing method NA Presence of the pseudogene (PROSP) causes difficulties

Significant elevation in the first 22 weeks of pregnancy –

– –

Decreased Decreased

Levels are significantly lower than adult values at birth and infancy Increases with age Lower in women Decreased Decreased Decreased Decreased


the Gla-domain or the propeptide, the functional clotting test gives a low value for activity, while the amidolytic (chromogenic) assay shows normal result. Mutations in the propeptide affecting the g-carboxylation process, substitution of a Gla residue, or mutations influencing phospholipid binding decrease PC activity as measured using the clotting test, while amidolytic activity remains unaltered (49–54). In general, PC deficiency caused by mutations in the serine protease domain can be diagnosed using both clotting and amidolytic assays. However, some mutations in this region (p.R229Q and p.S252N) result in abnormalities that can only be demonstrated with the clotting assay. It is estimated that approximately 5% of patients with type II PC deficiency have normal amidolytic activity, and the deficiency is detectable only by clotting assays. Initially PC antigen was measured using electroimmunoassay. However, this assay is now considered obsolete and no longer routinely used. Most laboratories perform commercially available ELISA testing using pairs of monoclonal or polyclonal antibodies against PC.

Laboratory tests of protein S deficiency Three types of assays are available for the determination of plasma PS: the functional PS activity assay, free and total PS antigen determinations. The test for PS activity measures the effect of PS as a cofactor on the degradation of FVa and FVIIIa by APC. Such clotting tests are the function of free PS concentrations; they are not influenced by PS in complex with C4bBP (Figure 3B). Commercially available tests use thromboplastin, APTT, RVV or FXa for initiation of coagulation. APC is added to patient or control plasma that has been pre-diluted with PS deficient plasma. Following this, the clotting time test is performed. The effect of APC on clotting time, i.e., the extent that the clotting time is prolonged, depends on the content of the cofactor PS in the plasma to be investigated. PS activity assays have a number of limitations (Table 1). First, in patients having FV Leiden mutation, significantly lower PS activity is measured, the results often overlap with the range for true PS deficiency. Such situations may lead to an incorrect diagnosis of type II PS deficiency (95). In the majority of assays, purified FVa is added to the test. This step decreases somewhat the interference from FV Leiden, but does not eliminate the problem completely. Different kits give highly variable results; they have different cut-off values and most of them are very sensitive to reagent handling. Pre-analytical variables are similar to those described for PC activity measurements; high FVIII may lead to underestimation, while the presence of lupus anticoagulant may lead to overestimation of PS activity. Plasma samples are sensitive to repeated freezing and thawing; in vitro activation of FVII may shorten the clotting time in assays using thromboplastin as activator, resulting in underestimation of PS activity. Commercially available PS activity assays give the correct diagnosis of PS deficiency in 97% of patients having PROS1 mutations (96). However, the specificity of the functional

tests is low due to the above-mentioned interfering factors that might lead to inappropriate interpretations of test results, resulting in a false-positive diagnosis. For this reason, some authors do not recommend the use of PS activity assays for diagnosis of PS deficiency, and instead favor free PS antigen (see below) determinations (97, 98). However, by omitting the functional assay, type II deficient patients would remain undiagnosed. Therefore, it has been suggested that both activity and free antigen assays be perfomed from the same sample (99). For the measurement of total PS concentrations, ELISA is the most frequently used method. Measurement of free PS antigen was originally performed from the supernatant following precipitation of C4bBP-bound PS by polyethylene glycol. This method was time consuming and poorly reproducible. Later, monoclonal antibodies against the C4bBP binding domain of PS were produced and measurement of free PS antigen became easier and faster using ELISA assays. Further development led to the introduction of latex enhanced immunoassays (LIA) which were easily adapted to automated coagulometers (100). In the latest ECAT exercise, 77% of laboratories used a LIA method for free PS antigen measurements. In addition to monoclonal antibodies specific for free PS, a ligand binding assay has also been developed. In this assay, C4bBP is used to capture free PS from the plasma (101). Assays for the free form of PS are preferred over total PS determinations since this has higher positive predictive value for PS deficiency (68, 69). In the latest exercise of the European Concerted Action on Thrombosis (ECAT) thrombophilia testing program, only 89 laboratories reported total PS antigen results and 225 laboratories performed free PS antigen measurements.

Conditions affecting protein C and protein S levels; acquired deficiencies Adult reference intervals for PC and PS are wide, and there may be overlap between values seen in healthy individuals and deficient patients (102). In infants, both PC and PS concentrations are lower than adult values. In a healthy full term infant, PC activity is 35% (17%–53%), PS activity is 36% (24%–48%); these reach the lower limit of the adult reference interval by 1 year of age. The PC concentration may remain below the adult reference interval until adolescence (103–106). It should also be noted that PS concentrations are influenced by age and gender; lower results are obtained in women compared with men, and PS values increase with age. PS concentrations may decrease markedly during pregnancy, to a mean level of 46%, and to a lesser extent in individuals using oral contraceptives or on hormone replacement therapy (107, 108). PS measurements in pregnant women can only be used as a test for exclusion, decreased PS concentrations cannot be considered as being deficient. During the first 22 weeks of pregnancy, PC concentrations show a significant increase. It has been postulated that this increase may play a role in maintaining early pregnancy by regulating both coagulation and inflammation (107).


Treatment of patients with vitamin K antagonist (VKA) therapy influences plasma PC and PS activity and antigen concentrations. Activities of PC and PS are markedly decreased and, depending on the assay, antigen may also be lower. Using typical therapeutic doses of VKA, PC antigen and activity decreases to approximately 50% and 25%, respectively (109). Patients should not receive VKA therapy for at least 10 days prior to testing, they should be switched to low molecular weight heparin therapy until collecting the sample for PC and PS measurements. As the half-life of PC is much more shorter than that of PS, it decreases faster following the initiation of VKA therapy and recovers more rapidly following discontinuation. Abnormal g-carboxylation due to vitamin K deficiency also results in decreased PC and PS activity and antigen concentrations. PC and PS deficiency can develop with DIC, severe infection, sepsis and acute excessive thrombosis due to consumption (110, 111). Decreased synthesis of PC and PS can be a consequence of liver disease or immaturity of the liver in preterm infants. Autoimmune syndromes can also be associated with acquired PC and PS deficiency due to the presence of autoantibodies. Postvaricella purpura fulminans is a rare complication in children caused by acquired PC or PS deficiency (112). PS deficiency has also been described in patients with AIDS (113, 114). Therapy with L-asparaginase may lead to decreased PC concentrations by decreasing its synthesis in the liver. Patients having nephrotic syndrome may also exhibit low PS concentrations.

larger gene alterations should be confirmed by other methods, such as quantitative PCR or long PCR.

Concluding remarks The diagnosis of PC and PS deficiency is not an easy task; the functional tests are influenced by several pre-analytical and analytical factors, and the molecular genetic diagnosis is also challenging. In the case of PC, the use of the chromogenic or the clotting functional test as screening tests is a matter of debate. A clinical guideline most recently issued by UK-based medical experts recommends the chromogenic PC assay as being the preferred test (116). In our experience, only the use of both chromogenic and clotting tests could cover the full range of PC deficiencies and reduce the problems arising from interfering conditions. In the diagnosis of PS deficiency, if a PS activity assay is used for initial screening, low results should be further investigated using a immunoreactive assay for free PS. Acquired deficiencies should be considered and looked for when establishing the diagnosis. In all cases, repeat testing is crucial for establishing the diagnosis. If the results of functional and antigenic assays do not confirm the diagnosis unequivocally, genetic testing is indicated.

Acknowledgements This work was supported by a grant from the Hungarian National Research Fund (OTKA K78386).

Molecular genetic diagnosis of protein C and protein S deficiencies Conflict of interest statement Since both PC and PS deficiencies may be acquired. Prior to suggesting a genetic defect, all the possible acquired conditions must be excluded. Equivocal cases require confirming the presence of a true inherited deficiency using mutation analysis. As multiple sites of mutation have already been described in PROC and PROS1 genes, and since no so-called hot spot could be identified within these genes, DNA sequencing is the most reliable method for establishing a genetic diagnosis. The molecular genetic diagnosis of PS deficiency represents a particularly difficult situation. The presence of the PS pseudogene makes the genetic diagnosis of PS deficiency rather complicated; careful design of primers are required to eliminate the amplification of pseudogene fragments. In a high number of individuals with PS deficiency, no mutation was found when using DNA sequencing. This discrepancy is due to larger gene alterations that are not diagnosed by this method (66, 75, 115). A recently developed and commercially available method for demonstrating large gene segment deletions or duplications is the multiplex ligationdependent probe amplification (MLPA) method. Re-analysis of DNA samples from ‘‘mutation negative’’ PS deficient patients using this method has revealed large deletions or duplications in a number of cases (74). The presence of such

Author’s conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Antithrombin deficiency and its laboratory diagnosis

La´szlo´ Muszbek1,2,*, Zsuzsanna Bereczky1,2, Bettina Kovaćs3 and Istvań Koma´romi2

Keywords: amydolytic assay; antithrombin, antithrombin deficiency; thrombophilia.

1

Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary 2 Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary 3 Borsod Abau´j Zempleń County and Teaching Hospital, Miskolc, Hungary

Abstract Antithrombin (AT) belongs to the serpin family and is a key regulator of the coagulation system. AT inhibits active clotting factors, particularly thrombin and factor Xa; its absence is incompatible with life. This review gives an overview of the protein and gene structure of AT, and attempts to explain how glucosaminoglycans, such as heparin and heparan sulfate accelerate the inhibitory reaction that is accompanied by drastic conformational change. Hypotheses on the regulation of blood coagulation by AT in physiological conditions are discussed. Epidemiology of inherited thrombophilia caused by AT deficiency and its molecular genetic background with genotype-phenotype correlations are summarized. The importance of the classification of AT deficiencies and the phenotypic differences of various subtypes are emphasized. The causes of acquired AT deficiency are also included in the review. Particular attention is devoted to the laboratory diagnosis of AT deficiency. The assay principles of functional first line laboratory tests and tests required for classification are discussed critically, and test results expected in various AT deficiency subtypes are summarized. The reader is provided with a clinically oriented algorithm for the correct diagnosis and classification of AT deficiency, which could be useful in the practice of routine diagnosis of thrombophilia. Clin Chem Lab Med 2010;48:S67–78. *Corresponding author: La´szlo´ Muszbek, MD, PhD, Clinical Research Center, University of Debrecen, Medical and Health Science Center, 98, Nagyerdei krt., Debrecen, 4032, Hungary Phone: q36 52 431 956, Fax: q36 52 340 011, E-mail: [email protected] Received July 30, 2010; accepted September 25, 2010; previously published online November 10, 2010

Introduction The stepwise discovery of antithrombin (AT, SERPINC1) is an exciting story. A detailed account and chronology of the most important events leading to the foundation of our present knowledge about this essential inhibitor of blood coagulation has recently been in an excellent review by Abildgaard (1), recommended for readers interested in this subject. Here, only the issue of nomenclature is mentioned. In the earlier literature, AT used to be termed antithrombin III to distinguish the protein from other proteins with antithrombin activity. Antithrombin I is the thrombin absorbing capacity of fibrin (2) and antithrombin II, more frequently termed heparin cofactor II (SERPIND1) which is another inhibitor of thrombin in plasma (3). Although this is not the only antithrombin present in plasma, we adopted the current nomenclature and use the terms antithrombin and AT throughout the article. AT is a single-chain glycoprotein with a molecular mass of 58,200 Da. The mature protein consists of 432 amino acids with three internal disulfide bonds. As much of the literature and the existing databases use traditional amino acid residue numbering, starting with the N-terminal residue of the mature protein, we used this system in the article. The numbering system recommended by the Human Genome Variation Society (HGVS) starts with the initiator methionine. In the case of AT, HGVS numbering can be calculated by adding 32, corresponding to the 32 amino acid residues of the leader sequence, to the traditional number. AT has two isoforms which differ only in the extent of glycosylation (4, 5). The a isoform is N-glycosylated on four Asn residues (95, 135, 155 and 192), while the b isoform lacks glycosylation on Asn135. The a variant is the major AT isoform (90%–95%) in the circulation, the b isoform represents only 5%–10% of AT in plasma. AT is synthesized in the liver, its half-life in the circulation is approximately 2.4 days. A prominent feature of AT is its high affinity binding to negatively charged glycosaminoglycans (GAGs) such as heparin or heparan sulfate which contain specific pentasaccharide units. Due to the lack of carbohydrate residue on Asn135, the b isoform binds to GAGs with higher affinity. Heparan sulfate in the form of heparan sulfate proteoglycane (HPSG) is present on the surface of vascular endothelium. Thus, a higher portion of the b isoform becomes cleared from the circulation and targets the vessel wall.

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The structure of antithrombin and structural changes during its interaction with active clotting factors The atomic 3D structure of native AT was resolved approximately 15 years ago (6, 7). Since then, numerous AT structures have been published which have helped in the understanding of how AT exerts its inhibitory function (8–14). AT belongs to the family of serine protease inhibitors (serpins), the largest family of protease inhibitors that consists of over 1500 members (4, 14, 15). These are single chain globular proteins that consist of 300–500 amino acid residues and show about 30% sequence identity. Serpins share a common tertiary structure; they contain three b-sheets (A-C) and eight to nine a-helices (A-I). A flexible peptide loop, reactive center loop (RCL) containing the reactive site, is exposed on the top of the molecule. RCL contains a sequence which is complementary to the active site of the target protease. All serpins feature significant structural flexibility, which allows dramatic structural changes upon reaction with the protease to be inhibited. These are so-called suicide inhibitors. The target protease cleaves a scissile bond in RCL and then it remains covalently linked to the inhibitor. Antithrombin is a misnomer, the inhibitory effect of AT is not restricted to thrombin. It is a polyvalent serpin that also inhibits activated factor X (FXa) and to a lesser extent a whole series of serine proteases involved in the hemostatic machinery, including FIXa, FXIa, FXIIa, plasmin and kallikrein (4, 5). AT inactivates FVIIa only when it is bound to tissue factor (16–18). AT is a co-called progressive inhibitor; the rate of its reaction with the active coagulation factors is slow, but in the presence of heparin or HPSG, the rate of inhibition is accelerated 500-fold. AT has a typical serpin secondary and tertiary structure. It consists of nine helices and three b-sheets (Figure 1). In the uncleaved form, it can exist in two main conformational states. In the native uncleaved form, the 24-membered RCL with the scissile P1-P1’ (Arg393-Ser394) bond is outside the main body of AT (Figure 1A). In the latent conformation, the RCL is inserted into the b-sheet. The latter conformation is thermodynamically more stable than the native form, which is kinetically ‘‘trapped’’ in a high energy state. AT circulates primarily in this kinetically trapped native form. The X-ray structure of this conformation revealed that the P1 residue (Arg393) points to the surface of the body of AT, and the P14-P15 residues are inserted into b-sheet A, which constrains the RCL and allows contacts between the P1 arginine side chain and the body of AT. Having such a rigid conformation of RCL, AT is a poor inhibitor of FXa or thrombin, and is unable to inhibit FIXa. The binding of pentasaccharide or heparin containing the pentasaccharide unit causes remarkable changes of the conformation of RCL and its close proximity (Figure 2B). The entrapped part of RCL is expulsed from b-sheet A, the end of the third b-strand of b-sheet A moves closer to the fifth b-strand and helix D becomes elongated. The interaction between AT and the pentasaccharide unit takes place in two steps (not shown on Figure 2); an initial weak binding inter-

mediate becomes transformed into a high binding state with 1000-fold higher affinity. The latter conformation is necessary for the effective formation of the Michaelis complex (Figure 2C) between AT and FXa or FIXa, in which Arg393 of AT and the region in its immediate vicinity is recognized by the protease as a substrate loop. The mechanism of Michaelis complex formation between thrombin and AT is somewhat different. In this case, the conformational change induced by the allosteric effect of pentasaccharide is not sufficient, and probably not even required. Thrombin also binds to heparin, and the bridging effect of heparin of 18 saccharide units or longer, which brings thrombin and AT together, is essential for effective interaction (11, 22, 23). After the rate-controlling Michaelis complex formation, the inhibition of active coagulation factors follows the general scheme of serpin action. In the first step of proteolytic reaction, an acyl-enzyme intermediate is formed through an ester bond between Arg393 and the active site serine of the protease. AT undergoes a rapid, drastic and irreversible conformational change where the P14-P3 part of RCL becomes incorporated into b-sheet A as an additional strand (Figure 2D), and AT assumes a cleaved relaxed form. This process is accompanied by a 1000-fold reduction in heparin affinity and by a large-scale conformational change in the acylenzyme complex. The protease which is covalently tethered to Arg393 becomes transported from the top to the bottom ˚ away from its original position. of AT, approximately 70 A Due to the distortion of the active site of the protease, the acyl intermediate becomes stabilized and the second step of proteolytic reaction, the release of the cleaved peptide, cannot take place. In this process the protease structure becomes disrupted and the catalytic triad distorted. Only very slow release of the inactive inhibitor and enzyme can be detected from the AT-protease complex (24).

The role of antithrombin in the regulation of coagulation AT serves as a highly important regulator of hemostasis; its absence is incompatible with life (5). The primary actions of AT are the inhibition of thrombin mediated fibrin clot formation and the generation of thrombin by FXa. As mentioned earlier, AT also inhibits activated clotting factors higher up in the intrinsic (FIXa, FXIa, FXIIa) and extrinsic (FVIIa-tissue factor complex) pathways. It also inhibits a series of other non-coagulant effects of these clotting factors, including platelet activation, vascular cell signaling, proliferation, cytokine production, etc. There are two paradoxes concerning the effect of AT and its importance in the regulation of clotting machinery: 1) it is a weak progressive inhibitor of activated clotting factors, 2) it fails to inhibit effectively fibrin-bound thrombin and FXa present in an activation complex on the platelet surface. As discussed in the previous section, interaction with heparin, or its in vivo ‘‘substitute’’ HSPG, significantly accelerates the inhibitory action of AT and makes it a highly effective inhibitor of thrombin, FXa and other active clotting

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Figure 1 Structural elements of antithrombin (AT) in its native (A) and latent (B) state based on the X-ray structure in the RCSB protein data bank (pdb ID: 2b4x). The b-sheets A, B and C are colored yellow, green and purple, respectively. Helical secondary structural elements are shown in orange, except for helix D that is shown in black. Helix D plays a crucial role in heparin pentasaccharide binding. (In the case of serpins, conserved helical structure elements are identified by capital letters.) The P3-P14 portion of the reactive center loop consisting of P1-P17 and P1’-P17’ residues is colored dark blue. The Arg393 (P1) residue is shown by a ball-and-stick representation. In the latent state, a substantial portion of the reactive center loop is inserted into b-sheet A as an additional b-strand. The figures were prepared using Chimera software (Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, CA, USA) (19).

factors (5). HSPG is widely available on the vascular endothelium and/or in the underlying subendothelial matrix and tissues. Only a minor portion of HSPG contains the 3-O-

sulfated pentasaccharide unit required for the acceleration of AT activity (25). This active HSPG (A-HSPG) species constitutes 5% of the total HSPG associated with rat microvas-

Figure 2 Schematic representation of the mechanism of activation and action of antithrombin (AT) on FXa. (A) The native (circulating) form of AT, (B) the pentasaccharide-activated AT, (C) AT-factor Xa (FXa) complex; only epidermal growth factor 2 (EGF 2) and serine protease domains of FXa are shown, (D) the acyl-enzyme complex in which FXa is covalently linked to AT. For the A, B and C parts of the figure the X-ray coordinates wpdb IDs are 2b4x, 2gd4 (AT only) and 2gd4 (AT-FXa), respectivelyx deposited in the protein data bank (20) were used. The construction of (D) was based on the structure of the a1-antitrypsin-elastase acyl-enzyme complex (pdb ID: 2d26) (21). a1-antitrypsin was replaced by AT and elastase was substituted by FXa. Next, energy minimization of the constructed complex was performed with Yasara software (Yasara Biosciences GmbH, Vienna, Austria) (www.yasara.org). b-sheets and helices are shown in magenta and orange, respectively. Peptide sections that undergo remarkable conformational changes wP1-P15 section of the reactive center loop (RCL), the third b-strand of b-sheet A (s3A) and helix D (hD)x are depicted in green. The Arg393 residue and the pentasaccharide (PeS) are shown by ball-and-stick representations. The figures was prepared using Chimera software (19).


cular endothelial cells. Studies of its distribution in different vessels (26, 27) suggest that A-HSPG present on the surface of endothelial cells provides a basic level of activated AT for anticoagulant activity, while the availability of a much larger pool following endothelial damage and vessel wall injury dramatically increases the anticoagulant potential of AT. The relative importance of progressive AT activity or AHSPG induced activity in the physiological regulation of blood coagulation is not clear. The fact that homozygous mutations at the heparin binding site, which render AT unable to bind GAGs, are compatible with life (see later for details), as opposed to other homozygous AT deficiencies, suggests the importance of progressive activity. On the other hand, the severe thrombophilia seen in such patients underlines the importance of heparin/HSPG induced AT activation. The association of active clotting factors with the surface of activated platelets and with the fibrin clot significantly modifies the inhibitory effect of AT or AT-heparin. When present in the prothombinase complex on platelets or phospholipid surfaces, FXa escapes the inhibition by AT (28–30). Thrombin bound to fibrin or to fibrin degradation products becomes refractive to inhibition by the AT-heparin complex. Fibrin, heparin and thrombin form a ternary complex, in which thrombin exosite 1 and exosite 2 are occupied by fibrin and heparin, respectively (31). This prevents AT-associated heparin from interacting with exosite 2 on fibrinbound thrombin, and the bridging action of heparin cannot operate. It is of interest that FVIIa becomes sensitive to inhibition by AT only when bound to tissue factor (16, 17). The above findings suggest a double role for the AT and AT-AHSPG complex in the physiological regulation of blood coagulation. It might control low-level thrombin formation that occurs physiologically in the unperturbed circulation by the inhibition of tissue factor-FVIIa complex, FXa and perhaps other active clotting factors. AT might also exert a scavenger function by neutralizing FXa and thrombin that have escaped from the clot and from the activation complex. The latter mechanism could prevent the propagation of the clot to areas away from the site of vascular injury. In vivo, the AT-protease complex is rapidly eliminated from the circulation by utilizing a common serpin-protease complex clearance pathway. Binding to members of the lowdensity lipoprotein receptor family, primarily to low density lipoprotein-related protein which is an important receptor in the liver, is the main pathway of the elimination of serpinprotease complexes (5, 32).

Gene structure of antithrombin The gene for human AT (SERPINC1) is located at the 1q23q25 position and contains seven exons producing a 1.4-kb messenger RNA (mRNA), and six introns (33, 34). All the exon/intron boundaries follow the GT-AG rule. Nine complete and one partial Alu repeats were identified in introns 1, 2, 3B, 4 and 5. There is a highly polymorphic trinucleotide repeat sequence in intron 4 which is useful for haplotype analysis in studies of recurrent mutations and for linkage

analysis in families with thrombosis (35). Primer extension analysis has mapped the AT transcriptional start site in liver cells to a position 72 bp upstream of the ATG translation initiator codon (36). A leader sequence of 32 amino acids is encoded by exon 1 and the 5’ end of exon 2 (37). The heparin binding site of AT is encoded by exon 2 and exon 3a. The reactive site, located in the carboxy-terminal part of the protein, is encoded by exon 6.

Epidemiology of antithrombin deficiency The prevalence of inherited AT deficiency in the general population is estimated to be between 1:2000 and 1:3000 (38). Most of the genetic defects result in type II (qualitative) deficiencies (39). The prevalence of AT deficiency in patients with venous thromboembolism (VTE) is much higher, between 1:20 and 1:200 (40). According to an Italian study of symptomatic patients and relatives, type I mutations (quantitative deficiencies) are more frequent than type II variants (41). In unselected patients with a history of VTE, the frequency of AT deficiency is 0.5%–1.1% (40, 42). In a cumulated analysis of 1705 selected patients with VTE, the frequency of AT deficiency was 2.4% (43). During a mean follow-up time of 2.3 years the incidence of venous thrombosis was high; being 12% in individuals with hereditary AT deficiency in a small Italian cohort (44). For comparison, the incidence of thrombosis in protein C (PC) and protein S (PS) deficiency was 2.8% and 3.3%, respectively. In the large prospective EPCOT study (European Prospective Cohort on Thrombophilia), the risk of first VTE in asymptomatic AT, PC or PS deficient individuals and in individuals with Factor V Leiden mutation was analyzed (ns575). During the 5.7 year of follow-up, 4.5% of these individuals developed VTE, the annual incidence of first VTE was the highest in those with AT deficiency (1.7%/ year) (45). Based on the prevalence data in the general population and in VTE patients, the relative risk of VTE in patients with AT deficiency was estimated to be approximately 25–50-fold (46). Since then, prospective and casecontrol studies have calculated the same magnitude of VTE risk conferred by AT deficiency in different ethnical groups (47–49). Based on the results of these epidemiological studies, it can be concluded that the risk of VTE conferred by hereditary AT deficiency is the highest among inherited thrombophilias. However, the risk of VTE seems to vary according to the subtypes of AT deficiency (see later). AT deficiency also represents an increased risk for development of PE in deep venous thrombosis (DVT), and an increased risk for recurrence of VTE. In an Italian study of patients with proximal DVT, the risk of pulmonary embolism (PE) was 2.4-fold (95% CI: 1.61–3.63) in AT deficient patients compared to individuals who developed DVT without inherited thrombophilia (50). In AT deficiency, the annual incidence of recurrent VTE was found to be 10% (95% CI: 6.1%–15.4%) in a recently released Dutch study (51). In


an Italian cohort, the adjusted hazard ratio for the recurrence of VTE was 1.9 (95% CI: 1.0–3.9) (52).

Molecular genetic background of antithrombin deficiency, genotype-phenotype correlations The first report on AT deficiency was described by Egeberg in 1965 (53). The first functional AT defect, AT Budapest, was reported by Sas et al. in 1974 (54). Since then, a high number of deficient patients have been identified, and the molecular genetic background was clarified in a significant number of cases. According to the recommendations of the International Society on Thrombosis and Haemostasis, AT deficiency is classified as type I (quantitative) and type II (qualitative) deficiency (55). In type I deficiency, AT activity and the antigen concentration are equally decreased, suggesting defective synthesis or secretion of the protein. In type II deficiency, the defect may involve the reactive site (type II RS), the heparin-binding site (type II HBS) or it can exert a pleiotropic effect (type II PE) (56). The inheritance of AT deficiency, in general, is autosomal dominant. However, in the case of type II HBS deficiency, it often shows incomplete penetrance or an autosomal recessive pattern. The majority of AT deficient patients are heterozygous for the defect with typical AT activity values approximately 50%. Homozygosity is incompatible with life, with the exception of type II HBS variant (described later). The molecular genetic background of AT deficiency is heterogeneous. The mutations are best summarized in the Antithrombin Mutation Database (http://www1.imperial.ac.uk/medicine/about/divisions/departmentofmedicine/experimentalmedicine/haematology/coag/antithrombin/) and in the database of human gene mutation data (HGMD) (http://www.hgmd.cf.ac.uk) (Figure 3). Almost 50% of the 215 different mutations that have been reported in the HGMD are missense mutations. Small deletions and insertions are also common, contributing 20% and 10%, respectively. Non-sense mutations and splicing site mutations represent 8% and 5% of all reported causative sequence variants, respectively. Whole or partial gene deletions are relatively frequent (5%), while complex rearrangements are rare. Type I AT deficiencies are most commonly caused by insertions or deletions leading to frameshift and premature stop codon, or less commonly by non-sense mutations. These mutations obviously explain the type I phenotype, primarily a result of unstable mRNA transcripts and/or the presence of truncated proteins. Large gene segment deletions also lead to type I deficiency. By screening ‘‘mutation negative’’ AT deficient cases using the multiplex ligation-dependent probe amplification (MLPA) technique, several gross deletions were identified. The breakpoints are often located within Alu repeat elements (57). Amino acid changes caused by single nucleotide substitutions within the coding region of SERPINC1 may also lead to type I deficiency. In this case, the absence of mutant protein in the circulation is due to misfolding or a secretion defect (4).

The type II AT deficiencies are most commonly caused by missense mutations. Among the mutations known to involve the reactive site domain, two regions are preferred: the hinge region (most frequently residues Ala382 and Ala384) and around the reactive domain at residues Gly392 (AT Stockholm), Arg393 and Ser394 (58). Most of the missense mutations leading to type II HBS deficiency affect residues Pro41 (AT Basel), Arg47 (AT Padua I), Leu99 (AT Budapest 3) and Arg129. Practically all patients with AT Budapest 3 (p.Leu99Phe) mutation described to date were of South Eastern European origin, which may suggest a founder effect (35, 59–61). Type II PE deficiency is caused by mutations involving residues 402, 404–407 and 429. This region is responsible for both the structural and functional integrity of AT. These mutations lead to impaired function of the reactive site and also to reduced secretion (58). A new pleiotropic mutant (AT Murcia, p.K241E) has been described recently in which altered glycosylation of the molecule led to impaired heparin binding and thrombin inhibition (62). Some of the missense mutations occurred in the mobile regions of AT, mainly at the hinges of the reactive center loop, or in the region involved in the shutter-like opening of the main b-sheet of the molecule. The latter is required for insertion of the reactive loop into the b-sheet. Even change in a single amino acid in these sensitive regions can lead to conformational changes, loss of stability that facilitates the formation of intermolecular linkages and lead to the formation of oligomers or transformation to the latent conformation (4). The secretion of these variants is also impaired which results in a circulating deficiency. Homozygous type I AT deficiency is not compatible with life and heterozygous patients usually suffer severe thrombosis at a young age. The same stands for type II RS and type II PE deficiencies. However, there is at least one notable exception. The heterozygous p.Ala384Ser mutation (AT Cambridge II) causes type II RS deficiency with a mild phenotype, and this mutation can also exist in homozygous form (63, 64). Type II HBS deficiency confers a lower risk of thrombosis compared with the other subtypes (65, 66). Homozygous type II HBS patients usually survive, thrombosis may develop even earlier (frequently in childhood) than in patients with heterozygous type I or other type II deficiencies. Symptoms of AT deficiency are DVT and/or PE which are often recurrent. DVT not infrequently develops at unusual sites, such as in the proximal extremities, and in mesenteric, renal, portal, retinal and cerebral veins (67–70). Intracardial atrial thrombosis has also been reported (71). The risk of thrombosis conferred by AT deficiency to pregnant women is significantly greater than in other deficiencies. The estimated risk is 1:2.8 for women with type I deficiency, which is approximately 350-times higher than the risk conferred by pregnancy alone (72, 73). In addition to venous thrombosis, occasionally, arterial thrombosis has also been reported in patients with AT deficiency (74, 75). AT Cambridge (p.A384S) mutation increased the risk of myocardial infarction 5.66-fold as reported by a Spanish study that enrolled 1224 patients and


Figure 3 Distribution of causative mutations in the SERPINC1 gene according to the database of human gene mutation data (HGMD) that have been published to date (www.hgmd.cf.ac.uk). Nucleotide numbering is given relative to the first nucleic acid of the initiator ATG codon. Amino acids are numbered according to the mature protein, where the first methionine is numbered –32.

1649 controls (76). In contrast, in a large cohort of relatives of VTE patients with PC, PS or AT deficiency (ns468), the risk of arterial thrombosis in those -55 year of age was increased only in PC and PS deficient patients, while AT deficiency was not associated with an increased risk (hazard ratio 1.1, 95% CI: 0.1–10.9) (77). According to a recent meta-analysis of studies involving children with arterial ischemic stroke and cerebral venous sinus thrombosis, the summary OR for the risk of arterial ischemic stroke in children having AT deficiency was 3.29 (95% CI: 0.70–15.48), while the OR for the risk of cerebral venous sinus thrombosis was 18.41 (95% CI: 3.25–104.29) (78). These findings do not support a significant contribution of AT deficiency to the risk of atherothrombotic events.

Acquired antithrombin deficiency In healthy full-term newborns, the concentration of AT is in the range of 51%–75% of adult average values (79). Due to severe immaturity of the liver, in preterm infants AT concentrations can be much lower than in full terms infants (80). AT concentrations reach adult ranges by the age of 1 year (81). Production of AT is reduced in liver disease with impaired hepatic function. In patients with nephrotic syndrome or other diseases associated with renal or enteral protein loss, the low AT concentration is due to increased elimination. Low concentrations of AT as a result of consumption are found in patients with sepsis, disseminated intravascular coagulation, large thrombus, thrombotic micro-


angiopathy, acute hemolytic transfusion reactions and malignancies (82). There are two notable examples of druginduced AT deficiency. Long-term therapy with unfractionated heparin is a common cause of moderate AT consumption, which is probably the result of greatly enhanced formation of thrombin-AT complex in plasma. Therapy with L-asparaginase leads to intracellular retention of AT within the endoplasmic reticulum, perhaps due to interference with folding of the molecule and with glycosylation of the protein (83, 84). Interestingly, co-administration of dexaamethasone with L-asparaginase increased the concentration of AT and reduced the risk of thrombosis. The effect of dexamethasone is possibly due to induced expression of heat shock proteins and endoplasmic reticulum-associated chaperons which prevent the conformational effect of L-asparaginase (85).

Laboratory diagnosis of antithrombin deficiency A first-line test for the diagnosis of AT deficiency should detect all deficiencies, i.e., AT deficiencies due to decreased AT concentration as well as to a defective molecule. Therefore, the first line test should be a functional assay. The original clotting methods where the inhibition of thrombin by diluted native serum or defibrinated plasma was measured by fibrinogen clotting are impractical and inaccurate and not in use any longer. With the modern chromogenic (amidolytic) assays, the inhibition of thrombin or FXa activity by AT is measured using thrombin/FXa specific tri, or tetra-peptide substrates which show sequential similarity to the P1-P3 or P1-P4 sequences of the natural substrates of these enzymes (Figure 4) (86, 87). The peptides conform to the active site of the respective active clotting factor and a para-nitroanaline (pNA) group is attached to their C-terminal end. Thrombin or FXa rapidly release the pNA group from their peptide substrate. Free pNA, as opposed to the peptide-bound form,

has strong light absorption at 405 nm and its release can be easily monitored spectrophotometrically. The assays can be performed in the presence of heparin (heparin cofactor activity) or without heparin (progressive activity). In the former assays, the inhibition of active clotting factors is very quick, while in the latter cases more time is required for ATII to exert its inhibitory action. As only the heparin cofactor activity is decreased in all subtypes of AT deficiency (Table 1), the assay measuring this activity is the generally accepted first line test for the diagnosis of deficiency. Unfortunately, as external quality control exercises reveal, the improper practice of using only an antigenic AT assay, which detects -50% of AT deficiencies, still exists in a few laboratories. Figure 4 demonstrates the assay principle of amidolytic AT assays. Heparin binds to AT making it highly reactive with thrombin and FXa (activated AT; AT*). Thrombin or FXa is added in excess of AT and a part of it becomes rapidly complexed with heparin-AT*, in the complex AT activity is abrogated. The extent of thrombin/FXa inhibition depends on plasma AT activity, and the residual free thrombin or FXa is inversely related to AT activity. The amount of free thrombin or FXa is measured using a chromogenic substrate described above. The increase in absorbance at 405 nm can be measured using a kinetic or end-point method (the former is preferred), and the change of absorbance is converted to AT activity using a calibration curve. Reference plasma of known AT activity is used to construct the calibration curve. A WHO international standard (2nd International Standard Antithrombin, Plasma, NIBSC code: 93/768) with an assigned potency of 0.85 International Units (IU) is available from the National Institute for Biological Standards and Control (NIBSC; Potters Bar, UK). This international plasma standard should be used by companies for the calibration of their reference plasma and this information should be stated on the application sheet. The chromogenic heparin cofactor AT assay has good reproducibility. Laboratories equipped with automated laboratory analyzers or coagulometer should aim for within-batch precision CVF2%, and for within-laboratory reproducibility (total error) CVF5%. According to a previous report based on the quality assessment program for thrombophilia screening by the ECAT Foundation which included 136 laboratories during the time period 1996–2001, the median long-term within-laboratory analytical CV was 7.6% with a 95% CI of 3.6–35.5 (88). These values suggest that for many laboratories, there is much work needed to improve the quality Table 1 Laboratory diagnosis and classification of antithrombin (AT) deficiencies.

Figure 4 Measurement principle of chromogenic antithrombin assays. Both anti-thrombin and anti-FXa heparin cofactor assays are demonstrated. AT, antithrombin; AT*, antithrombin activated by heparin; FXa, activated factor X; R, the peptide part of chomogenic thrombin or FXa substrates; pNA, para-nitro aniline. In the last line, the oligopeptide components of a thrombin (thr) substrate (S-2238) and a FXa substrate (S-2772) are shown.

Subtypes of AT deficiencies

Heparin cofactor AT assay

Progressive AT assay

AT antigen assay

Type Type Type Type

x x x x

x x n x

x n n n or subnormal

I II RS II HBS II PE

RS, reactive site; HBS, heparin binding site; PE, pleiotrop; n, normal.


performance of AT assays. Joining an international accredited external quality assessment program is highly recommended for laboratories routinely performing AT measurements. Human thrombin was used in previous thrombin inhibition assays. Human thrombin also reacted with heparin cofactor II and made the assay relatively insensitive for the detection of AT deficiency (89, 90). In most commercial kits, human thrombin has been replaced by bovine thrombin which shows minimal reaction with heparin cofactor II. FXa does not react with heparin cofactor II at all. Heparin cofactor AT assays based on bovine thrombin and FXa inhibition seem to function equally well, the sensitivity of both assays is close to 100%. As the reactive site of thrombin and FXa differs somewhat, one would expect that AT deficiencies caused by certain mutations around the reactive site are detected by the two types of assays with different sensitivity. Indeed, the Ala384Ser mutation (AT Cambridge II) which is a relatively prevalent variant in the general population, is not detected by the anti-FXa assay, but anti-thrombin activity is mildly, but significantly, reduced (63, 64). However, this mutation causes only a relatively mild thrombophilia, and even elderly homozygous individuals might lack the history of VTE. In contrast, according to our experience with a high number of AT Budapest 3 mutants, the anti-Xa assay is significantly more sensitive in detecting this type II HBS deficiency compared with the assay based on thrombin inhibition. Progressive AT assays are based on the same principle as heparin cofactor assays, but are performed in the absence of heparin on less diluted plasma samples. In addition, the incubation time is prolonged significantly. Unfortunately, the application sheets for the commercial AT activity assays do not provide a description on how to use the test kit as a progressive assay, the appropriate conditions need to be established by the user. These conditions may vary among commercial kits, and the user needs to experiment. As a starting point, 10-fold diluted plasma and a 15-min incubation time is recommended. Although this test is essential for the diagnosis of type II HBS deficiency (Table 1), for the reasons mentioned above, the progressive assay is very much under utilized in the diagnosis and classification of AT deficiencies (91). As discussed earlier, differentiation between heterozygous type II HBS deficiency causing mild thrombophilia and other type of functional defects causing a severe phenotype is of clinical relevance. In certain, clinical set-ups this might influence the decision concerning anticoagulant therapy. Distinguishing between homozygous type II HBS deficient patients who have a very severe phenotype, and heterozygotes is also of clinical relevance. Measurement of AT antigen concentrations is required for the classification of AT deficiencies. Traditional electroimmunodiffusion and radial-immunodiffusion techniques are too time consuming, imprecise and their use is no longer recommended. At present, latex-enhanced immuno nephelometry is the most frequently used method for measurement of AT antigen concentrations (92), and commercial kits for this purpose are available. It is rather surprising that no reference interval determined according to guideline (C28-A3) from the Clinical and Lab-

oratory Standards Institute (CLSI; Wayne, PA, USA) is available for AT activity and antigen. A number of different ‘‘normal’’ ranges, varying within a narrow interval have been reported in the literature and are available in manufacturer’s application sheets. Accepting 80% of the average normal (0.8 IU/mL) as the lower limit of reference interval for AT activity seems to be an acceptable compromise, and most laboratories use this value. The upper limit of the reference interval does not have any clinical relevance. AT antigen concentrations can be expressed as mass concentration (mg/L), although there are a rather wide variety of ‘‘normal’’ ranges for mass concentration. In addition, values for mass concentration are difficult to compare to AT activity values. For this reason most laboratories use the same principle and the same reference interval for AT antigen as for AT activity. It should be noted that by definition, 2.5% of the values obtained with normal, non-deficient samples are below the lower limit of the reference interval, and values between 70% and 80% should be interpreted with extreme caution. Platelet poor citrated plasma is used for both activity and antigen measurements and can be stored at –208C for up to 4 months. Measurement of AT activity and concentration is not recommended within 3 months of an acute event. During this period, if values are within the reference interval, the exclusion of AT deficiency is possible, but the diagnosis of AT deficiency cannot be confirmed. In a number of cases, AT determination is requested for patients who are on anticoagulant therapy. Oral anticoagulant therapy with vitamin K antagonists, such as warfarin or acenocoumarol might increase the level of AT (93–98), while administration of unfractionated heparin decreases the concentration of AT (58, 94, 97, 99). Low molecular weight heparins do not have such an effect (100). For these reasons, we do not recommend diagnosing AT deficiency in patients who are undergoing unfractionated heparin therapy. Also, we do not recommend attempting to exclude AT deficiency during oral anticoagulant therapy. In our experience switching from oral anticoagulant therapy to low molecular heparin for 10 days prior to blood collection is a good compromise which allows the measurement of valid AT values. The algorithm used in the authors’ laboratory for the diagnosis and classification of AT deficiency is demonstrated in Figure 5. If heparin cofactor AT activity is -80% we carefully look for and exclude acquired AT deficiencies, such as liver disease, renal or enteral protein loss, consumption coagulopathy, unfractionated heparin treatment or therapy with l-asparaginase. To establish the diagnosis, repeated tests are required from different blood samples collected from the same person. If feasible, we recommend a time interval of at least 3 weeks between the two blood collections. If the heparin cofactor activity is repeatedly equal to or -70% and acquired causes have been excluded, the laboratory diagnosis of inherited AT deficiency can be established. Between 70% and 80% of AT activity, it is highly recommended to confirm the diagnosis by molecular genetic testing. Once AT deficiency is diagnosed, the next step is its classification, which occurs in two steps. Plasma AT antigen concentrations are measured to differentiate between type I and type II deficiency. Decreased


Figure 5 Laboratory diagnosis and classification of antithrombin (AT) deficiency. Assays are shown in rectangles, diagnoses in italics are underlined. As stated in the text, we do not recommend diagnosing AT deficiency in patients receiving therapy with unfractionated heparin, and do not recommend excluding AT deficiency during oral anticoagulant therapy.

AT antigen implies type I deficiency, while AT antigen in the normal range indicates type II deficiency. Finally, it is clinically important to distinguish II HBS subtype from other type II variants by performing a progressive activity assay. As opposed to other type II subtypes, HBS variants have normal progressive activity. Table 1 summarizes the results of diagnostic and classification tests in different subtypes of AT deficiency.

Concluding remarks AT is a slow progressive inhibitor of active clotting factors, particularly thrombin and factor Xa (FXa). Glycosaminoglycans with a 3-O-sulfated pentasaccharide unit, like heparin or heparan sulfate, bind to AT with high affinity and greatly accelerate the reaction with active clotting factors. Even in the presence of heparin/heparan sulfate, AT poorly inhibits FXa in activation complex and fibrin-bound thrombin. It might control low level thrombin formation that occurs physiologically, and could also exert a scavenger function by neutralizing FXa and thrombin that have escaped from the clot and from the activation complex. In general, inherited AT deficiency causes severe thrombophilia. However, the phenotypic appearance varies with different subtypes. Homozygous type I AT deficiency caused by decreased synthesis or secretion is incompatible with life. In the heterozygous form, it is frequently accompanied by DVT, not infrequently

of unusual localization, or PE after the second decade of life. Functional defects (type II AT deficiencies) caused by missense mutations are classified according to the site that is affected by the mutation as reactive site (RS), heparin binding site (HBS) AT deficiencies or multiple site defect caused by mutations with pleiotropic effects (PE). Type II HBS subtype is less severe than other AT deficiencies. In the heterozygous form it presents with only mild thrombophilia, and homozygotes also survive, although usually with very early thrombotic complications. The diagnosis of inherited AT deficiency is important for establishing the risk of recurrent thrombotic events. The diagnosis might influence the clinical decision concerning the duration of anticoagulant therapy. The diagnosis is established by laboratory tests, although the exclusion of acquired deficiency requires careful clinical attention. The first line test is a functional chromogenic heparin cofactor assay, which measures the inhibition of thrombin or the inhibition of FXa in the presence of heparin. Both anti-thrombin and anti-FXa assays perform well and, with very few exceptions, detect all subtypes of AT deficiency. The reference interval is quite narrow and in most cases the range of 80%–120% of average normal is accepted. Measurement of AT antigen concentrations allows differentiation between type I and type II AT deficiencies, while progressive AT activity assays are required to make the clinically important distinction between type II HBS and other type II subtypes. Increased utilization of a progressive activity assay is desirable. The diagnosis might be confirmed by molecular genetic testing, which is important in the case of activities in the range of 70%–80%.

Acknowledgements This work was supported by a grant from the Hungarian National Research Fund (OTKA K78386).

Conflict of interest statement Author’s conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Factor V Leiden and FII 20210 testing in thromboembolic disorders

Tadej Pajicˇ* Department of Haematology, Division of Internal Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia

Abstract Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection are very important in order to define the increased relative risk for venous thromboembolism in selected patients. Use of DNA-based methods to detect both mutations has become widely available in clinical diagnostic laboratories, including fluorescence-based quantitative realtime PCR (qPCR). The latter is a rapid, simple, robust and reliable method to identify genotypes of interest. There are several chemistries used for qPCR; this article describes their principles and applicability for Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection. Clin Chem Lab Med 2010;48:S79–87. Keywords: Factor V Leiden; FII 20210; quantitative realtime PCR; thromboembolic disorders.

Introduction Venous thromboembolism (deep-vein thrombosis and pulmonary embolism, VT) is a major health problem in Western societies, with an incidence of about one per 1000 individuals, and increasing (1, 2). However, the risk for VT is agerelated, with the disease occurring primarily in older age groups (3). Although the major one, it is not the only risk factor related to VT. The pathogenesis of this complex disease involves circumstantial or acquired and genetic risk factors (4). Independent environmental factors contributing to VT include, but are not limited to: smoking, male gender, older age, immobilisation, surgery, malignant neoplasm, pregnancy, use of oral contraceptives, hormone-replacement therapy, and inflammatory conditions (3–6). Several genetic risk factors for venous thrombosis have been identified. Among them, antithrombin, protein C and protein S deficiency, dysfibrinogenemia and homozygous homocystinuria are rare, and account for only 5%–20% of patients with inherited thrombophilia. The situation changed remarkably in 1993 after the discovery of a defect in the *Corresponding author: Tadej Pajicˇ, Zalosˇka 7, 1000 Ljubljana, Slovenia Phone/Fax: q386 1 43 20 230, E-mail: [email protected] Received September 10, 2010; accepted October 19, 2010

protein C anticoagulant pathway, which resulted in resistance to activated protein C (APC) (5, 7). Factor V Leiden (FVL) which causes resistance to APC was discovered in 1994 (8) and is the most common genetic risk factor for venous thrombosis. A few years later, the second most frequent and important inherited risk factor for thrombosis, prothrombin (F2) c.20210G)A mutation was identified (9). Mutation to FVL can be identified by DNA-based tests or by functional APC-resistance (APCR) tests. A DNA genetic assay is required for the identification of the prothrombin (F2) c.20210G)A mutation.

Activated protein C resistance and Factor V Leiden mutation As described by Dahlback (6), the starting point for the discovery of APC resistance was unexpected behaviour in a functional assay for protein C observed in a plasma sample from a patient with thrombosis. The addition of APC to the patient’s plasma did not result in prolongation of the clotting time. Further work demonstrated that APC resistance, as a phenotypic description of the condition, was inherited (9–11), and factor V was purified (12). Factor V is an important component of the coagulation cascade, which, in association with factor Xa, activates prothrombin to thrombin. In 1994, Bertina et al. (8) from the city of Leiden, The Netherlands, and other laboratories, reported (13–16) the same causative mutation in exon 10 of the gene encoding factor V (F5). This mutation resulted in a G™A substitution at nucleotide 1691, producing a missense mutation that substitutes glutamine for arginine at amino-acid residue 506 (R506Q) in the factor V heavy chain protein product. Factor V has a 28-amino-acid leader peptide. Thus, this sequence variation has also been referred to as Arg534Gln wc.1601 G)A (p.Arg534Gln), FV Leiden mutation (FVL); rs6025x. The R506Q substitution in FVL involves one of the three sites on factor Va that is cleaved by APC; the other sites are at positions Arg306 and Arg679. The mutated protein is activated in a normal way and retains normal procoagulant activity, but is less susceptible to inactivation by APC and results in disposition to a hypercoagulable state (APC resistance) (17, 18). Moreover, the mutant factor V has diminished cofactor activity in the inactivation of factor VIIIa by APC (19, 20). Both these abnormalities result in the failure of APC to prolong the activated partial thromboplastin time, the classic APC resistance test. Depending upon the method used, the functional assay may pick up cases of APC resistance not due to FVL, although it accounts for more than

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90% of APC resistance (21–24). It is thought that the relatively common haplotype of the factor V gene, the HR2 haplotype (H1299R), might also cause resistance to APC and increase the risk of venous thrombosis when co-inherited with FVL (25, 26). There is also a rare mutation in the second of the three sites in factor Va that APC cleaves which probably causes resistance to APC (27, 28). The prevalence of FVL mutation varies, with a higher frequency in Caucasians (up to 15%) and being rare or absent in Asians and Africans and in Australian and American natives (29–33). The FVL mutation was found to have high prevalence (in up to 50%) in patients with thrombosis, depending on the selection criteria used (10, 11, 33–35). The relative risk for venous thrombosis associated with the FVL mutation in the absence of other acquired or environmental predispositions is approximately four- to seven-fold for heterozygotes and 80-fold for homozygotes (25, 36).

Prothrombin (F2) c.20210G)A mutation Prothrombin is an important component of the coagulation cascade. It is a vitamin K-dependent protein that participates in coagulation and its regulation. Prothrombin participates in the final stages of the blood coagulation cascade where it is converted to thrombin in the presence of factor Xa, factor Va, calcium ions, and phospholipids. A few years after identification of APC resistance and the causative FVL mutation, the second most frequent, and important inherited risk factor for thrombosis, was identified. The sequence alteration c.20210G)A is located in the 39untranslated region of the prothrombin gene (F2) and is associated with slightly increased plasma prothrombin concentrations (37). The c.20210G)A mutation in F2 represents a gain-of-function mutation, causing increased recognition of the cleavage site, increased 39 end processing, and increased accumulation of mRNA and protein synthesis (38). These changes can result in a hypercoagulable state. The relative risk for venous thrombosis associated with the mutation is two- to four-fold for heterozygotes (39, 40). Homozygosity for the c.20210G)A mutation is rare, but it increases the risk of thrombosis above that which has been observed for heterozygotes (5, 41). The prevalence of the mutation varies and is dependent on geographic location and ethnic origin. It is found in 0%–4% of the general population. The prevalence of the mutation in southern Europe is twice as high as in northern Europe. It is rare in Asian and African descendants, and in native Australians and Americans (33–35, 42). The mutation is also found in 6%–8% of patients with VTE (6, 37, 39, 43). It was found that 6%–12% of patients with VTE who were heterozygous for FVL also had the prothrombin mutation (F2) c.20210G)A (compound heterozygotes) (5, 44, 45). The relative risk for venous thrombosis in the individuals carrying both mutations are higher than in individuals without either mutation (25, 46, 47).

Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection The FVL mutation can be identified using a DNA-based test or by functional APC-resistance (APCR) tests. It is known that while errors can occur with genetic methods, testing for APC resistance can be helpful in assessing the presence of FVL, whether used initially as a screening test or if used in conjunction with molecular testing (48). A modification of the classic APC resistance test makes the test more sensitive to factor V and FVL mutations; with up to 100% sensitivity and specificity for the factor V mutation (49–53). A normal modified APCR test excludes the presence of FVL. However, when it is abnormal, the FVL genotype needs to be confirmed by genetic testing. A DNA assay is required for the identification of the prothrombin (F2) c.20210G)A mutation. As both mutations are very important in defining the increased relative risk for VTE in selected patients, the DNA-based methods that detect both mutations have become widely available in clinical diagnostic laboratories. Most assays are capable of using genomic DNA prepared from blood using a variety of extraction protocols. The various molecular methods in use for detecting the FVL and the prothrombin c.20210G)A mutations are also very sensitive, robust, accurate and reliable in identifying the mutation of interest (25, 48, 54). The ‘‘gold standard’’ method for detection of mutations is bidirectional sequencing of the specific genetic region of the gene of interest. However, there are many technologies that can be used to detect FVL and the prothrombin c.20210G)A mutation. The polymerase chain reaction (PCR), restriction-fragment-length polymorphism (PCRRFLP) and allele-specific PCR are acceptable methods and used in many studies (48). However, in recent years the fluorescence-based quantitative real-time PCR (qPCR) approach (55–57) has become widely available in many clinical diagnostic laboratories. It has the advantage that it can detect and measure minute amounts of nucleic acids in a wide range of samples from numerous sources. In addition to its use as a research tool, many diagnostic applications have been developed (58), including FVL and prothrombin (F2) c.20210G)A mutation detection. This approach enables simple, fast, sensitive and specific nucleic acid quantification and mutation detection, particularly single base mutations (59–61). Some of the principle advantages and disadvantages of each will be discussed further. However, errors in DNA-based testing occur due to analytic mistakes as well as inadvertent sample mix-ups and transcription errors; an issue that has been discussed elsewhere (48, 62). Laboratory personnel need to be aware of the potential for these failures and for the rare silent mutations within the F5 gene (1692A)C, 1689G)A and 1696A)G) and rare sequence variations at or near the prothrombin (F2) c.20210G)A mutation (i.e., the mutations 20209C)T, 20207A)C, 20218A)G and 20221C)T). These will influence genetic analysis in a manner dependent upon the test system used (63). However, when these sequence variations

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are rare in the population their influence on the assay may be considered negligible (63).

Real-time PCR There are several real-time PCR instruments and several different fluorescence detection technologies: DNA-binding agents, fluorescent primers and fluorescent probes. In the most common formats, the PCR reaction includes locus-specific primers in addition to a single or pair of fluorescencelabelled oligonucleotide probes. The specially designed primer systems amplify and detect the mutant and normal alleles using sequence-specific hybridisation-based assays. The most common probes that are used are dual hybridisation (LightCycler probes) or hydrolysis probes (58). One of the chemistries developed after the hydrolysis probes is Scorpions (64), and new chemistries are coming (59, 65). The experiment protocol usually combines purified genomic DNA, master mix (Taq DNA polymerase, buffer solution, deoxyribonucleotide triphosphates, and salts), primers and probe(s). This is followed by thermal cycling, reading, and analysis of the results.

Mutation detection with melting curve analysis using dual hybridisation probes The DNA segment of interest is amplified with locus-specific primers, and the amplicon is detected by fluorescently labelled oligonucleotide probes (dual hybridisation probes) (58). Probes hybridise to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. One of the probes is labelled at the 59 end with LightCycler (LC) Red 640 or LC 705 (acceptor dye) (Roche Diagnostics, Mannheim, Germany). The second probe is labelled at the 39 end with fluorescein (donor dye). The 39 end of each probe is blocked with either a dye or a phosphate group to prevent extension during PCR. Only after hybridisation to the target sequence do the probes come in close proximity. In this situation, the energy emitted by the excitation of fluorescein is transferred to the acceptor dye, which then emits fluorescence at a longer wavelength, showing that fluorescence resonance energy transfer has occurred. The emitted fluorescence is then measured by the instrument (25, 66–68). Genotyping is performed using melting curve analysis with detection of the rate of melting of a wild-type probe from the amplicon after the amplification cycles when it is present at increased concentrations. Usually, the fluoresceinlabelled probe spans the mutation site (mutation probe). The stability of each probe/target complex is indicated by the melting temperature (Tm). Melting from the mutant allele occurs at a lower temperature than that from wild-type allele. The software plots the negative derivative of the fluorescence with respect to temperature. The peaks that are generated occur at Tms specific for the wild-type and mutant alleles (Figure 1). At a minimum, the analysis requires a heterozygous control and a no-template control (25, 63, 66–68).

Interpretation of results is based on the presence or absence of peaks with a Tm that is specific for the wild-type or mutant allele. It is also very useful to check for the DTm wTm (wild-type)–Tm (mutant)x which is less variable than the Tm values. In some circumstances, it helps to identify additional sequence variations (63) that need to be confirmed by other tests, frequently with sequencing. This approach in detecting FVL and the prothrombin (F2) c.20210G)A mutation is simple, allowing the result to be obtained in approximately 2 h and making analytical errors unlikely (63, 67, 68). The instrument software could have embedded algorithms to identify the genotype, making interpretation of the results easier. Although the instrument is relatively expensive and the method is prone to contamination with the amplicon or DNA (25, 63), it is a robust and reliable method for identifying the mutation of interest in the sample type to be used clinically.

Mutation detection using hydrolysis probes Some systems use only a single fluorescently labelled oligonucleotide probe (TaqMan hydrolysis probes) that provide very sensitive and specific detection of DNA. The systems require a pair of PCR primers and a probe with both a reporter and a quencher dye attached (59, 69, 70). The quencher dye attached at the 39 end of the probe could be a fluorescent or non-fluorescent dye with a minor groove binder (MGB) at the 39 end (58, 59, 71). The latter increases the Tm of probes, allowing the use of shorter probes and, with the non-fluorescent quencher dye attached, provides more accurate allelic discrimination (72). The probe is designed to bind to the sequence amplified by the locus specific-primers (59). If the target sequence is present during the qPCR, the probe is hydrolysed with the 59-nuclease activity of the Taq DNA polymerase. Cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal and removing the probe from the target strand, allowing primer extension to continue to the end of the template strand. The fluorescent signal is generated and increased with each cycle (59, 71, 73). The number of PCR cycles necessary to detect a signal above the threshold is called the quantification cycle (Cq), and is directly proportional to the amount of target present at the beginning of the assay (58, 59, 71, 73). The change for signal corresponds to the increase in fluorescence intensity when the plateau phase is reached. Usually, the ROX (6carboxy-X-rhodamine) fluorophore is added to the qPCR master mix as the passive reference for normalisation of the fluorescence. The use of ROX improves the results by compensating for small fluctuations in fluorescence due to things, such as bubbles and well-to-well variation that may occur in the plate (25, 71, 73). For genotyping, this technology uses two allele-specific hydrolysis probes and a PCR primer pair to detect the specific sequence variation. One probe is designed to detect the mutated allele and the other detect the wild-type. The allele-


Figure 1 Factor V Leiden (A) and prothrombin (F2) c.20210G)A (B) mutation detection using melting curve analysis and dual hybridisation probes. The generated peaks occur at melting temperatures (Tms) specific for the mutant and wild-type alleles. (A) The first peak represents the mutant allele (578C"2.58C), the second relates to the wild-type allele (658C"2.58C), DTm wTm (wild-type)–Tm (mutant)x is 88C"1.58C. (B) The first peak represents the mutant allele (498C"2.58C), the second one relates to the wild-type allele (598C"2.58C); DTm wTm (wild-type)–Tm (mutant)x is 108C"1.58C. Red lines represent a heterozygous, light and dark green lines relate to a wild-type genotype. Blue line represents the no-template control. Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection were performed using the Factor V Leiden Kit (66) and Factor II (Prothrombin) G201210A Kit, respectively (both Roche Diagnostics, Mannheim, Germany) (67). All reactions were performed in LightCycler capillaries using the LightCycler 2.0 instrument (Roche) with the LightCycler Software Version 4.1. The manufacturer (Roche) claims that genotyping with the LightCycler Software 4.1 will classify the rare silent mutations 1692A)C, 1689G)A and 1696A)G within the F5 gene as false-positive for Factor V Leiden (66). Within the F2 gene, in addition to the prothrombin (F2) c.20210G)A mutation, four known mutations at positions 20209, 20207, 20218 and 20221 exist (i.e., the mutations 20209C)T, 20207A)C, 20218A)G and 20221C)T), and these additional mutations are spanned by the mutation probe. These rare mutations will lead to an unknown or wild-type result after performing genotyping (67).

specific hydrolysis probes have different reporter dyes. After PCR amplification, an endpoint plate reading is performed using a real-time PCR system. Allelic discrimination is based on the fluorescence measurements made during the plate reading to plot fluorescence values based on the signals from each well. The plotted fluorescence signals indicate which alleles are in each sample (25, 71). The other approach used to determine the specific allele is by using the quantification cycle (Cq). The principle of qPCR is the same as that described above, but genotyping of the sample relies on the Cq values of the specific probes for the mutated and normal alleles obtained during qPCR. The Cq values for the samples and controls are compared with the pre-defined Cq values of the specific probes for the mutated and normal alleles provided by the manufacturer (Figure 2) (74, 75). At a minimum, the analysis requires a heterozygous control and a no-template control. Using a 59-nuclease assay chemistry protocol is a simple way to obtain results in approximately 2 h. The advantage of the platform is that a large number of mutations can be detected simultaneously. The assays for FVL and F2 c.20210G)A mutation detection can be performed on the same microplate using the same thermal cycling conditions

(71, 74, 75). The use of the constant and the qPCR appropriate amount of DNA extract from the samples and amplification controls in the amplification reaction is important, in order to prevent the problem of non-specific hybridisation or inhibition of fluorescence emission (71). Care must be taken with interpretation of results, as genotype are assigned manually and as additional sequence variation may suggest different genotypes (76) that need to be confirmed by another method.

Mutation detection using Scorpion primers Scorpion primer technology allows precise discrimination between different alleles of a target nucleotide (64, 77–80). Scorpion primers combine a primer and a probe in a single molecule. There are two formats for Scorpions: uni- and bimolecular. The uni-molecular Scorpion format consists of a specific probe sequence that is held in a hairpin loop configuration by complementary stem sequences on the 59 and 39 sides of the probe. The fluorophore is attached to the 59 end and is quenched by a moiety coupled to the 39 end of the loop (64, 80). The hairpin loop is linked to the 59 end of


Figure 2 Factor V Leiden (A) and prothrombin (F2) c.20210G)A (B) mutation detection using hydrolysis probes. In the assay, genotyping of the sample relies on the quantification cycle (Cq) values of the specific probes for the mutated (VIC probe) and normal alleles (FAM probe) obtained during qPCR (74, 75). The horizontal green line represents a threshold, set at a value of 0.2. It is specified by manufacturer Nanogen Advanced Diagnostics S.r.L., Torino, Italy. (A) The blue lines represent the amplification of the normal alleles (FAM probe). The red line represents amplification of the mutated alleles (VIC probe). The Figure show heterozygous and wild-type genotypes. The heterozygous genotype is identified when both probes (FAM and VIC) are amplified and the differences between Cq of the probes are -2 Cqs. The wild-type genotype is identified when the specific probe for the wild-type genotype is amplified. (B) The green lines represent amplification of the normal alleles (FAM probe). The blue line represents amplification of the mutated alleles (VIC probe). The Figure shows the heterozygous and wild-type genotype. The heterozygous genotype is identified when both probes (FAM and VIC) are amplified and the differences between Cq of the probes are -2 Cqs. The wild-type genotype is identified when the specific probe for the wild-type genotype is amplified. For the other pre-defined Cq values of the specific probes for the mutated and normal alleles, see references (74, 75). Delta Rn is difference in the intensity of the fluorescence signal of the interest and background. Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection were performed using FACTOR V Q-PCR Alert Kit (74) and FACTOR II Q-PCR Alert Kit (75), respectively (both Nanogen Advanced Diagnostics S.r.L.). All reactions were performed in Micro-Amp optical 96-well plates using an ABI Prism 7000 Sequence Detection System with the Sequence Detection Software version 1.2.3. (Applied Biosystems, Forster City, CA, USA). The manufacturer (Nanogen Advanced Diagnostics S.r.L.) does not specify the performance of the assay when rare mutations are present.


a primer via a non-amplifiable monomer (blocker). After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon, thus opening up the hairpin loop. The fluorescence is no longer quenched, which results in an increase in fluorescence from the reaction tube. The non-amplifiable monomer prevents non-specific PCR products (59, 64, 79, 80). Elements of the bi-molecular Scorpion are a PCR primer, blocker (a non-amplifiable monomer), and a probe with a fluorophore on one, and a dark quencher on a separate oligo. After extension of the Scorpion primer, the denaturation step is performed and the quencher oligo disassociates. When cooling is performed, the extended Scorpion with the specific probe sequence is able to bind to its complement and begins to fluorescence. The unextended primer is quenched (64, 79). The ability to multiplex greatly expands the power of qPCR analysis, particularly when applied to the simultaneous detection of sequence variations (58, 60, 61, 81). Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Allelic discrimination can be achieved through hybridisation of the probe to the target sequence or by using the Amplification Refractory Mutation System (ARMS), where the primer is sited over the polymorphic site rather than the probe (79). This technology is used in a new assay kit for simultaneous FVL and F2 c.20210G)A mutation detection with the GeneExpert Dx System (Cepheid, Sunnyvale, CA, USA) using qPCR. The system automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in a single use disposable cartridge that contains the PCR reagents and hosts the PCR process. Each cartridge contains freeze-dried beads with all the necessary components for PCR: DNA polymerase, nucleotides, primers and Scorpions, that have to be rehydrated before use. The sample is added to the corresponding well in the cartridge and the analysis starts. Each Scorpion sequence is labelled with a specific fluorophore. Using the PCR cycles, the specific binding of the Scorpion sequence to the target mutation is detected by the system and the genotype is reported using algorithms imbedded in the software (Figure 3) which makes interpretation of the results straightforward (82). The newest instrument software version allows one mutation detection only, either FVL or F2 c.20210G)A. However, it is a simple and rapid assay because Scorpions have a fast reaction mechanism (59). The analysis can be performed using as little as 50 mL of fresh or frozen sodium citrate or EDTA anticoagulated whole blood. In our test, we obtained the result in approximately 30–40 min. A disadvantage could be that the systems do not allow for high throughput sample processing. However, the analyses can be performed shortly after samples come to the laboratory; this could also be suitable for many coagulation laboratories that perform other thrombophilia testing. It is a one cartridge, one sample approach, and controls are not analysed at the same time. However, the assay includes a probe check control (Figure 3) which measures the fluorescence signal from the rehydrated probes, filling of

Figure 3 Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection using Scorpion primers and the GeneExpert Dx System (Cepheid, Sunnyvale, CA, USA). Factor V Leiden and prothrombin (F2) c.20210G)A mutation detection were performed using Xpert HemosIL FII&FV kit (Cepheid, Sunnyvale, CA, USA and Instrumentation Laboratory, Bedford, MA, USA). Following the PCR cycles, the specific binding of the Scorpion sequence to the target mutation is detected by the system and the genotype is reported using embedded algorithms wGeneXpert Dx System version 2.1 (Cepheid)x (81). The manufacturer (Cepheid) states that rare factor V mutations (1692A)C, 1689G)A and 1696A)G) and any additional sequence variations in the probe binding region may interfere with detection of the target and produce an invalid result (81).

the reaction tube, probe integrity and dye stability. The probe check passes the procedure if all criteria meet the assigned values (82). The external controls consisting of normal and abnormal whole blood samples may be used for quality control, including testing variability between different reagents. At the moment, I cannot find in the literature the performance of the assay when the rarely present additional sequence variations in the sample occur. Nevertheless, it is advisable to check any questionable results by other methods.

Conclusions FVL and prothrombin (F2) c.20210G)A mutation detection are very important for defining the increased relative risk for VTE in selected patients. The DNA-based methods to detect both mutations have become widely available in clinical diagnostic laboratories. qPCR is a rapid, simple, robust and reliable method for identifying genotypes of interest. The newer instrument software with embedded algorithms to identify genotypes makes interpretation of the results easier, and possibly reduces the potential for error. However, care must be taken in interpreting questionable data which must be confirmed by others methods, preferably sequencing. Because the results of genetic analyses might have important clinical and family implications, it is important to perform


internal quality control measures (25) and participate in external quality assurance programmes, an issue discussed elsewhere (48, 62). In conclusion, it is the responsibility of the laboratory to evaluate and validate any new methodology, before it is utilised for clinical testing (25).


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Review

Hyperhomocysteinemia and thrombophilia

Mojca Bozˇicˇ-Mijovski* Department of Vascular Diseases, University Medical Centre, Ljubljana, Slovenia

by vitamin supplementation (3–5). Certain patient populations may benefit from tHcy measurements, but controversy currently exists as to which patient and asymptomatic family members to test (5, 6).

Abstract It is now widely accepted that hyperhomocysteinemia (HHC) is a risk factor for thrombophilia. HHC is the result of either impaired enzyme function or a deficiency of vitamin B (folate, B6, B12), or both, and can be treated with vitamin supplements. Measuring plasma total homocysteine (tHcy) is included in the routine thrombophilia panel performed in many laboratories, despite having a limited value to the clinician. Many methods are available for tHcy measurements. High-pressure liquid chromatography (HPLC) with fluorescence detection is a widely used method, but is being replaced by more convenient immuno- or enzyme assays. In this paper a general overview on homocysteine is given, with an emphasis on laboratory methods. Clin Chem Lab Med 2010;48:S89–95. Keywords: homocysteine; methods; thrombophilia; venous thromboembolism.

Introduction Homocysteine is an amino acid formed as an intermediate in the methionine cycle (1). Increased homocysteine concentrations in plasma (hyperhomocysteinemia, HHC) and severe HHC, result in increased concentration of homocysteine in the urine (homocystinuria) and increase the risk of thrombosis (2). Several chromatographic, immunological and enzyme methods are available for measurement of homocysteine and are described in this article. HHC is considered to be a mild thrombophilic risk factor that increases the risk of venous thromboembolism (VTE) about 2.5-fold (2). As a recognized thrombophilic factor, detection of HHC is included in the routine thrombophilia test panel performed in many laboratories. However, some experts doubt the benefit of routine homocysteine measurements as part of the thrombophilia panel, especially after intervention trials showed no reduction in risk of VTE following reductions in plasma homocysteine *Corresponding author: Mojca Bozˇicˇ-Mijovski, Department of Vascular Diseases, University Medical Centre, 1000 Ljubljana, Slovenia Phone: q386 1 522 8032, Fax: q386 1 522 8070, E-mail: [email protected] Received June 3, 2010; accepted September 7, 2010

Thrombophilia Thrombophilia is defined as the tendency to develop thrombosis due to predisposing factors that may be genetically determined, acquired, or both (7). HHC can be both genetically determined and acquired as described in detail below. Historically, the association between homocystinuria, which occurs in 1 in 200,000–300,000 of the general population, and atherosclerosis was recognized 40 years ago (8). Not long afterwards, several case control studies confirmed an increased risk of cardiovascular disease, peripheral artery disease and cerebrovascular disease in subjects with mild HHC (9–11). The first case control studies on the association between mild HHC and VTE, performed 20 years later, showed conflicting results (12, 13). Finally, a meta-analysis confirmed that mild HHC causes thrombophilia, increasing the risk of VTE about two-fold (OR: 2.5, 95% CI: 1.8–3.5) (2). A subsequent meta-analysis confirmed these findings (OR: 3.0, 95% CI: 2.1–4.2) and identified a greater risk in patients younger than 60 years of age (14). Mild HHC is found in approximately 15% of patients with VTE. The prevalence of HHC in patients with VTE depends on the defined reference values and cut-off used for total plasma homocysteine (tHcy), as described below (1). Despite the different cut-offs that have been used in published studies, HHC is among the most frequent thrombophilic factor in patients with VTE. The role of thrombophilia in arterial thrombosis remains a controversial issue. Several case control and prospective studies were performed to confirm or exclude mild HHC as a risk factor for arterial thrombosis. However, these studies reported conflicting results. An atherothrombotic endpoint, such as myocardial infarction, is generally a result of both atherosclerosis and thrombus formation. Thus, it is difficult to draw conclusions about separate atherogenic and arterial thrombotic effects of increased tHcy. Overall, there appears to be a weak positive association between arterial thrombosis and HHC (1). Whether HHC is a marker or a cause of thrombosis has long been debated. Prospective studies showed that subjects with HHC are at an increased risk of future VTE, thus strengthening the assumption of a causal relationship between HHC and this disease (15, 16). tHcy measurements

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were therefore widely adopted in clinical laboratories and included in the thrombophilia panel (17). It was later shown that HHC is associated with a low absolute risk of VTE, and concomitant thrombophilic defects are likely the main determinants of VTE risk, rather than HHC itself. For example, in subjects with HHC and Factor V Leiden, the relative risk of VTE is increased markedly (RRs22) (18). Although HHC can be treated easily with vitamin supplementation (folate, vitamin B6 and vitamin B12) (19), reduction of tHcy was not shown to reduce the risk of recurrent VTE (20). Furthermore, a prospective study on subjects 55 years of age or older with known cardiovascular disease or diabetes mellitus showed a similar incidence of VTE in the vitamin therapy group and the placebo group (0.35 per 100 person-years) (21). Since lowering of tHcy showed no reduction in risk of VTE, and also due to the fact that HHC is considered a mild risk factor for VTE, some experts doubt the value of tHcy as a component of thrombophilia testing (3–5). Certain patient populations may benefit from tHcy measurements, but controversy still exists as to which patient and asymptomatic family members to test (5, 6). When a patient is diagnosed with an increased tHcy, consideration should be given to the possibility that HHC is secondary to another underlying condition, such as a vitamin deficiency, renal impairment or hypothyroidism. It is particularly important to rule out vitamin B12 deficiency, since the initiation of high dose folate therapy may precipitate acute neuropathy (19).

Biochemistry Homocysteine is an amino acid with a thiol (-SH) group. It is not a building block of proteins, but an intermediate product in the methionine cycle, the function of which is to generate one-carbon methyl groups for transmethylation reactions essential to all life forms. In the liver and kidney an alternative remethylation pathway utilizes betaine as the methyl donor. Homocysteine can be diverted from remethylation to the transsulfuration pathway where it is metabolized to cysteine (Figure 1) (1). The word homocysteine has a precise chemical meaning, referring only to the reduced form of the molecule (Figure 2). In plasma, there are several forms of homocysteine, and the reduced form comprises a small proportion (1%–2%) (22). Approximately 75% of homocysteine is bound by disulfide bonds to cysteine in proteins, mostly to albumin (23). Homocysteine can auto-oxidize to form homocystine and can also oxidize with other thiols, such as cysteine (homocysteine-cysteine mixed disulfide) and glutathione (homocysteine-glutathione mixed disulfide) (1). The proportion of different homocysteine forms in plasma is stable (24). Since no evidence from clinical trials exists about the superiority of measuring one form over another, total plasma homocysteine (tHcy) is usually measured as a sum of all forms of homocysteine in plasma. The term homocyst(e)ine has also been used for unspecified forms of homo-

Figure 1 Homocysteine metabolism.

cysteine, but has become less popular since parentheses cannot be pronounced (1).

Causes of hyperhomocysteinemia Severe HHC (homocystinuria) is an inherited disease due to homozygous or compound heterozygous mutations in cystathionine-b-synthase (E.C.4.2.1.22) and other enzymes regulating methionine metabolism. In patients affected by homocystinuria, over 130 mutations of the cystathionine-bsynthase gene have been reported (25). Mutations in the 5,10-methylene tetrahydrofolate reductase (MTHFR) (E.C.1.1.1.68) and methionine synthase (MS) (E.C.2.1.1.13) genes are less frequent, but can also cause homocystinuria (26). A number of relatively common polymorphisms in the genes of homocysteine metabolic pathway enzymes have also been investigated with respect to their influence on mild HHC. The most common polymorphism is a C to T transition at nucleotide 677 in the MTHFR gene which results in a thermolabile variant of the enzyme, with impaired activity (27). The MTHFR 677TT genotype is present in up to 20% of the general population (28, 29). However, it has been shown that HHC is present only in subjects with the 677TT genotype and concomitant folate deficiency (30), which often occurs in the elderly (31). Meta-analysis show that the MTHFR polymorphism is neither a risk factor for VTE or arterial thrombosis (4). However, an association between this polymorphism and VTE was found in subjects over 55 years of age (28). It is unlikely that the relationship between HHC and VTE is mediated by this gene defect to a substantial degree, and there is no reason to obtain this genetic test for the assessment of thrombophilia (4). Another polymorphism in the same gene (A1298T) affects the activity of MTHFR. Although neither homozygous nor heterozygous subjects have HHC, the compound C677T and A1298T polymorphisms appear to influence tHcy concentrations (32). No association of the A1298T polymorphism with cardiovascular diseases has been established (33). Inconsistent results have been obtained regarding the influence of polymorphisms of methionine synthase reductase and cys-

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Figure 2 Structure of different homocysteine forms.

tathionine-b-synthase genes on tHcy concentrations, and no association with cardiovascular diseases have been observed (34–36). Acquired causes of HHC include deficiency in folate, cobalamin (B12) or B6. Five-methyltetrahydrofolate is a cosubstrate for remethylation of homocysteine, while B12 and B6 act as enzyme cofactors (Figure 1). Vitamin deficiency often occurs in the elderly and is probably the cause of the increased prevalence of HHC seen with age (31). Vitamin supplementation significantly reduces tHcy concentrations in subjects with HHC as well in subjects with normal tHcy concentrations (19, 37, 38). In spite of this, clinical trials have failed to show a protective effect of vitamin supplementation against cardiovascular diseases (39–41), including recurrent VTE (20).

chromatography with tandem mass spectrometry (51). Regardless of the type of chromatography used, all tHcy forms need to be reduced to homocysteine with a free thiol group (Figure 2). The reducing agents used most often are dithioerythritol, tri-n-butylphosphine, sodium borohydride, potassium borohydride and 2-mercaptoethanol (24). After separation, homocysteine is detected directly or after derivatization. The advantages of chromatographic assays include their wide analytical range, simultaneous determination of other compounds (e.g., other sulfur amino acids and methylmalonic acid) and low cost. However, they require skilled staff, are labor-intensive and have relatively low throughput (52). Chromatographic assays are reproducible and precise, but the agreement between different chromatographic methods is not sufficient to enable them to be used interchangeably (53–55).

Methodology

Enzyme-linked immunosorbent assay

Homocystinuria can be detected in urine using the simple silver-nitroprusside test in which homocysteine reacts with sodium nitroprusside to produce a pink-purple color (42). This qualitative method is still used as a screening test in neonates (43) in some parts of the world. However, it should be used with caution because it lacks the sensitivity required of a screening test. In the 1970s, when second generation amino acid analyzers became available, free homocysteine was detected in the acid-soluble fraction of plasma or serum. This method was soon abandoned because the procedure was inconvenient and impossible to use in most clinical settings (44). As mentioned earlier, measuring free homocysteine has no benefit over measuring tHcy, and a number of other methods were developed. Quantitative methods for measuring tHcy were introduced in the 1980s (23, 45). They can be categorized into chromatographic, immunochemical and enzymatic methods.

Frantzen et al. (56) developed an enzyme-linked immunosorbent assay (ELISA) that allowed tHcy measurements to become more accessible to clinical laboratories. The method is based on the enzymatic conversion of reduced homocysteine to S-adenosyl-L-homocysteine (SAH) by the action of SAH hydrolase (E.C.3.3.1.1), followed by quantification of SAH in a competitive immunoassay by the use of a monoclonal anti-SAH antibody (57). This assay is slightly less reproducible than chromatography (54, 55) and allows batch processing only. However, it does not require specialized equipment and different formats on various analytical platforms can be used. The same principle as ELISA has been used in commercially available automated immunoassays, which differ in their mode of detection for SAH. These detection modes include the fluorescence polarization immunoassay (FPIA) on an Abbott IMx (58) or AxSYM analyzer (59), the chemiluminescence immunoassay on an ADVIA Centaur (Bayer Healthcare) (60) or Immulite 2000 (Diagnostic Products) analyzer (61) and an immunonephelometric assay on the BN II or BN ProsSpec (Siemens). All immunoassays provide results with small bias compared to chromatographic assays (55, 56, 60, 62). Also, they are accurate, reproducible, simple to perform and do not require skilled technical staff (54, 63). Values obtained from different laboratories that use the same commercial assay can be compared directly (54).

Chromatographic techniques

A wide variety of chromatographic techniques combined with a variety of detection systems are used to measure tHcy, including gas chromatography-mass spectrometry (45), highpressure liquid chromatography with fluorescent (12, 22), ultraviolet (46) or electrochemical detection (47), amino-acid analyzers (48, 49), capillary electrophoresis (50) and liquid


Colorimetric enzyme assay

In the last five years colorimetric enzymatic assays have become available that can be run on routine clinical chemistry analyzers. As for chromatographic and immunoassays, the first step in all enzymatic assays involves the reduction of all tHcy forms to homocysteine, followed by an enzymatic conversion (64, 65). Enzymatic methods offer a rapid and simple means of analysis. Moreover, they can be implemented on a much wider variety of automated platforms. Although their total imprecision does not meet the criteria for desirable performance at all concentrations – based on the within-subject coefficient of variation – enzymatic methods seem to perform better than chromatographic methods (64). Of all the available methods, liquid chromatography electrospray tandem mass spectrometry with a deuterated internal standard is the most accurate and precise (66), and should be considered the definitive method. Gas chromatographymass spectrometry with a deuterated internal standard should be considered as the reference method and all other methods as field methods if they are accurate, robust and practical (61). In order to reduce laboratory measurement variability and improve the accuracy and reliability of tHcy measurements, the National Institute of Standards and Technology (NIST) in collaboration with the Centers for Disease Control and Prevention (CDC) developed a Standard Reference Material (SRM 1955 Homocysteine and Folate in Human Serum). SRM 1955 was shown to be commutable with most tHcy assays (67). At present, there are no reports on SRM 1955 implementation and its effect on intra- and interlaboratory variability.

Biological determinants and preanalytical factors A small meal will not influence tHcy concentrations in healthy subjects, whereas intake of a large protein-rich meal may increase plasma tHcy concentration up to 15% after 6–8 h. This is probably why the lowest tHcy concentrations are found in the morning and the highest in the evening. Seasonal variations in tHcy concentrations have not been observed (52). Blood samples collected in the supine position have approximately 10% lower mean tHcy concentrations compared with those collected in the sitting position. This is probably due to the fact that 75% or more of homocysteine is bound to albumin, and albumin concentrations are lower in the supine position (44). Renal function and creatinine synthesis also affect tHcy concentrations, as well as other lifestyle determinants (e.g., vitamin intake, smoking, coffee, ethanol intake, exercise) and clinical conditions described in detail elsewhere (52). Optimal procedures for blood collection and handling are critical when tHcy is used for cardiovascular risk assessment. This is because inadequate procedures may result in artificial increases in tHcy which may be interpreted as increased risk. Little data concerning possible effects of tourniquet application are available. The effect of using a tourniquet for

3 min did not appear to affect tHcy concentrations (1). Plasma is the preferred sample type for tHcy measurements because the use of an anticoagulant allows immediate sample processing and helps minimize the ongoing temperature dependant release of tHcy from blood cells. The absolute increase is independent of plasma tHcy concentrations, thus sub-optimal sample handling tends to reduce the difference between high and low tHcy. Selection of an appropriate anticoagulant depends on the tHcy assay, e.g., EDTA improves the fluorescence yield, but is not compatible with some of the gas-chromatographic assays (52). Heparin, citrate or EDTA are usually used. tHcy values depend on the anticoagulant, therefore only one type of collection tube should be used in each laboratory (1). Several stabilizers may prevent the formation or release of tHcy from blood cells. Sodium fluoride inhibits cell metabolism, but also causes hyperosmosis: intracellular water is drawn into plasma and thus all plasma constituents are diluted. Tubes with sodium fluoride and heparin as anticoagulant are readily available Adenosine analogs are effective, but not compatible with assays based on SAH hydrolase. Acidic citrate stabilizes tHcy concentrations at room temperatures for up to 6 h (52, 68). Recently, commercially available collection tubes for stabilization of tHcy have been introduced, but have not yet been fully validated (69). Because stabilizers cause systematic deviations in tHcy concentrations and thus require different reference ranges (68), most experts still recommend plasma be prepared optimally by immediate centrifugation (within 1 h from blood collection) or, when immediate processing is not possible, by storing blood samples on ice immediately after collection until centrifugation (52). Adhering to these instructions is of utmost importance, because the production of tHcy by cells after blood sampling is a major source of error when measuring tHcy. After removal of blood cells, tHcy is stable. No changes were observed for several weeks following refrigeration (0–28C) or for several years when frozen at –208C (44). Repeated freezing and thawing does not affect tHcy concentrations, but thorough mixing of the samples is required after thawing (52). Moderate hemolysis does not influence tHcy concentrations in plasma (44). Methionine loading test

An oral methionine load has been used to identify subjects with heterozygous cystathionine-b-synthase deficiency with normal basal tHcy concentrations. Methionine loading involves ingestion of 100 mg methionine/kg of body weight and collection of blood after 4–6 h. In adults, the mean postmethionine-load tHcy is about 3 times the fasting value (52). This procedure has largely been abandoned since its clinical value remains uncertain. This procedure is inconvenient for clinicians and patients, and the reference values for post-load results have not been adequately established (70).

Reference range tHcy concentrations between 5 and 15 mm/L are usually considered normal, but due to poor standardization, each laboratory should establish its own reference range (13). In


addition to the different tHcy assays used, reference ranges differ because of different sample preparation procedures, varying definitions of the cut-off and subject selection (24). The use of gender-specific reference values has been recommended (71) because tHcy concentrations are higher in men compared with pre-menopausal women (72–74). In children, tHcy concentrations are half of those seen in adults, and do not differ between girls and boys. At puberty, tHcy markedly increases and the distribution becomes as skewed as in the adult population (71). Therefore, gender specific reference ranges have also been recommended (31, 73). Also, it has been suggested that reference ranges should be determined in a population with adequate vitamin status, because such individuals have markedly lower tHcy concentrations compared with the general population (71).

Conclusions Inherited or acquired HHC is recognized as a thrombophilic factor that increases the risk of VTE about two-fold. This thrombophilic defect is found in approximately 15% of patients with VTE depending on the reference values used. Several methods available for measuring tHcy, including the proposed definitive and reference method, have been described in this article. With the recently introduced standard reference material, standardization of all field methods should be improved in the near future. However, an improvement in the quality of laboratory results will not resolve the issue of the benefits of routine tHcy determinations in VTE patients screened for thrombophilia. The value of tHcy diminished after intervention trials showed no reduction in VTE risk after reduction in tHcy by vitamin supplementation. Studies that can elucidate which patient populations would benefit from tHcy determinations are needed.

Highlights • tHcy is an independent risk factor of VTE, but decreasing tHcy does not lower the risk • tHcy is not recommended for inclusion in the routine thrombophilia panel. Rather, it should be used in specific patient populations • tHcy is usually measured as a sum of all forms of homocysteine in plasma • liquid chromatography electrospray tandem mass spectrometry with a deuterated internal standard should be considered the definitive method • Standard Reference Material SRM 1955 was shown to be commutable with most of the tHcy assays, and should contribute to better standardization of tHcy measurements • Each laboratory should establish its own gender- and agespecific reference range in a population with adequate vitamin status.

Conflict of interest statement Author’s conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.

Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Pediatric thrombosis

Alenka Trampus-Bakija* University Children’s Hospital, University Medical Centre, Ljubljana, Slovenia

Abstract Thrombotic risk factors and thrombosis in children has been receiving increased attention. True idiopathic thrombosis is extremely rare in children. Most patients have a significant underlying medical condition and the presence of a central catheter is the most important risk factor. Children are more likely than adults to have one or more significant genetic abnormality or coagulation deficiency. This review discusses problems concerning the heterogeneity of thrombotic states in children and highlights the importance of understanding the concept of developmental hemostasis. Issues regarding step-wise test selection and the interpretation of results are addressed, as well as basic monitoring of anticoagulant drug effects. Clin Chem Lab Med 2010;48:S97–104. Keywords: hemostasis; interpretation of results; laboratory tests; pediatric thrombosis; screening; thrombophilia.

Introduction Thrombotic events (TE) are infrequent in children, but have become an increasingly common problem, particularly in tertiary care pediatric hospitals. Despite the very low rate of spontaneous events as compared with adults, TE may result in significant morbidity. A mortality rate of 2.2% has been reported in the Canadian Childhood Thrombophilia Registry due to TE, with the majority due to pulmonary embolism (PE) (1). The true incidence of pediatric thromboembolic events is not known. It is significantly less than that in adults (30–500/ 10,000 adults), although increasing. Different international registries (Canada, USA, Netherlands and Australia) report an annual incidence of 0.07–0.14/10,000 for children in the general population. The incidence increases to 5.3/10,000 in hospital populations (2–4). The peak age for thrombotic risk *Corresponding author: Alenka Trampus Bakija, University Children’s Hospital, University Medical Centre, Ljubljana, Bohoricˇeva 20, 1000 Ljubljana, Slovenia Phone: q386 1 522 47 75, Fax: q386 1 522 39 57, E-mail: [email protected] Received June 2, 2010; accepted September 7, 2010; previously published online November 10, 2010

is found in those younger than 1 year of age (as many as 45% of all cases) and in the adolescent group, with a higher frequency in females (5, 6). The clinical manifestation of thromboses differs from that in adults where superficial and deep veins of the leg are affected. Neonates have a high rate of catheter-related thrombosis and renal thrombosis. Further locations of thromboembolism in young infants are cerebral sinovenous thrombosis and mesenteric vein thrombosis (7–9). Pediatric stroke is among the 10 most frequent causes of death in childhood in Western societies, and represents a significant cause of morbidity and mortality in children (4, 10–12). Because of the lack of clinical trials concerned with testing, prevention or treatment of pediatric thrombosis, most recommendations and guidelines have been extrapolated from adult trials. Children with thrombosis are a heterogeneous group and it is unlikely that a single approach to testing or treatment is optimal for all. Adult population-based studies have identified many prothrombotic factors, of which five have good clinical utility. Inherited antithrombin (AT) deficiency, protein C and protein S deficiency, Factor V Leiden mutation and prothrombin 20210 mutation were associated with increased risks of thrombophilia. Two mutations are far more prevalent than deficiency of anticoagulant proteins and have caused a significant increase in thrombophilia testing over the last two decades. The clinical usefulness of testing has recently been actively debated, and the utility of testing varies greatly, especially in child populations (13, 14). The concept of developmental hemostasis is universally accepted and has specific implications in the diagnosis of hemostatic disorders in children. Healthy children are relatively protected from thromboembolism due to the relationship between age and the plasma concentrations of coagulation proteins. Mechanisms include a reduced capacity to generate thrombin, good antithrombotic potential of the undamaged vessel wall and increased capacity of a2-macroglobulin to inhibit thrombin (15). On the contrary, newborns with any disturbance in the hemostatic system are at particularly high risk of thrombotic events due to their immature coagulation system. Vitamin K-dependent coagulation factors and anticoagulant factor activity, including AT, protein C and protein S, are decreased significantly. Fibrinolytic activity due to lower plasminogen concentrations is also decreased in infants. Concentrations of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) and a2-antiplasmin are different in newborns compared with adults. These levels rapidly increase/decrease during the first weeks of life. By 6 months of age, almost all coagulations factors reach adult values and the risk of thrombosis decreases (5, 16–18).

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Article in press - uncorrected proof S98 Trampus-Bakija: Pediatric thrombosis

Thrombotic risk factors in the pediatric population Risk factors in children are for the most part the same as those identified in adults, and include known genetic factors. As many as 50% of adult patients with venous thromboembolism (VTE) have no identifiable risk factors, but 70%–90% of affected children show at least one recognized risk factor for VTE. An average of two, and up to four predisposing factors, are found in the pediatric population. In children, 95% of VTE are secondary complications of serious disease (19–23). Inherited risk factors

Prothrombotic disorders are well-recognized risk factors of VTE in adults. The frequency of congenital prothrombotic disorders in children varies between 15% and 60% in different studies according to the clinical classification and the population studied (22, 24, 25). The distribution of risk factors also varies with respect to the ethnic background and number of patient controls investigated. It is becoming increasingly evident that pediatric patients with thrombosis are not a homogeneous group. Inherited prothrombotic disorders are usually suspected in children with an unexplained cause of thrombosis, a positive family history, a history of recurrent thrombotic events or thromboses in an unusual site (11, 26). Inherited thrombophilia is described as an additional thrombotic risk factor in children with idiopathic thrombophilia and those with an underlying disease or condition. The most common inherited thrombophilias are listed in Table 1. The odds ratios that are presented are similar when compared to adults, but are derived from a very small number of pediatric studies (13, 19, 20, 30–33). Table 1 Inherited thrombophilia and VTE. Risk factor

Factor V leiden mutation Prothrombin 20210 mutation Protein S deficiency Protein C deficiency Antithrombin deficiency Elevated lipoprotein (a) Elevated factor VIII ()150%) (OR rises with higher FVIII:C) Hyperhomocysteinemia/ MTHFR C677T mutation a

Odds ratio (OR) (95% CI) Children

Adult

3.56a (2.57–4.93) 2.63a (1.61–4.29) 5.77a (3.07–10.85) 7.75a (4.48–13.38) 8.73a (3.12–24.42) 4.50a (3.19–6.35) 6.1c (2.1–17.7) ND

5.7b (3.5–9.3) 2.8b (1.2–6.6) 1.8b (0.7–4.7) 1.8b (0.5–6.6) 5.7b (0.8–43.3) 3.2b (1.9–5.3) 4d (1–16) 0.7b (0.4–1.2)

According to Young et al. (20); baccording to von Depka et al. (27); according to Goldenberg et al. (28); daccording to Kraaijenhagen et al. (29). OR are for heterozygous population; ND, not determined; FVIII:C, factor VIII activity.

c

Factor V Leiden mutation in children is associated with primary and secondary VTE. The frequency in healthy populations is about 5%. Heterozygous or homozygous children with FV Leiden mutation have an increased risk of thrombosis of 5–10-fold or 80-fold, respectively. They usually experience their first thrombotic event following puberty (34–36). The prothrombin 20210 gene mutation is estimated to be the second most prevalent prothrombotic genetic defect, with a frequency of 2%–3% in the Caucasian population. Most children with this mutation do not develop thrombosis until adult life (37, 38). Congenital antithrombin deficiency (type I – quantitative, and type II – qualitative) is associated with a high risk of thrombosis. The level of AT is about 50%, and is less in heterozygous AT deficiency patients. Thus, the value overlaps with physiological levels in infants and young children. AT deficiency is found in approximately 4%–6% of young patients with venous thrombosis. Thrombosis is uncommon in the first decade, but the risk rises sharply between the ages of 15 and 30 years, and is manifested as idiopathic thrombosis or recurrent venous thromboembolism. The homozygous condition for AT type I deficiency is lethal in utero or results in severe complications at birth in type II deficiency (39–42). Homozygous protein S and protein C deficiency occurs in approximately 1 in 500,000 livebirths. It usually manifests as purpura fulminans in the newborn period and represents a life threatening disorder in the neonatal period. This clinical condition is characterized by extensive subcutaneous thrombosis and disseminated intravascular coagulation. Heterozygous protein C and S deficiency usually manifest as TE in adulthood, and protein S deficient young women or adolescent girls should exercise special care when using oral contraceptives (30, 43). The mutation of the methylenetetrahydrofolate reductase (MTHFR) gene causes mild to moderate increases in homocysteine. Gene polymorphism by itself is not associated with VTE, and the odds ratio for increased homocysteine in children has not been established (13, 44). Concentrations of homocysteine could also be increased due to nutritional deficiency of vitamin B6, B12 or folate, or deficiency of cystationine b-synthase (CBS) (31). Patients with homozygous CBS deficiency manifest an early tendency to thrombosis (45). Increased plasma concentrations of lipoprotein (a) wLp(a)x ()300 mg/L) in children have been reported to be a risk factor of VTE in childhood. The prevalence is the same but the risk is even higher (odds ratio 7.2) than that found in heterozygous children for the FV Leiden mutation (32, 46). Published data for adults is conflicting (27, 47). Increased plasma Factor VIII activity ()150%) is accepted as an independent marker of increased thrombotic risk, and patients have recurrent TE (28, 29, 48). The mechanism underlying the increase remains unknown. There are some specific clinical settings which represent a distinct inherited risk factor for thrombosis in children. Congenital heart disease includes a state of low flow stasis,

Article in press - uncorrected proof Trampus-Bakija: Pediatric thrombosis S99

polycythemia and in vivo platelet activation. Patients with sickle cell disease have an increased risk of PE, and approximately 11% of children with sickle cell disease will experience stroke at a young age (19). Acquired risk factors

True idiopathic thrombosis is extremely rare in children, but its frequency increases in puberty. This peak is associated with high PAI-1 activity and decreased t-PA antigen concentrations, mostly due to hormonal status, use of contraceptives, obesity and smoking (46, 49). The pathogenesis is multifactorial and a single cause predisposes, but is not sufficient to trigger thrombosis. Acquired factors include pathological processes and/or provoking effects. The mechanisms involved in pathogenesis include all elements of Virchov’s triad, which include abnormalities in the endothelium (vessel wall injury), abnormalities in hemorheology (blood flow/stasis) or abnormalities in platelets, as well as coagulation and fibrinolytic pathways (blood constituents). Acquired risk factors for VTE and their contribution to changes in Virchow’s triad elements are listed in Table 2 (50, 51). Central venous lines (CVL) are important in the medical and supportive management of various diseases. However, they represent the most common risk factors of VTE in the pediatric population, with an incidence of 5.3 per 10,000 hospital admissions; representing one third of hospital TE. CVLs are associated with )90% of neonatal VTE (2, 22, 52). Thrombus growth occurs near the catheter implantation site, thus approximately 50% of VTE occur in the upper extremity (53–56). TE in patients with malignancy is the result of complex interactions of disease, chemotherapy and side effects of treatment. Most reports concern acute lymphoblastic leukemia (ALL) and the high risk of TE during L-aspariginase therapy (57, 58). Knowledge of childhood TE in other malignancies is limited. The presence of antiphospholipid antibodies and/or lupus anticoagulants in children also predisposes to risk of deep Table 2 Acquired hypercoagulable states in children with the elements affected in Virchow’s triad (50, 51). Cause of thrombosis

Abnormal element of Virchow’s triad

Central lines Surgery/trauma Infection (sepsis, meningitis, otitis) Malignancy Dehydration Polycytemia Congenital heart disease Non-infectious inflammation Autoimmune disorder (SLE, APS) Renal disease/nephrotic syndrome Drugs (asparaginase, prednisone, factor concentrate, heparin, oral contraceptives...)

I I S/I/B S/I/B S S S S B B B

I, vascular wall injury; S, circulatory stasis; B, blood constituent.

venous thrombosis, PE and cerebral venous sinus thrombosis. Arterial thrombosis appears to be less common in children with APS. Pediatric patients with APS have a very high risk of recurrent thrombotic events (59–61). Transient LA are often detected in children with infection and do not represent a risk of TE (60). Other acquired conditions are systemic infections, congenital heart disease, asphyxia and hypovolemia in the neonatal period. Trauma, surgery, immobilization, oral contraceptives, nephrotic syndrome, corticosteroid therapy and some other chronic disorders contribute to prothrombotic risk factors in older children (22, 62, 63).

Thrombophilia screening in children Thrombophilia screening is a matter of debate, and there are different approaches to screening guidelines. In 2002, the Subcommittee for Perinatal and Pediatric Thrombosis of the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis (ISTH) group recommended a panel for multilevel testing for thrombophilia in children (64, 65). However, it has become obvious that in the heterogeneous pediatric group of thrombosis, a single optimal recommendation cannot be applied. Since the physiology of children and the types and manifestation of diseases differ from that of adults, it has been recommended that symptomatic patients undergo comprehensive screening for even very rare genetic and prothrombotic factors. Detection of one thrombophilic factor does not exclude the existence of a second or third. Data presented for the subgroup of pediatric patients suffering from multiple combined prothrombotic risk factors shows an enhanced risk of recurrent TE following an initial spontaneous event. The same principle for testing should be taken into account in testing asymptomatic siblings and first-degree relatives of symptomatic children (24, 66). The groups of pediatric patients with prothrombotic risk who might benefit from testing are listed in Table 3. These recommendations are based on the current understanding of thrombosis as well as ethical issues. The primary guide for patient benefit is to identify combined defects or homozygotes (possibility of therapy), to provide counseling to family members or when the risk of recurrence of TE exists, to provide counseling for adolescent females (high estrogen), to consider thromboprophylaxis, to promote a healthy lifestyle and to collect more data on genetic risk factors (for research). The cost-benefit should not be ignored, as well as potential psychological damage in over- or misinterpretation of results. There is still a lack of epidemiological studies and evidence on the benefit of thromboprophylaxis in the pediatric population. The decision to perform thrombophilia testing should be made on an individual basis when considering benefits and risks. The goal of testing is by no means for diagnostic purposes, but rather for counseling. A limited number of guidelines apply directly to thrombophilia testing in asymptomatic children. Multi-step evaluation regarding


Table 3 Recommendations for thrombophilia screening in the pediatric population waccording to Raffini and Manco (13, 64, 67)x. Population group

Level of recommendation

Adolescents with spontaneous thrombosis Children with non-catheter related VTE or stroke Children with symptomatic catheter related VTE Asymptomatic children with a positive family history Children with asymptomatic catheter related VTE Asymptomatic children – high risk (CVL, ALL, OC) – routine screening Children participating in thrombosis research

2 2 1 1 0 0 2

2-Testing is recommended; 1-testing is made on individual basis/ not enough data; 0-testing is not recommended. CVL, central venous line; ALL, acute lymphoblast leukemia; OC, oral contraceptives.

the clinical picture and anamnesis may be performed as shown in Table 4 (44, 64, 65, 68–70). On behalf of the ISTH subcommittee, Manco et al. suggested that pediatric patients should be tested for the full panel of genetic and acquired prothrombotic factors. Basic

tests at level 1 (complete blood count, erythrocyte sedimentation rate, CRP) should help exclude myeloproliferative disorders, infections and inflammation, including sickle cell anemia, by the examination of a blood smear. These tests are helpful in deciding on long-term therapy. Level 2 tests should be performed if the tests at level 1 are negative, and if the affected child has a strong positive family history, recurrent thrombosis or severe thrombosis. There are prothrombotic risk markers that are still under investigation, including von Willebrand factor antigen and the composition of multimers, platelet receptor polymorphism, tissue plasminogen activator, thrombin activatable fybrinolysis inhibitor and some others. Their utility is a mater of debate (64, 65, 68). To assess underlying hypercoaguability, an extensive laboratory evaluation should be done at the time of an acute TE. The patient should be screened in a specialized coagulation laboratory for prothrombotic defects, and this is especially true for children. It is necessary to obtain the appropriate samples before therapeutic intervention with anticoagulation therapy or fresh frozen plasma. Genetic testing is not influenced by any therapy. Sample quality is crucial. For coagulation testing, the right anticoagulant must be chosen. Some assays are sensitive to storage at room temperature, especially functional coagulation factor level determinations. Sample storage at –208C or below for a limited period of time prior to testing can pro-

Table 4 Laboratory testing in children with thrombosis. Level

Test

Assay principle

Level 1 Basic initially

CBC (include platelet count) Erythrocyte sedimentation rate, C-reactive protein Screen for sickle cells

Factor V Leiden Prothrombin 20210 mutation Fasting homocysteine level Lipoprotein (a) Lupus anticoagulants (LA) ACL and anti-b2GPI antibodies Factor VIII

Hematology analyzer Westergren method Immunological Blood smear examination (Hemoglobin electrophoresis) Clotting assays Functional: heparin cofactor activity against F Xa Functional: chromogenic activity Immunologic: free protein S antigen APTT based with predilution of test plasma in FV depleted plasma DNA based DNA based HPLC or immunological Immunological Functional: clotting assay Immunological assays Clotting-based test

Euglobuline clot lysis time Plasminogen activity Fibrinogen concentration D-dimer PAI Test for PNH Factors IX, XII, XI

Functional Functional or immunological Clauss method Immunological assays Functional or immunological Flow cytometry Clotting-based test

APTT, PT, thrombin time Antithrombin Protein C Protein S Activated protein C resistance (APCR)

Level 2 Extended

APTT, activated partial thromboplastin time; PT, prothrombin time; ACL, anticardiolipin; anti-b2GPI, anti-b2-glycoprotein I; PNH, paroxysmal nocturnal hemoglobinuria.


long stability. Antigen assays are unaffected by freezing and thawing of samples prior to analysis, but functional assay results may vary. There are methods (APCR, LA) that are influenced by platelet contamination; double centrifugation and freezing of samples is recommended. Effective use of an internal quality control scheme with normal and borderline abnormal samples is encouraged to control the assays at different measurement levels. External quality control may expose methodological errors (69, 71, 72).

subjects should be included. The absolute minimum number needed for a normal Gaussian distribution is 40. This number is very high for neonates or very small children who are difficult to recruit. Not only do the well known pre-analytical variables need to be considered, but a number of additional problems in obtaining a representative blood sample, such as obtaining blood via heparinized catheters or syringes, the presence of polycythemia in many neonates (9:1 citrate-toblood ratio), under-filled collection tubes, etc. need to be considered (13, 77). Laboratories with no in-house reference ranges should at least validate the manufacturer’s range.

Interpretation of results The interpretation of thrombophilia test results is difficult and errors are not infrequent. Genetic testing using the polymerase chain reaction is now available for several genetic variants commonly associated with thrombosis (FV Leiden mutation, prothrombin variant mutation and MTHFR polymorphisms). Results are not age dependent and should not be repeated. Interpretation should be based on identification of gene mutations/polymorphisms with respect to the individuals ethnic background (73). APCR, protein C activity, free protein S antigen and activity, antithrombin assays, fibrinogen concentrations, homocysteine concentrations, plasminogen, Lp(a), coagulation factor activity, and antiphospholipid antibody assays could be affected by acute phase reactants at the time of onset of thrombosis. For all plasma based assays, a clotting abnormality is confirmed as a defect only if the plasma concentration/activity of protein is outside the normal range in at least two different samples. Tests giving abnormal results should be repeated 3–12 months after an acute TE. Patients should not be on anticoagulant therapy at least 14–30 days before testing (46, 74, 75). Many laboratories compare their pediatric results to either adult reference values or published pediatric reference ranges. The incorrect interpretation of results based on reference ranges adopted from the literature has been shown to lead to errors in diagnosis (71). A high standard of clinical care requires the interpretation of laboratory results based on ageappropriate, analyzer- and reagent-specific reference values. Reference ranges published 20 years ago by Andrew et al. (18) should be used with caution. Instruments, assays and reagents used today are different from those used 25 years ago. The concept of developmental hemostasis was confirmed in Australian neonates 4 years ago. However, some normal ranges differ from those used previously (16). With an understanding that reference values define the 95% confidence intervals in the healthy population, borderline values are problematic. The use of absolute cut-off values often is not useful because there is overlap between the normal range and the results of heterozygous individuals. Therefore, special care is necessary, particularly in infants, when interpreting values for protein C, protein S and AT. Transient acquired loss of procoagulant factors can lead to concentrations that are close to congenital deficiency (67, 76). Parental testing may help to exclude these cases. The establishment of a laboratory’s own age-related normal values is laborious and expensive. Ideally, for local reference ranges (mean"2 SD), 120 healthy adult ‘‘normal’’

Monitoring of therapy The responsiveness of the child’s hemostatic system to antithrombotic drugs and thrombolytic agents differs markedly from adults. Because of this specificity, therapy monitoring tests are performed more frequently. Unfractionated heparin (UH) has been used for decades as an anticoagulant. The pharmacokinetic properties of UH in small children are quite distinct from that seen in adults. The prolongation of APTT is often measured, but the anti-Xa assay is currently the preferred monitoring option, with a target range of 0.35–0.7 U/mL (78, 79). Oral anticoagulant therapy with vitamin K antagonists is monitored via the prolongation in the PT. Typically, an international normalized ratio (INR) of 2.0–3.0 must be achieved. A higher range of 2.5–3.5 is recommended for patients with mechanical heart valves (76) and APS with recurrent TE (59). Low molecular weight heparin (LMWH), in clinical use since 1980, has advantages over UH because of its more predictable pharmacokinetics and pharmacodynamic properties. LMWH monitoring is performed exclusively via anitXa, with a target range 0.1–0.4 U/mL for prophylaxis and 0.5–1.0 U/mL for treatment doses (69, 76, 78). The guidelines recommend that for adults, laboratory monitoring of LMWH is not generally necessary. However, trials to determine the proper dose have not been performed for special populations, such as patients with severe obesity or very low body weight, renal failure, pregnancy, infants and children. New anticoagulants, classified as factor Xa and thrombin inhibitors, are increasingly being used in clinical practice. Coagulation monitoring has not been recommended for this type of therapy, although it is wise to have an appropriate method in case of an overdose or for monitoring the specific patient groups mentioned above. Factor Xa direct inhibitors can be monitored by anti-Xa chromogenic or clot-based methods, while APTT and ecarin clotting time are useful for direct thrombin inhibitors (69, 76). Patients who require concomitant thrombolytic therapy (e.g., tissue plasminogen activator) with anticoagulation as the initial therapy should be monitored before therapy and every 24 h. To check the concentration of platelets and fibrinogen and to determine the fibrinolytic potential and activation, PT, APTT, fibrinogen, platelets, plasminogen concentration and D-dimer concentrations should be obtained (80).


Conclusions Thrombosis in children is a serious condition. Better predictors in relation to risk factors would prevent additional complications in the child’s further development. There still is a need to investigate the problem of TE, the association between risk factors, prophylaxis and therapy. No universal guideline is available at present, and there is still an urgent need for pediatric-specific studies to validate new, incoming assays and to develop appropriate clinical algorithms. The list of new anticoagulant drugs is growing, and potential benefits for easier treatment of children are in sight. Meaningful research and clinical studies of platelet function and the role of platelet vessel wall interactions in infants are limited by the large volumes of blood required for the classical aggregation studies. Close cooperation of the laboratory (reproducible abnormal results) and clinical team (family history and clinical phenotype) is important in pediatric patients, and it seems necessary to improve the diagnostic process or improve ordering practices (81). Unfortunately, as some of the requesting clinicians have little understanding of the factors that influence thrombophilia testing methods and results, especially when a child is in question, laboratory comments should be reported with the test results. The laboratory and genetic test interpretation should be made done with caution as a definitive diagnosis can markedly influence a patient’s life. The usefulness of tests in pediatric patients should always be considered from the cost-benefit effect, from the view of the low overall absolute risk of thrombosis compared to adults.

Acknowledgements The author would like to thank Majda Benedik Dolnicˇar, MD for help in editing this manuscript.


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Review

Thrombophilia screening – at the right time, for the right patient, with a good reason

Mojca Stegnar* Department of Vascular Diseases, University Medical Centre, Ljubljana, Slovenia

Abstract Thrombophilia can be identified in about half of all patients presenting with venous thromboembolism (VTE). Thrombophilia screening for various indications has increased tremendously, but whether the results of such tests help in the clinical management of patients is uncertain. Here, current recommendations for thrombophilia screening in selected groups of patients, and considerations whether other highrisk subjects should be tested are reviewed. The methods for determination of the most common thrombophilic defects (antithrombin, protein C, protein S deficiencies, Factor V Leiden and prothrombin G20210A) associated with strong to moderate risk of VTE are described, indicating the timing and location of thrombophilia screening. Circumstances when a positive result of thrombophilia screening helps clinicians decide if adjustments of the anticoagulant regime are needed are discussed. Finally, psychological, social and ethical dilemmas associated with thrombophilia screening are indicated. Clin Chem Lab Med 2010;48:S105–13.

be investigated are not well-defined, and the appropriate timing for screening and the type of testing to be performed is still a matter of debate. Moreover, it has not been settled whether the results of screening help in the clinical management of patients investigated. Thus, clinical laboratories spend considerable amounts of time and resources performing laboratory investigations that do not seem to be completely justified (2). Until recently, thrombophilia was investigated almost exclusively by means of plasma based assays (phenotype). More recently, genetic assays have also become available commercially, and in some cases, genotyping can now be used in combination with, or instead, of phenotyping. Both types of analyses have advantages and disadvantages. Plasma based assays are typically not demanding and can be performed with relatively simple instruments. However, these assays are not standardized and results are therefore variable. On the other hand genetic tests provide unambiguous results. The disadvantages of genetic analysis are that with some thrombophilic defects (e.g., antithrombin, protein C and S deficiencies), the phenotype may be attributable to numerous different mutations, making the genetic analysis laborious and less helpful (2).

Who should be screened? Keywords: pregnancy complications; screening; thromboembolism; thrombophilia.

Introduction Thrombophilia is defined as a tendency to develop thrombosis as a consequence of pre-disposing factors that may be genetically determined, acquired, or both (1). Knowledge about thrombophilia has increased considerably in the last two decades. This has lead to widespread screening of thrombophilia and therefore increased pressure on clinical laboratories. Demands for screening are increasing dramatically, although this is not always justified because the patients to *Corresponding author: Prof. Mojca Stegnar, PhD, Department of Vascular Diseases, University Medical Centre Ljubljana, 1525 Ljubljana, Slovenia Phone: q386 1 522 8032, Fax: q386 1 522 8070, E-mail: [email protected] Received May 19, 2010; accepted July 23, 2010; previously published online November 6, 2010

There are no absolute indications for diagnostic thrombophilia screening. Potential relative indications could include general population screening, selected screening of populations where thrombophilic defects are expected to be more prevalent (e.g., asymptomatic family members of patients with known thrombophilia), populations at increased risk of venous thromboembolism (VTE; e.g., pregnancy, oral contraceptive use, high-risk surgery) and screening symptomatic patients with VTE. With the exception of screening of the general population, which is not recommended, all of these potential indications are controversial and must be considered in the context of clinical presentation. According to the International Consensus Statement (3), screening for thrombophilia is recommended for the groups of patients described below (Table 1). Patients with VTE

VTE is considered to be a multi-causal disease. The principal clinical conditions that pre-dispose to VTE are immobiliza-

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Table 1 Thrombophilia screening: who should be screened, which tests should be performed and when to screen? Patients to be screened are listed according to the International Consensus Statement on Thrombophilia and VTE (3). Tests are limited to those showing high to moderate association with VTE (4). Who should be screened? Patients with VTE

First unexplained VTE Recurrent VTE VTE at young age (-50 years) associated with transient pre-disposing condition (e.g., surgery) VTE at unusual site VTE associated with use of hormonal contraception, hormone replacement therapy or pregnancy Recurrent superficial thrombophlebitis without cancer

Women with pregnancy failure or complications of pregnancy Asymptomatic first degree relatives of patients with VTE and thrombophilia All children with thrombosis Patients with warfarin-induced skin necrosis Neonates with purpura fulminans Which tests should be performed? Antithrombin Protein C Protein S APC resistance Factor V Leiden Prothrombin G20210A Lupus anticoagulants

Antiphospholipid antibodies When to screen? Any time Not during an acute event Not during treatment with heparin Not during oral anticoagulant treatment Not during pregnancy, oral contraception and hormone replacement therapy One month after cessation of oral anticoagulant treatment

Heparin cofactor activity against factor Xa (amidolytic assay) Activity (amidolytic) assays with snake venom as the activator ELISA for free protein S antigen APTT-based method, pre-diluting patient plasma with factor V deficient plasma Genotyping, several molecular methods Genotyping, several molecular methods Screening tests: DRVVT and APTT Mixing test(s): 1:1 proportion of patient plasma with PNP Confirmatory test(s): performed by increasing PL of the screening test(s) Solid-phase ELISA for anti-cardiolipin and anti-b2-glycoprotein I antibodies Factor V Leiden, prothrombin G20210A Antithrombin, protein C, protein S Antithrombin, lupus anticoagulants Protein C, protein S, lupus anticoagulants Antithrombin, protein C, protein S All tests

VTE, venous thromboembolism; APC, activated protein C; ELISA, enzyme-linked immunosorbent assay; APTT, activated partial thromboplastin time; DRVVT, diluted Russell viper venom time; PNP, pooled normal plasma; PL, phospholipids.

tion, trauma, surgery, malignancy, infection, the postpartum period and use of oral contraceptives or hormone replacement therapy. For the majority of patients, VTE can be attributed to at least one of these clinical conditions (secondary VTE), while in others, VTE occurs spontaneously (primary or idiopathic VTE). In the background of all these clinical conditions can be the pre-disposition to VTE due to thrombophilic defects. At least one defect can now be demonstrated in approximately half of patients with VTE (4, 5). However, screening for thrombophilia is recommended only for selected subgroups of patients (3). This includes all patients with a first episode of primary (idiopathic) VTE, patients with recurrent VTE irrespective of the presence of a pre-disposing condition, young patients (age-50 years)

with VTE associated with a transient pre-disposing condition (e.g., surgery), patients with VTE at an unusual site (e.g., cerebral, hepatic, mesenteric, or renal vein thrombosis under the age of 50 years), women in whom VTE is associated with the use of oral contraception, hormone replacement therapy or pregnancy, and patients with recurrent superficial thrombophlebitis without cancer and in the absence of varicose veins. Women with pregnancy failure or complications of pregnancy

Many studies investigating the relationship between thrombophilia and pregnancy failure and complications suggest

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that both acquired and inherited thrombophilic defects are associated with an increased risk of pregnancy failure (i.e., recurrent miscarriage, late pregnancy loss), as well as complications of pregnancy such as pre-eclampsia and HELLP (haemolysis, elevated liver enzymes, low platelet count) syndrome. A meta-analysis of studies published between 1975 and 2002 showed that Factor V Leiden, resistance to activated protein C (APC), prothrombin G20210A mutation and protein S deficiency were associated with foetal loss, while methylenetetrahydrofolate reductase C677T gene mutation, protein C, and antithrombin deficiencies were not. The magnitude of these associations varied according to the type of foetal loss and type of thrombophilia (6). It appears that hypercoagulability with thrombosis of the placental vasculature is only one of the pathophysiological mechanism by which thrombophilia increases the risk of pregnancy failure (7). Asymptomatic first degree relatives of patients with VTE and thrombophilia

Because of the beneficial effect that screening may have on asymptomatic individuals, laboratory investigations should include all available first degree family members of the proband. Screening is particularly important for women of childbearing age. Affected members who have been identified should be informed of their future risk and counselled about appropriate prophylaxis at the time of exposure to challenging events (e.g., surgery, immobilization, and pregnancy) (2). Children with thrombosis

Thrombosis in children is a rare event when compared with thrombosis in adults (8). In contrast to adults, children are more likely to have one or more significant genetic or acquired thrombophilic defects that may require specific therapy for management of acute thrombosis (9). In 2002, the Subcommittee for Perinatal and Pediatric Thrombosis of the International Society of Thrombosis and Haemostasis recommended that all paediatric patients with venous or arterial thrombosis be tested for a full panel of genetic defects of thrombophilia (10). The rationale for this recommendation was that pediatric patients often have several risk factors for thrombosis, and even if several acquired risk factors are present, screening for genetic thrombophilic defects should also be performed (10). Pediatric patients who develop VTE are a heterogeneous group, and as more data has become available, recommendations for thrombophilia screening have been recently revised (11), but are beyond the scope of this review. Patients with warfarin-induced skin necrosis

Warfarin-induced skin necrosis is a rare, but serious complication of oral anticoagulant therapy. This condition has been associated with protein C deficiency, but only rarely reported in patients with protein S deficiency. The syndrome typically occurs during the first few days of warfarin therapy, often in

association with the administration of a large initial loading dose of the drug (12). Neonates with purpura fulminans not related to sepsis

Purpura fulminans is a rare syndrome of intravascular thrombosis and haemorrhagic infarction of the skin that is rapidly progressive and accompanied by vascular collapse and disseminated intravascular coagulation. In neonates, purpura fulminans is associated with a hereditary deficiency of the natural anticoagulants, protein C and protein S, as well as antithrombin (13).

Should other (high-risk) subjects be screened for thrombophilia? General population

The prevalence of thrombophilic defects that carry considerable risk of VTE (antithrombin, protein C and S deficiency) is very low (14). Although the prevalence of Factor V Leiden and prothrombin G20210A is considerably higher, if one considers the relatively low risk of VTE associated with these two conditions (15, 16), it must be concluded that screening of the general population is not justified. Women using oestrogen preparations (oral contraceptives or hormone replacement therapy)

Oral contraceptives containing third generation progestogens are associated with a six- to nine-fold risk of VTE when compared to those not using oral contraceptives (17). The combination of oral contraceptive use with any of the genetic or acquired thrombophilic defects may further increase the risk of VTE. The relative increase in risk associated with use of oral contraceptives combined with Factor V Leiden (odds ratios34.7) (18) and prothrombin G20210A (odds ratios 16.3) (19) would suggest screening of women, at least for these two thrombophilic conditions, before they are started on oral contraceptives. However, thrombophilia screening that is currently available is not cost effective because the number of laboratory tests needed to be performed to avoid one thrombotic event would be extremely high (20). Regardless, before starting use of oral contraceptives each patient should be screened carefully to identify those with an increased risk of thrombophilia (17). With hormone replacement therapy, there is a two- to fourfold increased risk of VTE compared to non-users, as demonstrated in randomized studies. The risk is greatest with oral preparations and seems to be greater at the start of therapy and in women with thrombophilic defects, in particular Factor V Leiden. Although the relative risk of VTE is similar in users of oral contraceptives and those undergoing hormone replacement therapy, the absolute risk is higher in the latter due to old age. However, due to the low incidence of


VTE, the absolute risk is low and general screening strategies are not likely to be effective (21). Pregnant women

In pregnant women, the risk of a first VTE event increases approximately four-fold compared to non-pregnant women, and increases up to 14-fold during puerperium (22). As suggested by a recent meta-analysis, pregnant women with genetic thrombophilic defects, with the exception of methylenetetrahydrofolate reductase C677T, were found to have increased risk of VTE (23). However, due to the lack of studies, gaps still exist in our knowledge of the risk of pregnancy-related VTE associated with thrombophilia. Therefore, thrombophilia screening is indicated only in selected patients with previous VTE, when antiphospholipid syndrome is suspected, or if a positive family history in first generation relatives is present (24). Surgical and other hospitalized patients

VTE is one of the most common complications of hospitalization and is associated with substantial short- and longterm morbidity, deaths and health care costs. Mobile medical patients and patients after short (-30 min) surgical procedures with no additional risk factors have a low risk of VTE, most non-orthopaedic surgical patients (major general, gynaecological, urologic, and thoracic surgery) and sick medical patients have moderate risk, while patients who have undergone hip or knee arthroplasty, or surgery for hip fracture have a high-risk for VTE. In the moderate and high VTE risk groups of patients, thromboprophylaxis with various anticoagulant drugs (unfractionated heparin, low-molecular weight heparin, new anticoagulant drugs) is administered routinely after risk stratification (25). Screening for thrombophilia with a positive result will not modify the prophylactic strategy in the majority of patients. Therefore, routine pre-operative screening before surgery and in other hospitalized patients is not recommended. Pre-operative screening for thrombophilia should be reserved only for patients with a personal or family history of VTE of unknown aetiology and who have not been investigated (3). Patients with arterial thrombosis

The association between thrombophilic abnormalities and the three main complications of atherothrombosis (myocardial infarction, ischaemic stroke, and peripheral arterial disease) is modest. Several studies have shown that Factor V Leiden (26), APC resistance (27) and prothrombin G20210A (28) may be contributory risk factors for myocardial infarction or ischaemic cerebrovascular disease in select groups of patients. However, the most comprehensive prospective study, performed on American physicians, showed that Factor V Leiden does not increase the risk of myocardial infarction or stroke (29). Due to the very low prevalence of deficiency of antithrombin, protein C and protein S, it is difficult to reveal any underlying association, if one is indeed present, in arterial thrombosis. Therefore, routine screening

for thrombophilic conditions is not warranted in most cases of arterial complications, and may only be informative in a very restricted population with arterial events, such as young persons, particularly when associated with smoking or oral contraceptive use (30). Thrombophilic conditions also seem to play a role in the risk of premature occlusion after revascularization procedures (31).

Which tests should be performed and how? On the basis of the evidence provided by studies, in principle it is plausible to include all markers that may help to identify thrombophilic defects that are firmly associated with an increased risk of VTE. These encompass the genetic thrombophilic defects of antithrombin, protein C and protein S deficiency, APC resistance, Factor V Leiden and prothrombin G20210A, as well as acquired thrombophilic defects lupus anticoagulants and antiphospholipid antibodies. Other thrombophilic defects, such as hyperhomocysteinaemia, high fibrinogen, increased factors VIII, IX, XI, dysfibrinogenaemia, reduced tissue factor pathway inhibitor and factor XIII polymorphisms could also be included in the investigation. However, there is less supportive data on their association with VTE and thus measurement of these analytes is not necessarily useful (14). Laboratory investigation could also be extended to fibrinolysis proteins, since decreased overall fibrinolytic potential and high plasma concentrations of thrombin-activatable fibrinolysis inhibitor have consistently been associated with the risk of VTE, whereas little evidence exists for a role of other proteins: plasminogen, antiplasmin, tissue plasminogen activator, and plasminogen activator inhibitor 1 (32). This review is limited to laboratory methods used to detect genetic (antithrombin, protein C, protein S, Factor V Leiden, prothrombin G20210A and APC resistance) and acquired (APC resistance, lupus anticoagulants and antiphospholipid antibodies) thrombophilic defects that are associated with a strong to moderate risk of VTE (4) (Table 1). Antithrombin

In the evaluation of thrombophilic individuals, a functional antithrombin assay should be used (33). A subsequent antigenic antithrombin assay result can help differentiate between type I and type II deficiency, but this is typically not performed for clinical purposes (34). The functional antithrombin assays comprise amidolytic (chromogenic) assays with excess thrombin or factor Xa. Assays that use factor Xa instead of thrombin do so to decrease potential interference from accelerator proteins other than antithrombin, such as heparin cofactor II. Furthermore, newer commercial assays have incorporated protease inhibitors, such as aprotinin to prevent cleavage of substrate by other serpins. Because factor Xa inhibition based assays appear to be the most widely used routine antithrombin activity measurement methods, it is important to realize that there is a possibility of not detecting all type II deficiencies with this technique (35).


Protein C

Lupus anticoagulants/antiphospholipid antibodies

For thrombophilia screening, protein C should be measured using a functional (clotting or amidolytic) assay, while measurement of antigen should be used only for further characterization of defects identified by one or more functional assays. Functional tests are commercially available and may be easily adapted to automation in many coagulometers, but are potentially susceptible to APC resistance (36) or high factor VIII (37). These problems may be circumvented by use of amidolytic assays with snake venom as the activator. Functional assays may leave undetected cases of subtle protein C dysfunction where the defect is restricted to the active site responsible for inactivation of the natural substrates. However, these cases are very rare and only a limited number of affected patients would be missed using amidolytic assays only (2).

Guidelines for the detection of lupus anticoagulant (46) have been updated recently (47). This update recommends that the diluted Russell viper venom time (DRVVT) be measured first as this is the most robust test for detecting lupus anticoagulants (48). Following this test, a sensitive APTT, performed with silica as an activator and a low phospholipid content, should be performed (49). Lupus anticoagulants should be considered as positive if one of these two tests gives a positive result. For the mixing test, pooled normal plasma (PNP) should be mixed with patient plasma in a 1:1 ratio, and the results are suggestive of lupus anticogulant if the clotting time is longer than the local cut-off threshold. Confirmatory test(s) must be performed by increasing the concentration of phospholipids (PL) in the screening test(s) (47). Thrombophilic patients should be screened also by assaying antiphospholipid antibodies using a solid phase enzymelinked immunosorbent assay (ELISA) to detect anticardiolipin and anti-b2-glycoprotein I antibodies. The presence of medium to high titres of anti-cardiolipin and antib2-glycoprotein I antibodies of the same isotype, most often IgG, is consistent with a positive lupus anticoagulant test and identifies patients at high-risk of VTE (50).

Protein S

Due to the assumption that dysfunctional protein S can be detected in patients with thrombophilia, functional assays should be the logical choice. However, the available functional assays are based on the APC cofactor activity of protein S (38), and these assays are not very specific and are affected by APC resistance (39). Therefore, diagnoses based on these tests must be considered with caution. A reasonable alternative to screening thrombophilic patients for protein S deficiency is measurement of antigen, either total or free protein S. It is matter of debate whether it is necessary to assay both antigens, or either. Perhaps free protein S antigen, being closely related to the functional form of protein S, is more useful than total antigen to detect patients with protein S deficiency (40). APC resistance/Factor V Leiden

APC resistance assessed in plasma using activated partial thromboplastin time (APTT)-based methods, with and without addition of APC (41), is a simple, inexpensive and a sensitive technique to detect the ‘‘APC resistance syndrome’’, as well as Factor V Leiden mutation. The original method, modified by pre-diluting patient plasma with Factor V deficient plasma (42), also enables measurement of APC resistance in patients on oral anticoagulant therapy. As APC resistance in the majority of cases is associated with Factor V Leiden, DNA analysis to detect this genetic mutation (43) is another possibility (44). However, it should be emphasized that Factor V Leiden accounts for most, but not all, cases of APC resistance (45), suggesting that DNA analysis, if used alone, would not identify all patients at risk. Prothrombin G20210A

Several molecular methods are commonly used in genetic testing for prothrombin G20210A, all are generally robust and reliable (44). An increasing number of kits for separate determination of prothrombin G20210A or in combination with other thrombophilic defects (e.g., Factor V Leiden) are becoming commercially available.

When should screening be performed? An acute VTE event with or without concomitant therapy may influence laboratory investigations, with the exception of DNA analysis, or make interpretation of the results difficult (2). In acute VTE, plasma antithrombin and occasionally proteins C and S may decrease transiently. In general, thrombophilia screening should be delayed for at least 6 weeks to allow acute-phase reactant proteins to return to baseline. Heparin therapy can lower antithrombin concentrations and impair interpretation of clot-based assays for lupus anticoagulants. Oral anticoagulants affect the results of screening for protein C and protein S, and some tests for APC resistance. Therefore, it is preferable to defer the laboratory investigation until after discontinuation of oral anticoagulant treatment (at least 2 weeks). In those for whom temporary discontinuation of anticoagulation is not practical, heparin can be substituted for warfarin when screening for protein C and S. However, the effect of warfarin on protein S levels may not disappear for 4–6 weeks. DNA testing for Factor V Leiden and prothrombin G20210A mutations is unaffected by an acute VTE event and/or anticoagulation therapy (14) (Table 1). In children, it is often useful to screen for thrombophilia acutely, rather than waiting until long-term anticoagulant therapy has been discontinued. This may involve repeated screening for one or two proteins, but will not substantially increase costs. In addition, antiphospholipid antibodies and their effect on coagulation may be transient and may be missed if screening is delayed for 3–6 months. Finally,


genetic screening may be very important in managing future pregnancies (10).

Does thrombophilia screening help in the clinical management of patients? In principle, laboratory screening should be performed whenever the results may influence decisions on management of the patient (therapy or prevention). Specifically, with respect to thrombophilia, the relevant issue is under what circumstances a positive result would help the clinician decide if adjustments in the anticoagulant regime in a thrombophilic patient is needed. Besides, many patients and clinicians are motivated to find an explanation for their disease, although it should be realized that the existence of a thrombophilic defect does not exclude other risk factors, given the multicausal aetiology of VTE. The results of thrombophilia screening are unlikely to influence the management of acute VTE. This is because, at present, treatment of acute VTE is not dependent on its cause. VTE has a tendency to recur and, in principle, results of thrombophilia screening might influence decisions on prevention of its recurrence (secondary prophylaxis), and may help clinicians decide how long and how intensively to treat patients. However, the evidence on the ability of laboratory screening for thrombophilia to predict the risk of recurrent VTE is much more questionable than its association with first VTE (51). Recently, large follow-up studies suggested no clear increased risk of recurrent VTE in carriers of Factor V Leiden, prothrombin G20210A and increased factor VIII (52). Only a deficiency in one of the anticoagulant proteins, antithrombin, protein C and protein S showed a mildly increased risk (52, 53). Also, combination of these deficiencies with Factor V Leiden or prothrombin G20210A did not greatly increase the risk of a recurrent VTE event (54). To date, there is also no conclusive evidence from clinical studies that would suggest that patients with genetic thrombophilic defects should be treated for a longer time or more intensively than patients with idiopathic VTE (55, 56). However, persistently positive lupus anticoagulants in the setting of a recent history of VTE is a strong risk factor for recurrence, and qualifies the patient for long-term oral anticoagulation therapy. Long-term anticoagulation is not devoid of haemorrhagic risks, and the accuracy of the laboratory diagnosis of lupus anticoagulants plays a crucial role in making decisions on whether, and for how long, anticoagulant therapy should be undertaken (57). Screening for thrombophilia may be beneficial for those family members of a proband who are carriers of the thrombophilic defect, but are still asymptomatic. The risk of a first episode of VTE in relatives with thrombophilia is increased two- to 10-fold. Nevertheless, the overall absolute risk in thrombophilic families has been assessed in many studies and is generally low, even in high-risk situations such as pregnancy, puerperium, surgery, immobilization, trauma and use of oral contraceptives. It is clear that the 2% annual risk

of major bleeding associated with continuous anticoagulant treatment outweighs the risk of VTE (58, 59). In pregnancy failure and hypertensive pregnancy complications which are associated with the presence of acquired (lupus anticoagulants and antiphospholipid antibodies) or genetic thrombophilic defects, guidelines suggest a combination of low doses of aspirin and low molecular weight heparin (60).

Where should tests be performed? The role of the clinical laboratory in thrombophilia screening can be divided into two parts: screening capacity and characterization of the thrombophilic defect (61). Both parts of the investigation of thrombophilia require considerable expertise, skill and experience in performing more sophisticated coagulation tests, as well as considerable expertise and experience in interpretation of the results. Therefore, specialized coagulation laboratories seem more suited for this purpose. However, it should be emphasized that a close connection between the clinic and the laboratory is an essential pre-requisite for the effective management of thrombophilic patients. In this respect, laboratories from general hospitals could be more suited for thrombophilia investigation than specialized laboratories in remote clinics. Probably the choice should rest on considerations that involve the abovementioned factors, but also on the best allocation of the economic resources provided by local health services (2).

What are the psychological, social and ethical dilemmas associated with thrombophilia screening? As with any genetic test, a potential drawback in screening for thrombophilia can be the social consequences such as problems with acquiring life or disability insurance (62). Moreover, the results of genetic tests may also have appreciable psychological consequences, including depression, anxiety and persistent fear (63, 64). However, not all the psychological consequences after genetic testing are negative, as testing may also lead to relief and reduction in fear (65). As the benefits and disadvantages of screening for thrombophilia are not fully clear, the psychological and other consequences of screening should be a consideration when deciding whether screening for thrombophilia should be performed (66).

Conclusions Knowledge about thrombophilia has increased considerably in the last two decades, and this has lead to widespread screening for thrombophilia for various indications. However, according to our present knowledge, screening for thrombophilia should be limited to selected groups of patients, mainly to young VTE patients, patients with spon-


taneous and recurrent VTE, women with complications of pregnancy and children with VTE. However, with few exceptions, testing for thrombophilia generally does not alter the clinical management of these patients. It could be expected that in future, indications for thrombophilia screening might also be extended to other patients at risk of VTE because of pre-disposing conditions, such as pregnancy or high-risk surgery, when the results of further clinical studies and cost effectiveness analyses become available.

Conflict of interest statement Author’s conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Review

Methodological issues of genetic association studies

Ana-Maria Simundic* University Department of Chemistry, University Hospital ‘‘SESTRE MILOSRDNICE’’, Zagreb, Croatia

Abstract Genetic association studies explore the association between genetic polymorphisms and a certain trait, disease or predisposition to disease. It has long been acknowledged that many genetic association studies fail to replicate their initial positive findings. This raises concern about the methodological quality of these reports. Case-control genetic association studies often suffer from various methodological flaws in study design and data analysis, and are often reported poorly. Flawed methodology and poor reporting leads to distorted results and incorrect conclusions. Many journals have adopted guidelines for reporting genetic association studies. In this review, some major methodological determinants of genetic association studies will be discussed. Clin Chem Lab Med 2010;48:S115–8. Keywords: genetic association study; methodology; polymorphism; population.

Introduction Genetic association studies explore the association between disease, predisposition to disease or some other well-defined state/characteristics and one or more genetic polymorphisms (1). Such studies compare genotype and allele frequencies in selected groups of affected and unaffected individuals. The association exists only if the frequency of an allele or genotype differs significantly between groups. Many different designs may be employed to study this association including cross-sectional, cohort, case-control, family studies, extreme values and others. Genetic association studies may be either genome-wide association studies or candidate gene association studies. A genome-wide association study is a nonhypothesis driven examination of genetic variation across a given genome, designed to identify genetic associations with a disease, state or some measurable trait. Such studies are made possible due to completion of the Human Genome Project and marked development in technology and bio-infor*Corresponding author: Assist. Prof. Ana-Maria Simundic, PhD, University Department of Chemistry, University Hospital ‘‘SESTRE MILOSRDNICE’’, Zagreb, Croatia E-mail: [email protected] Received June 10, 2010; accepted July 12, 2010; previously published online November 6, 2010

matics. Genome-wide association studies may help identify genome regions that harbour genetic variations contributing to some specific phenotype. Candidate gene association studies are hypothesis-driven and usually focus on genes in some specific candidate pathway known to be associated with development of the disease. Case control studies are one of the most commonly performed genetic association studies and their number is constantly increasing in the biomedical literature (2). Unfortunately, many genetic association studies fail to replicate their initial positive finding in an independent population (3, 4). This raises serious concerns about the methodological quality of these reports. Case-control genetic association studies are quite often compromised by some major methodological flaws in the study design and data analysis, and are often reported poorly (5–8). Inappropriate methodology and poor reporting may lead to distorted results and incorrect conclusions. The scientific contribution of such studies is not trustworthy and could be misleading. This is not only a waste of valuable resources, but is also highly unethical (9–11). Many journals have therefore adopted some existing guidelines (12) or proposed their own recommendations for reporting genetic association studies (13–16). Some highly ranked journals have even adopted the policy of not publishing genetic association studies related to complex disorders, unless there exist some exceptional reasons (17). However, others welcome only submission of association studies of high quality (18). The following characteristics are attributes of high quality genetic association studies: • • • • • • • • • • •

study is sufficiently powered, sample size is large, p-value is small, cases and controls are well-phenotyped, only associations that make biological sense are being analyzed, results are replicated at least once in an independent population, gene-dose relationship is present, corrections for multiple statistical testing are performed, quality issues have been addressed, genotyping error rate is quantified and is low, alternative causes for false-positive association are investigated, methods for controlling stratification have been discussed.

Listed above are only some of most commonly reported quality characteristics of genetic association studies. The complete list is quite comprehensive and new standards are continuously emerging. In this review, some major methodological determinants of genetic association studies will be discussed.

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Issues related to the phenotyping and genotyping Proper definition of phenotype

Accurate phenotyping of cases is essential to the quality of a genetic association study. In the last two decades, technological progress in genotyping and the development of genome wide genetic maps has substantially facilitated the research of complex traits and multifactorial diseases. Multifactorial diseases are inherently complex and affect different organs and organ systems, therefore resulting in quite heterogeneous phenotypes. Due to this inherent phenotypic complexity, multifactorial diseases may therefore be quite challenging for researchers (19). A key step to successful phenotyping is the choice of trait or disease being studied. Traits may be discrete (qualitative, dichotomous) and continuous (quantitative). Studying discrete traits is much more clinically relevant, but difficult to perform because of the issues related to proper definition of the disease. Diagnostic criteria for some disease are relatively non-specific, imprecise and may even require a somewhat intuitive approach, such as for example some psychiatric disorders (20) or a metabolic syndrome (21). Classification of cases into disease groups should preferentially be made according to the histopathology or molecular findings, rather than on clinical examination to ensure the homogeneity of the group being studied. There should be an acceptable rationale for the classification of cases. Focusing on some particular disease subtypes may be more efficient, provided the incidence is not too low. Continuous (quantitative) traits are much more convenient to measure and should always be preferred when diagnostic criteria are not unambiguous for a certain disease. Quantitative traits are also sometimes referred to as intermediate phenotypes. They are either some measurable risk factors, such as blood pressure, body mass index (BMI) or fasting triglyceride concentrations, or parameters which enter the diagnostic criteria, such as cardiac troponin T (cTnT) or D-dimer concentrations. Continuous traits are obtained easily from a large number of individuals with high precision and accuracy, allowing for more powerful statistical analysis of the data. It should be noted that the collection of quantitative traits might be compromised by therapy or treatment, for example, with antihypertensive drugs. If this was not specified and considered, it may be a potential source of bias. It should also be noted that the relationship between a genetic variant affecting a certain risk factor (e.g., low-density lipoprotein-cholesterol or fasting plasma glucose) and a disease or some clinical condition (e.g., coronary artery disease or type 2 diabetes) is not always unambiguous. Studies investigating continuous traits may not necessarily provide evidence for association of the genetic variant with the relevant disease, and establishing a connection with the disease requires further confirmation. The source for patient recruitment and method for the selection of (sub)groups should always be specified, as well as the exact exclusion and inclusion criteria. Quite often, the Material and method section of a genetic association study

is lacking complete information concerning the way that cases were recruited into the study. It could be that the study was done properly, but only poorly reported. However, it could also mean that it was not done properly. By carefully following the reporting guidelines, such important details would never be omitted from the manuscript. The control group is just as important as the patient group. Proper selection of adequate controls is also of utmost importance to the quality of the study. Ideally, controls should be matched to cases for all relevant characteristics. Cases and controls should come from the same or genetically similar population. It is well-known that genotype and allele frequencies for many susceptibility genes differ between ethnic groups (22–24). Therefore, ethnicity should be defined precisely for both controls and cases. Population stratification

Population stratification occurs when two populations having different genetic ancestries and/or different genetic mechanisms underlying the disease are merged into one study (25). If population stratification is present in the study, the observed effect may not occur due to a real association of the disease with the gene variant, but due instead to the differences in genotype frequencies and disease prevalence among populations in the total group. Population stratification may spuriously inflate or diminish the observed association of the genetic variant and trait which may be especially important if the observed effect is moderate or small. Several statistical methods have been developed to compensate for potential population bias (26–28). Another way to overcome the problem of population stratification is to use the family-based study and transmission disequilibrium test (TDT) (29). Hardy-Weinberg equilibrium

In the absence of evolutionary forces, allelic frequencies in the population remain unchanged from one generation to the next. This was postulated over a century ago, and is wellknown and widely accepted among geneticists as the HardyWeinberg equilibrium (HWE) (30). Five conditions are required in order for a population to remain at HardyWeinberg equilibrium: 1. 2. 3. 4. 5.

No change in allelic frequency due to mutation. A large breeding population. Random mating. No immigration or emigration. No natural selection.

Deviations from HWE should be explored first in controls. If conditions for HWE are met and there is a deviation from HWE, the most probably reason for this is genotyping error (31). Other possible reasons for deviations from HWE are population stratification and random chance (32). If controls are in HWE, cases should also be tested for HWE deviations. Modest deviation in cases could provide evidence for some

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weak associations, even in the absence of significant differences between genotype frequencies (33). Unfortunately, according to some original reports, HW values are not always presented and are often calculated incorrectly (7, 34, 35). Genotyping error

Genotyping error is inherent to almost all analytical systems and might arise due to many reasons, such as for example, failure to recognize the triallelic nature of the SNP (36), preferential amplification of one allele when there exists disproportion in the size of the respective alleles (37–39), existence of a mutation in close proximity to the polymorphic site (40), partial digestion in PCR-RFLP genotyping assays (41), heteroduplex formation (42) and other causes. Genotyping error may cause misclassification of up to 30% of samples (12) and may lead to serious over transmission of common alleles, thus contributing to an inflated false positive rate in genetic association studies (43). Authors should therefore aim to increase genotyping fidelity, by careful assay design as well as incorporating various quality check steps, such as avoiding batch – related biases, mixing the sample sets (controls and cases) within batches, double genotyping using the same or another assay format, using random blanks for minimizing contamination, etc. With experienced personnel and stringent quality control measures, the estimated error rate is small and has no impact on the quality of the study (44). When reporting genetic association studies, authors should properly describe what was done to detect and minimize genotyping errors.

Issues related to the study design Sample size

Sample size has long been recognized as one of the major determinants of the quality of a genetic association study. The sample size depends on the desired power of the study, level of significance and effect size (45). Additionally, in genetic association studies, the sample size that is necessary depends on the frequency of the predisposing allele (33, 46). The sample size that is necessary decreases with increasing frequency of the susceptibility allele and effect size (allelic odds ratio). In genetic association studies of multifactorial diseases, odds ratios are usually low because contribution of the genetic variant to the odds of this particular event is small in comparison to the overall risk in the individual (47). For small effect sizes and rare alleles, several 10s of 1000s of subjects need to be enrolled in the study to detect significant associations. Even with alleles as common as those present in up to 20% in controls, more than 10,000 subjects are needed to detect an odds ratio of 1.1 (33). Finally, in studies that report negative findings, adequate power of the study to detect an effect, if it truly exists, is of great importance. Unfortunately, at present, underpowered studies with negative results are not unlikely to be published in many journals.

Replication

For the reasons described in the previous paragraph, many preliminary studies report positive findings of significant associations, which are not successfully replicated in subsequent studies. The issue of replication has therefore recently received much attention and has become an unconditional criterion for publishing genetic association studies in many journals. Replication is needed to check for errors and bias and to strengthen the confidence in individual studies. Replication reduces type I errors (a error) where an effect is found when none is actually present (48). Each genetic association study should have a clear statement about whether the particular study is the first report or replication in an independent population, of the same allele, identical phenotype and same direction of the effect (12).

Conclusions Genetic association studies often suffer from inappropriate methodology and poor reporting. The low quality of genetic association studies may not only lead to distorted results and incorrect conclusions, but also are misleading and highly unethical. Journal editors, reviewers and authors should adhere to published guidelines and recommendations for the successful design and reporting of genetic association studies. It might be quite difficult to overcome all limitations, but one needs to try to implement as many of the methodological criteria as possible. With increasing availability of large data sets, technological improvements and bio-informatics, this task will be much easier.


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Review

Pharmacogenetics guided anticoagulation

Raute Sunder-Plassmann and Christine Mannhalter* Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

Abstract Advances in the field of human genetics has made it possible to develop prevention strategies for rare genetic disorders and to tailor pharmacotherapeutic approaches to anticoagulation and certain cancers. However, it is still not clear how genetic variations influence the risk and outcome of common diseases. Data from genome-wide association studies is just beginning to answer these questions. In contrast, pharmacogenetic knowledge is frequently not yet translated into clinical practice, even though in some cases, particularly regarding drugs used in treatment of thrombotic diseases (e.g., coumarines, platelet aggregation inhibitors), it is already known that testing for genetic variants prior to pharmacotherapy may help to prevent severe adverse drug reactions or avoid therapeutic failure. In this review, we address the potential impact that genetic alterations in the genes for vitamin K epoxide reductase and some cytochrome P450 variants may have on therapeutic strategies in anticoagulation. Clin Chem Lab Med 2010;48:S119–27. Keywords: anticoagulation; CYP2C9; CYP2C19; pharmacogenetics; vitamin K epoxide reductase.

Introduction Oral anticoagulants are the primary drugs used for the prevention of thromboembolic diseases in patients with deep vein thrombosis, atrial fibrillation, and mechanical heart valve replacement (1). Several clinical trials have demonstrated that anticoagulation with warfarin in patients with atrial fibrillation is more than twice as effective in prevention of stroke compared with any other medication, including all other antithrombotic drugs or surgical interventions (2). It is well known that the pharmacokinetics of warfarin are subject to variability due to interactions with external factors, *Corresponding author: Christine Mannhalter, Department of Laboratory Medicine, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, Vienna 1090, Austria Phone: q43 1404002085, Fax: q43 1404002097, E-mail: [email protected] Received July 30, 2010; accepted October 5, 2010; previously published online November 10, 2010

such as other drugs and foods. The narrow therapeutic index of warfarin requires that anticoagulation is maintained within the international normalized ratio (INR) range of 2.0–3.0 to optimize efficacy, while minimizing the risk of bleeding. Close monitoring of coagulation and frequent dose adjustments are necessary to prevent morbidity and mortality from thromboembolic or bleeding events. Even with careful monitoring, the initiation of warfarin dosing as well as chronic oral anticoagulation with warfarin is difficult to maintain within the therapeutic range. In addition to external factors, genetic factors, in particular variations in the genes encoding for vitamin K epoxide reductase (VKORC1) and cytochrome P450 2C9 (CYP2C9), have been associated with inter-individual differences in warfarin metabolism (3). Platelet activity can be inhibited by several drugs that interfere with platelet adhesion or platelet aggregation. Thienopyridines, including clopidogrel, are currently used for acute and long-term prevention of thrombotic events in acute coronary syndrome (ACS) wreviewed in (4)x. Clopidogrel is the cornerstone of antithrombotic therapy. After metabolic activation of the prodrug by the cytochrome P450 (CYP) enzyme system, metabolites of this drug bind and irreversibly block the platelet P2Y12 adenosine diphosphate (ADP) receptor (5, 6). In cases with decreased plasma concentrations of the active clopidogrel metabolites, poor response or clopidogrel resistance has been reported. Several CYP isoenzymes, including CYP2C19, CYP3A4, CYP3A5, CYP1A2, CYP2B6 and CYP2C9 are involved in this process. Previous studies have shown pronounced inter-individual variability in the metabolism of clopidogrel where CYP2C19 seems to be particularly important. Recent studies suggest that carriers of a CYP2C19 loss of function mutation who receive clopidogrel are at increased risk for recurrence of cardiovascular events (7–17).

Warfarin Warfarin (Figure 1) is a coumarin derivative used for oral anticoagulation and prevention of thromboembolic events. It is the most widely prescribed anticoagulant worldwide. Its discovery dates back to the early 1940s. Clinically available warfarin consists of a racemic mixture of two active optical isomers, (R)- and (S)-isoforms, the pharmacokinetic and pharmacodynamic properties differ considerably between the two. The (S)-enantiomer is three times more potent than the (R)-enantiomer and is metabolized predominantly by CYP2C9. Warfarin acts as an antagonist to VKORC1 and thus the carboxylation of vitamin K dependent proteins.

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Figure 1 Chemical structure of warfarin.

In 2004, Ansell et al. (18) published guidelines suggesting initial warfarin dosing of 5–10 mg/day for people receiving their first treatment. The different dose requirements for individuals is largely due to single nucleotide polymorphisms (SNPs) in the VKORC1 gene (19). Additionally, for the two major allelic variants, CYP2C9*2 and CYP2C9*3, there is good evidence that these alleles are linked to altered warfarin metabolism (20).

Genes and polymorphisms involved in warfarin dosing variability The prevalences of CYP2C9 and VKORC1 polymorphisms vary among different ethnic groups, and can account for over 30% of the variance in warfarin dosing. In the study by Meckley et al. (3), CYP2C9 alone, VKORC1 alone, and a combination of genetic and clinical factors explained 12%, 27%, and 50%, respectively, of the variation in warfarin maintenance dosing. Based on several retrospective studies, it was suggested that a pharmacogenetic-guided dosing algorithm can predict warfarin dosage and might reduce adverse events. To date, several pharmacogenetic-guided dosing algorithms for coumarin derivatives, predominately for warfarin, have been developed. However, the potential benefit of these dosing algorithms, in terms of their safety and clinical utility, is still not clear. The European pharmacogenetics of anticoagulant therapy (EU-PACT) trial will assess, in a singleblinded and randomized controlled trial with a follow-up period of 3 months, the safety and clinical utility of genotype-guided dosing in daily practice for the three main coumarin derivatives used in Europe (21).

Vitamin K epoxide reductase complex subunit 1 (VKORC1, OMIM 608547) and the vitamin K cycle VKOR activity was first reported in 1970 by Bell and Matschiner (22), however, it took more than 30 years to purify VKORC1. In 2002, Fregin et al. (23) found that humans with hereditary deficiency in several vitamin-K-dependent clotting factors had disorders in VKOR activity that mapped to human chromosome 16. VKORC1 is involved in the regeneration of vitamin K and vitamin K hydroquinone (KH2) from vitamin K 2,3-epoxide (KO) (Figure 2). KH2 is an essential co-factor for the posttranslational carboxylation of glutamic acid residues (Glu) to g-carboxy glutamic acid

(Gla). The enzyme is expressed in the liver, kidney, heart and skeletal muscle (24), and plays a central role in g-carboxylation. The vitamin K-dependent proteins that need to be g-carboxylated include the coagulation factors VII, IX, X, prothrombin, protein S, protein Z and protein C, osteocalcin and matrix Gla-protein, proteins active in bone metabolism, and Gas 6 (growth-arrest-specific protein 6) which is involved in cell proliferation and apoptosis (25). The VKORC1 gene (GenBank accession number AY587020) is located on chromosome 16p 11.2, spans 5126 bp and consists of three exons and two introns. A large family of homologues with significant sequence similarities exists among plants, archaea, bacteria, vertebrates and arthropods (26). The gene codes for a transmembrane protein, located in the mammalian endoplasmatic reticulum, and consists of 163 amino acids (27). Goodstadt and Ponting (26) found four cysteine residues and one serine/threonine residue which are absolutely conserved (Cys 43, Cys 51, Cys 132, Cys 135, Ser/Thr 57) and are considered to form the active site of VKORC1. In 2005, Geisen et al. (28) reported 28 SNPs in the VKORC1 gene: three silent SNPs in the coding region of VKORC1, and 25 SNPs in non-coding regions: 7 SNPs in the promoter region, 11 in introns, and 5 in 59UTR (untranslated region). For 14 SNPs, the frequency of the minor allele was at least 1%, the other half of polymorphisms were rare in the study cohort. Six SNPs form three main haplotypes (VKORC1*1, VKORC1*2, VKORC1*3) which comprise more than 99% of the genetic variability of the VKORC1 gene in Europeans (28). Rieder et al. (29) described 5 SNPs that are clearly associated with variability in warfarin dose requirement in European Americans: –4931T)C, –1639G) A, 1173C)T, 1542G)C, and 2255C)T. The different variants in this gene determine low-, intermediate- and highwarfarin dose requirements. Particularly, two polymorphisms significantly influence the time required to reach the first therapeutic range. D’Andrea et al. (30) found that people carrying the T allele of the polymorphism 1173T)C require less warfarin than those carrying the C allele, and Montes et al. (31) observed that the A allele of –1639G)A is associated with a low-dose requirement of the anticoagulant acenocoumarol. Both SNPs are in tight linkage disequilibrium. Using a reporter gene assay, Yuan et al. (1) detected 44% higher VKORC1 promoter activity for the –1639GG genotype compared to the –1639AA genotype. This polymorphism in the promoter region of the VKORC1 gene at position –1639 influences the warfarin dose needed for adequate anticoagulation. This is due presumably to the effect on transcription of VKORC1. Other genes mediating the action of warfarin have little or no impact on dose requirements. The SNPs –4931T)C, –1639G)A, 1173C)T, 1542G) C, and 2255C)T define two major haplotype groups designated A and B. Haplotype A carries the minor alleles and is associated with lower mRNA expression of VKORC1, as well as a lower warfarin maintenance dose. Haplotype B represents the major alleles, has higher mRNA expression and a higher warfarin dose requirement (29). Today, more than 40 different SNPs are known. It has been demonstrated that the VKORC1 rs9923231 SNP shows

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Figure 2 The vitamin K cycle. http://www.inchem.org/documents/ehc/ehc/ehc175.htm.

extensive geographic differentiation, with the T allele present in very high allele frequencies in East Asian populations. This unusual distribution may be the result of positive selection in East Asia. For future application of pharmacogenomic-based algorithms in different population groups, an understanding of the worldwide distribution of markers that determine warfarin dosing is important (32).

Cytochrome P450 2C9 (CYP2C9) For the CYP2C9 wOnline Mendelian Inheritance in Man (OMIM) 601130x gene, 37 different alleles and more than 300 variations in the DNA sequence have been reported. To date, the functional impact of many polymorphisms in

CYP2C9 has not been clearly established in clinical studies. However, for the two major allelic variants, CYP2C9*2 and CYP2C9*3, there is good evidence linking these alleles to altered warfarin metabolism (20). Deficiency of CYP2C9 enzyme activity leads to an increased half-life of S-warfarin which alters the time required to reach steady state concentrations (Table 1). Patients with CYP2C9*2 and *3 variants need longer times for stabilization of dose, and are at a higher risk of serious bleeding. The cSNP defining the CYP2C9*2 allele is the c.430C)T transition that leads to the amino acid exchange p.R144C. This amino acid substitution decreases CYP2C9 enzymatic activity and decreases the intrinsic clearance of warfarin. Heterozygosity for CYP2C9*2 decreases the dose for S-warfarin to 78% of that needed in homozygous *1/*1 individ-


Table 1 Genotypes of CYP2C9 and their impact on warfarin metabolism and dose requirement. Genotype

Warfarin metabolism

Warfarin dose, %

*1/*1 *1/*2 *1/*3 *2/*2 *2/*3 *3/*3

Normal metabolism Intermediate metabolism Intermediate metabolism Slow metabolism Slow metabolism Slow metabolism

100 78 57 66 – 24

uals. Homozygosity for *2/*2 decreases the dose to 57%. The cSNP defining the CYP2C9*3 is a transversion at c.1075A)C that results in the amino acid substitution p.I359L. This amino acid substitution decreases enzymatic activity by 95%. Heterozygosity for CYP2C9*3 leads to a decrease in the S-warfarin dose to 66% of that required in homozygous *1/*1 individuals. Homozygosity for *3/*3 decreases the dose to 24% (33). CYP2C9*2 and *3 are found in all major ethnic groups, but with different allelic frequencies. Some functionally relevant rare CYP2C9 alleles, such as *5, *6 and *11, are found preferentially in African populations and are not detected in Asian populations (34). In retrospective studies, it has been shown that INR values above 3.0 are twice as frequent among CYP2C9*2 or CYP2C9*3 heterozygotes, and are more likely to occur in the first and second week after starting warfarin or acenocoumarol therapy (35, 36). Thus, the determination of CYP2C9 status and VKORC1*2 genotyping may be helpful for identifying patients at risk of being outside the target range during initial anticoagulation with acenocoumarol. Although observational associations of pharmacogenetic variants with dose and adverse drug events are of value, they do not indicate whether the testing is able to reduce the rate of adverse events experienced by patients taking warfarin, while maintaining the efficiency of the drug. Nevertheless, testing can be valuable in specific settings such as, for example, when patients require very low maintenance therapy, or when the INR achieved during the induction phase is unusually high.

Clopidogrel Clopidogrel (Figures 3, 4) is a prodrug that requires in vivo biotransformation; primarily by CYP3A4. The drug is a second generation inhibitor of platelet aggregation. After metabolic activation of the prodrug through the CYP enzyme system, its metabolites bind and irreversibly block the platelet P2Y12 ADP receptor. Clopidogrel has lower rates of side effects, including neutropenia, which was a major concern in first generation thienopyridine ticlopidines. Clopidogrel is administered orally and has rapid effects on platelet aggregation. Usually, high loading doses of 300–600 mg are used (37–42). Platelets play a major role in atherosclerotic complications in patients with ACS and in stent thrombosis (43, 44). Thus,

Figure 3 Chemical structure of clopidogrel.

inhibitors of platelet activation (aspirin/acetylsalicylic acid and thienopyridines) are currently used for acute and longterm prevention of thrombotic events in ACS wreviewed in (4)x (45–47). While aspirin irreversibly blocks cyclooxygenase-1, preventing thromboxane A2 production (48, 49), thienopyridines (including clopidogrel) inhibit the P2Y12 ADP receptor subtype (5, 6). ADP-mediated P2Y12 receptor signalling leads to calcium mobilization, platelet degranulation, thromboxane A2 generation and glycoprotein IIb/IIIa receptor activation which is important for cross-linking of platelets by fibrin. P2Y12 receptor triggering amplifies and stabilizes platelet aggregation (5, 50–52).

Genes involved in clopidogrel function – CYP2C19 (OMIM 124020) Today, dual therapy with aspirin and clopidogrel is used in many clinical situations, and the benefits of dual therapy have been demonstrated in several large clinical trials wreviewed in (4)x. However, ‘‘resistance’’ to clopidogrel treatment, such as substantial inter-individual variability in the platelet inhibitory response has been frequently reported. This can manifest as a recurrence of cardiovascular events (10, 53–56). Recently, the underlying mechanism for the observed inter-individual variability in the response to clopidogrel treatment was suggested to involve hepatic CYP enzymes (57–61). On platelets, the active thiol acid metabolite of clopidogrel causes a breakup of lipid rafts containing oligomeric disulfide bound P2Y12 receptors. Via a disulfide exchange process with the thiol acid, non-functional P2Y12 dimers and monomers are then localized outside the rafts blocking P2Y12 receptor function irreversibly for the remaining lifespan of the platelet (approx. 7–10 days) (62–65). One of the hepatic CYPs supposedly involved in the biotransformation of clopidogrel is the enzyme CYP2C19. This enzyme appears to be particularly important in mediating clopidogrel resistance. CYP2C19 is a polymorphic enzyme with genetic variants that encode for proteins with reduced

Figure 4 Chemical structure of the active metabolite of clopidogrel.


to absent enzyme activity (poor metabolizer), or variants that are associated with increased transcription and high protein concentrations and enzymatic activity (66). Recent studies suggest that CYP2C19 loss of function mutation carriers (mainly CYP2C19*2) that receive clopidogrel are at increased risk for recurrence of cardiovascular events (7–17) depending on the ‘‘gene-dose’’. Patients carrying enzyme defective variants of CYP2C19 are 1.5–3.5 times more likely to die or have cardiovascular complications compared with patients with alleles coding for the fully functional enzyme (8, 12, 67). In carriers with the CYP2C19*17 variant, increased transcriptional activity with enhanced metabolism of CYP2C19 substrates has been observed. In these patients, the effect of clopidogrel on platelet aggregation was stronger than in wild-type patients (17). The mechanism of how CYP2C19 loss of function alleles are associated with clopidogrel ‘‘resistance’’ is still being debated, and the involvement of CYP2C19 in clopidogrel bioactivation is controversial. Some authors claim CYP3A4, not CYP2C19, is responsible for generation of the active thiol metabolite of clopidogrel, and that CYP2C19 loss of function alleles are instead associated with increased sensitivity to the aggregating effects of ADP. According to these authors, the reduced response to clopidogrel in the presence of CYP2C19 gene variants might come from alterations in the P2Y12 receptor that need to be identified wreviewed in (68) and (69, 70)x. Indeed, polymorphisms in the platelet P2Y12 receptor, GP IIb/IIIa, GP Ia, and protease-activated receptor-1 have been described previously. However, there is no clear evidence for their involvement in clopidogrel ‘‘resistance’’ (71–78). Interestingly, the platelet response to prasugrel, a third-generation thienopyridine and irreversible inhibitor of the P2Y12 receptor, is not influenced by CYP2C19 genotypes (7, 16, 79–84). Compared to clopidogrel, prasugrel is more effectively converted to its active metabolite and has a more rapid onset of action, but is associated with a higher frequency of bleeding complications (85–90).

Analytical methodologies Currently, a number of analytical tests for the identification of alleles of interest are commercially available. Most are based on anticoagulated blood as specimen, and a number of these tests are certified for in vitro diagnostic use. For these tests, the DNA extraction method is usually not included in the validation. Therefore, attention needs to be paid to the suitability of the extraction methods and the DNA concentration needed for accurate testing. Laboratories should follow standardized procedures to guarantee analytical performance of the tests. In addition, laboratories should participate in proficiency testing programs for pharmacogenetics.

Summary Since the first report on warfarin pharmacogenetics in 1999, there now is consent that genetic variants represent an impor-

tant predictor of warfarin maintenance dose before initiation of therapy. Only a few small studies relate the use of pharmacogenetics-guided dosing to initiation of warfarin therapy. At present, no significant improvements in outcomes with respect to safety or efficacy of therapy have been shown. Much of the information on warfarin sensitivity due to genetic variants is reflected by early INR values, traditionally used to guide titration of the dose. Even though the incorporation of genotype information improves the accuracy of dose prediction, the benefits for anticoagulation control or reduction in hemorrhagic complications has not been demonstrated convincingly. The inclusion of early INRs in prediction models reduces the contribution of genetics. Moreover, in large population cohorts, genetics explained only 20%–30% of variance in warfarin dosing. Finally, even pharmacogenetic prediction models did not predict doses reliably in at-risk patients who have warfarin requirements at the low or high end of the dose range. However, one needs to recognize that pharmacogenetic tests will be performed. Therefore, certain recommendations of expert consensus from the working group about modification of practice should be considered (33): • use for orthopaedic patients: test results could be provided before dosing • for other implications: genotyping results should be available within 3 days to allow for dosing adjustment before stabilisation of the INR • there is no need for VKORC1 testing once stable dosing has been achieved • confirmatory testing is not necessary except when a laboratory error is suspected It is known that platelet response to antithrombotic therapy can be influenced by several factors, and some patients may experience failure of platelet inhibition while others may suffer from bleeding complications. It should be acknowledged that genetic variations in enzymes involved in the bioactivation/metabolism of antithrombotic drugs explain some variability in the response to antiplatelet therapy. Knowledge of the individual patient’s genotypes may help decide on a specific therapeutic regime. Additionally, platelet function tests, able to determine platelet inhibition by antiplatelet drugs, may support adjustment of antiplatelet therapy if required. Many studies reported that genetic factors contribute to the phenotype of drug response, but only partial information is currently available on how the interplay between genetics and environment affects the drug response. Therefore, the contribution of genetic factors to drug response phenotype, and the translation of pharmacogenetic outcomes into drug discovery, drug development or clinical practice has proven to be disappointing. There are several gaps that limit the application of pharmacogenetics. Pharmacogenetic tests frequently do not adequately consider the multigenic nature of phenotypes of drug disposition and response. Also, the selection of gene variants has sometimes not been optimal. Effects on the regulation of protein expression including transcriptional/posttranscriptional/pretranslational processes that


impact the quantitative level of expression, the conversion of the mRNA blueprint into protein, and posttranslational/ degradative processes are usually not considered. In addition, it seems to be necessary to combine pharmacogenomics with nutrigenomics, epigenetics, and systems biology to move from disease treatment to disease prevention and optimal health. An integrative understanding, including the correlation between structure and function, genotype and phenotype, and systematic interactions among various proteins, nutrients, drugs, and the environment is needed. In addition, one must not forget that economic, commercial, political and educational barriers, among others, must be overcome in order for clinically useful information to be effectively communicated to practitioners and patients (91, 92). In general, better clinical evidence on the beneficial effects on patient outcome, particularly at the extremes of the dose requirements in geographically and ethnically diverse patient populations, has to be collected before the role of a pharmacogenetic approach in general, and also to oral anticoagulation therapy in particular, can be established in clinical practice. Future research will have to focus on accurately defining the possible differences, especially for predicting bleeding events in general and intracranial hemorrhages in particular. The large prospective trials currently under way in various settings will provide important data to evaluate the clinical utility and cost-effectiveness of pharmacogenetic-based dosing.


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