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Clopidogrel and Aspirin in Cardiovascular Medicine: Responders or Not -- Current Best Available Evidence C.E. Tourmousoglou* and C.K. Rokkas Department of Cardiothoracic Surgery, University of Athens School of Medicine, Attikon Hospital, Athens, Greece Abstract: Dual antiplatelet therapy represents an important advance for patients with established coronary artery disease. It is an important strategy for patients with acute coronary syndromes and those undergoing percutaneous transcatheter coronary interventions. Clopidogrel effectively inhibits ADP-induced platelet activation and aggregation by selectively and irreversibly blocking the P2Y12 receptor on the platelet membrane. Aspirin works by irreversibly acetylating the cyclooxygenase (COX-1) enzyme, thus suppressing the production of thromboxane A2 (TxA2) and inhibiting platelet activation and aggregation. Variable platelet response and potential resistance to therapy has emerged with aspirin and clopidogrel. The definitions of antiplatelet agents variability in responsiveness and nonresponsiveness are discussed. Clopidogrel and aspirin responsiveness as they are measured in the laboratory by various techniques (platelet aggregometry and point-of-care assays such as platelet function analyzer [PFA-100] and rapid platelet function assay [RPFA]) are evaluated. The mechanisms responsible for variations in responsiveness to antiplatelet agents such as clinical, cellular and genetic factors are defined. Aspirin and clopidogrel resistance are emerging clinical entities with potentially severe consequences such as myocardial infarction, stroke or death. The therapeutic interventions to deal with nonresponsiveness are reported, although specific recommendations are not clearly established. In the future, routine measurement of platelet function in patients with cardiovascular disease may become the standard of care. Personalized antithrombotic treatment strategies may be determined by ex-vivo measurements that identify critical pathways influencing thrombotic risk in the individual patient.
Key Words: Clopidogrel, aspirin, responders, nonresponsiveness, antiplatelet therapy. INTRODUCTION Activation and aggregation of platelets play a central role in the propagation of intracoronary thrombi after disruption of atherosclerotic plaque and results in myocardial ischemia or infarction [1]. At the site of vascular lesion, the initial step in thrombus formation is the adhesion of platelets to the exposed matrix. Then, platelets adhere to exposed subendothelium through interactions with a variety of platelet surface receptors. One of these receptors is the glycoprotein (GP) Ib-IX which is the main receptor for von Willebrand factor which is a subendothelial protein ligand and circulates in plasma. During their contact with collagen, platelets are activated via GP VI and 21 integrin and release thromboxane A2 and adenosine diphosphate, which result in stimulation and recruitment of additional platelets. This event is accompanied by clustering and activation of the glycoprotein GP IIb/IIIa receptor. Additional platelets are recruited via fibrinogen bridges between GP IIb/IIIa (IIb3) receptors. Tissue factor, which is locally concentrated, initiates the clotting cascade and leads to coagulation, the generation of more thrombi and the propagation of a fibrin clot [13]. Dual antiplatelet therapy represents an important advance for patients with established cardiovascular disease. There are a lot of clinical trials that have documented that aspirin is efficient in primary and secondary prevention of myocardial infarction, cardiovascular death and stroke [4, 5]. The ISIS-2 (The Second International Study of Infarct Survival) trial showed that the acute use of aspirin reduced mortality by 23% in acute ST-elevation myocardial infarction [6]. The Antithrombotic Trialists’ Collaboration found that stroke, myocardial infarction and cardiovascular death was reduced by 25% [5]. Aspirin had an additive benefit if used in combination with streptokinase for thrombolytic therapy. Clopidogrel is a second generation thienopyridine with a better tolerability profile than first generation thienopyridines (ticlopi*Address correspondence to this author at the Department of Cardiothoracic Surgery, University of Athens School of Medicine, Attikon Hospital, 1 Rimini St, 12462, Haidari, Athens, Greece; E-mail:
[email protected]
1871-5257/08 $55.00+.00
dine). In CAPRIE (Clopidogrel Versus Aspirin in Patients at Risk of Ischemic Events) trial, clopidogrel alone was modestly superior to aspirin alone in the prevention of stroke, MI and vascular death [7]. But more important benefits are demonstrated with the combination of clopidogrel and aspirin for the treatment of acute coronary syndromes especially in CURE (Clopidogrel in Unstable Angina to Prevent Recurrent Events) trial, in PCI-CURE (Percutaneous Coronary Interventions-CURE) trial, in CREDO (Clopidogrel for the Reduction of Events During Observation) trial [810], in CLARITY-TIMI 28 (Clopidogrel as Adjunctive Reperfusion Therapy-Thrombolysis in Myocardial Infarction 28) trial and in COMMIT-CCS 2 (Clopidogrel and Metoprolol in Myocardial Infarction Trial Second Chinese Cardiac Study) trial [11, 12]. Despite the obvious benefits with dual antiplatelet therapy, a considerable number of patients continue to have cardiovascular events. This paper reviews clopidogrel and aspirin responses, mechanisms of action of both drugs, definitions of nonresponsiveness or resistance, laboratory measurements, mechanisms of resistance, therapeutic interventions of the nonresponsiveness and future directions. We searched EMBASE, MEDLINE and the Cochrane Library to November 2007.We used the following search terms: clopidogrel, aspirin, acetylsalicylic acid, responders, not responders, resistance, variability in responsiveness. After reviewing the abstracts, we obtained and reviewed the full texts of relevant articles and their reference lists. MECHANISMS OF ACTION Clopidogrel Clopidogrel inhibits ADP-induced platelet aggregation as well as collagen and thrombin-induced aggregation. Clopidogrel indirectly inhibits the effect of these agonists via the attenuation of ADP-mediated amplification of the platelet response [13]. It is rapidly absorbed from the intestine and converted by hepatic cytochrome P450 isoenzymes (CYP3A4, CYP3A5, 2C19) to an active thiol metabolite [14]. This active metabolite binds to the P2Y12 receptor via a disulfide bridge between the reactive thiol © 2008 Bentham Science Publishers Ltd.
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group and two cysteine residues (cys17 and cys270) present in the extracellular domains of the P2Y12 receptor. The binding of ADP to the P2Y12 is permanently inhibited. Clopidogrel is a pro-drug administered orally. Approximately 85% of the pro-drug is hydrolyzed by esterases in the blood to an inactive carboxylic acid derivative and only 15% of the pro-drug is metabolized by the cytochrome P450 in the liver to an active metabolite. Clopidogrel also attenuates the formation of plateletleukocyte aggregates, the levels of CPR, p-selectin and CD 40L as well as the rate of thrombin formation [15]. Aspirin Aspirin reduces the activation of platelets by irreversibly acetylating cyclooxygenase-1 (COX-1) at serine residue at position 529. The COX-1 enzyme catalyses the conversion of arachidonic acid (AA) to prostaglandins G1/G2 which are converted by thromboxane synthase to thromboxane A2 (TXA2). TXA2 is a potent vasoconstrictor that activates platelet aggregation, through specific binding to the TXA2 receptor. TXA2 also activates mitogenesis, hypertrophy, proliferation of vascular smooth muscle cells and regulates the constriction of vascular and bronchiolar smooth muscle [16, 17]. The inhibition of COX-1 is rapid, saturable at low doses, irreversible and permanent for the life of the platelet because platelets lack the biosynthetic machinery to synthesize new protein. DEFINITIONS OF NONRESPONSIVENESS OR RESISTANCE Medical reports have used terms such as “variability of response”, “nonresponse”, “hyporesponse”, “adequate response”, “poor response”, or “resistance”. These terms have been applied to different results from different analyses of platelet function with varying cutoffs as well as the prevention of ischemic cardiovascular events [18]. This inconsistent nomenclature leads to confusion regarding variability of response or resistance and its clinical importance. Gubrel et al. [19] and Michelson et al. [20] observed that resistance to a specific agent is best defined by the persistent activity of the agent’s target despite treatment with the specific antiplatelet agent. This definition implies that patients are receiving sufficient doses of the drug to produce optimal levels for inhibition of the target. But there are multiple pathways of platelet activation and factors other than platelets during the process of thrombosis so the occurrence of clinical events during treatment with a specific antiplatelet agent should not be regarded as “resistance” to the therapeutic agent. Earlier studies have defined clopidogrel responsiveness according to the absolute differences between pre- and post-treatment platelet reactivity [21]. Muller et al. [22] defined clopidogrel nonresponsiveness as an inhibition of ADP (5 and 20 mMol/L)induced platelet aggregation that was less than 10% of baseline values at 4 hours after clopidogrel intake (600 mg single dose), semiresponse as an inhibition of 10% to 29% and normal response as an inhibition greater than 30%. Thus, the authors defined clopidogrel responsiveness according to the degree of inhibition of platelet aggregation obtained at baseline and after treatment. These studies have shown that clopidogrel-induced inhibition of platelet aggregation is highly variable and that a considerable number of patients may have poor or no antiplatelet effects. There are also other investigations that have empirically used different doses of agonists, different cut-off values and different assays to define clopidogrel-induced antiplatelet effects resulting in a highly variable prevalence of poor clopidogrel responders [23, 24]. The term “aspirin resistance” has been used in a clinical and laboratory context. “Clinical aspirin resistance” refers to the inability of aspirin to protect individuals from cardiovascular thrombotic events such as myocardial infarctions (MIs). However, this problem is more accurately referred to as “aspirin treatment failure”.
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Furthermore, many cardiovascular events that occur to patients treated with aspirin may not be prevented by aspirin. But it must be emphasized that “clinical aspirin resistance” is discovered retrospectively when an ischemic event first occurs. The term “laboratory aspirin resistance” refers to the lack of an anticipated effect of aspirin on a laboratory measure of its antiplatelet effect that is associated with a higher rate of future cardiovascular events [25, 26]. It is obvious that clinical aspirin resistance can be diagnosed before a cardiac event happens, making it possible that the patient receives more effective antiplatelet treatment. The issue of early identification of aspirin-resistant individuals is very important as “laboratory aspirin resistance” predicts cardiovascular events [27, 28]. The reported incidence of aspirin resistance ranges from 5% to 61% [29, 30], depending on the population studied and the laboratory test used [31-33]. There are many conflicting reports on the incidence and clinical relevance of aspirin resistance because this term refers to different criteria. These are: the inability of aspirin a) to protect patients from clinical arterial thrombotic complications, b) to cause a prolongation of bleeding time and c) to inhibit platelet TX biosynthesis [34, 35]. Aspirin resistance is a misleading term because in some situations aspirin successfully inhibits TX synthesis even though platelet aggregation persists. The term aspirin “nonresponce” encompasses the failure of aspirin to inhibit TX synthesis and reduce platelet aggregation [27]. Weber et al. [35] described three distinct groups using biochemical tests and functional in vitro studies. Type 1 (pharmacokinetic type) is the inhibition of platelet TX formation in vitro but not in vivo. Type 2 (pharmacodynamic type) is characterized by the inability of aspirin to inhibit platelet TX formation in vivo and in vitro. Type 3 (pseudoresistance) involves TX-independent platelet activation. This classification is limited to collagen induced aggregometry and sensitive to limitations of this method. It is important to mention that the definition of aspirin resistance based on inhibition of TXA 2 production considers only the pharmacologic action of aspirin and not platelet aggregation. LABORATORY DIAGNOSIS OF CLOPIDOGREL AND ASPIRIN RESISTANCE The prevalence of individuals with an inadequate response to clopidogrel varies between 4% and 34%, depending on the method of testing and the definition of “resistance” or “hyporesponsiveness” used in the individual analyses. Clopidogrel specifically inhibits one of the two ADP receptors so the most common laboratory method that is used for evaluation of clopidogrel responsiveness is ex vivo measurement of ADPinduced aggregation by light transmittance. Maximum platelet aggregation is calculated in platelet-rich plasma before and after treatment with clopidogrel and is considered the gold standard [36]. Patients with an absolute difference in aggregation of less than 10% are classified as nonresponders, those with a 10-20% difference are semiresponders and those with a greater than 30% difference are responders. The measurement of late platelet aggregation at 6 min after stimulation with ADP rather than maximum aggregation has been suggested to be a better measure of clopidogrel response. There is no clear rationale for the choice of 6 min as the cut-off time because the relation between any parameter and in vivo condition is unknown [37]. The most widely reported method in clinical trials and in basic research is maximum aggregation [3840]. Whole blood thromboelastography and the VeryfyNow P2Y12 receptor assay, using ADP as the agonist, measure clopidogrel responsiveness as point-of-care assays [38, 41]. In addition, there are flow cytometric determination of P-selectin and expression of activated IIb3 receptor following ADP stimulation, to assess platelet inhibition by clopidogrel [42, 43]. Flow cytometry also is used for measuring the phosphorylation state of vasodilatorstimulated phosphoprotein. Hopefully, this technique will be the
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most specific indicator of residual P2Y 12 activity in patients treated with a P2Y12 inhibitor [44, 45]. The estimates of the prevalence of laboratory aspirin resistance varies between 5.5% and 61%. Aspirin resistance is diagnosed by measuring platelet activation with methods either reflecting in vivo or ex vivo platelet activity. In-vivo tests include the measurement of urinary thromboxane B 2 , the expression of P-Selectin and soluble P-Selectin. Urinary 11-dehydro thromboxane B 2 concentrations provide a measure of in-vivo thromboxane production from both platelet and extra platelet sources but, if it is used alone, it cannot distinguish the source of thromboxane. This is a simple and inexpensive test. The test on expression of P-Selectin is expensive and requires controlled conditions. The selectins are adhesion proteins that are expressed on blood cells [46]. P-Selectin moves to the plasma membrane when platelets are activated [47]. A third in vivo test is the measurement of plasma soluble P-Selectin. Increased platelet activation is indicated by increased levels of this protein. Samples are stable for many months making them suitable for large epidemiological studies [48]. Ex vivo tests include optical platelet aggregation and the PFA100 (platelet function analyser). Optical platelet aggregation measures optical changes in plasma caused by platelet aggregation, induced by ADP or collagen. Although this is a widely used method, it does not correlate well with other indicators of platelet function, such as in vivo platelet activity, it is not specific and the results depend on the operator and the interpreter [49, 50]. The PFA-100 system has been regarded as an in-vitro bleeding time recorder [51-53]. The PFA-100 uses whole blood and the combination with erythrocytes and high shear rate may be more clinically relevant than conditions of platelet aggregometry. PFA100 is an expensive test and must be performed within 4 hours after blood collection. The Ultegra RPFA-ASA (rapid platelet function assay) is a rapid bedside test which measures agglutination of fibrinogencoated beads in response to propyl gallate or arachidonic acid stimulation. If aspirin produces the expected antiplatelet effect, fibrinogen-coated beads will not agglutinate and light transmission will not increase. The result is expressed as aspirin reaction units [54]. Point-of-care assays (PFA-100, RPFA) have important advantages compared with platelet aggregometry because they are simple and provide rapid results. But there are limitations, too. Gum et al. [28, 55] evaluated a cohort of patients with stable cardiovascular disease and showed that there was no relation between aspirin resistance measured by PFA-100 and subsequent adverse cardioTable 1.
Tourmousoglou and Rokkas
vascular events in contrast to an increased risk of MI, stroke or death among patients who were initially found to be resistant to aspirin by platelet aggregometry. The lack of such a relation might be due to interference by von Willebrand factor. It is clear that in-vitro tests of platelet function (PFA-100, RPFA, turbidimetric aggregation) for diagnosing aspirin resistance measure the effect of aspirin resistance in a non-physiologic setting. The fact that production of thromboxane from extra-platelet sources can activate platelets in-vivo is not taken into account. Urinary 11dehydro thromboxane B2 concentrations reflect in-vivo thromboxane production from both platelet and extra-platelet sources. Besides, the levels of urinary thromboxane B2 is affected by the dose of aspirin [56, 57]. It is known that higher doses of aspirin result in greater inhibition of COX-2 and lower concentrations of urinary thromboxane B2 but this finding is at odds with evidence from clinical trials that aspirin is effective in preventing cardiovascular events independent of its dose [57]. Specific limitations of optical transmission and impedance aggregometry are the following: a) the results vary by age, sex, race, diet, haematocrit, b) the reproducibility and accuracy of these techniques are poor, c) the tests are poorly standardized so the results are not comparable, d) the tests do not take into account interactions between cells that arise in vivo so platelet aggregometry is not ideal for testing platelet sensitivity to aspirin as it does not reproduce the high shear conditions that occur in vivo and the aggregation response is not exclusively modulated by thromboxane A2 [58]. The limitations of the Ultegra RPFA is that its result for aspirin resistance is based on the comparison with optical platelet aggregation [59]. MECHANISMS OF CLOPIDOGREL NONRESPONSIVENESS
AND
ASPIRIN
Clopidogrel Multiple factors contribute to variability in clopidogrel responsiveness including interindividual variability in the metabolism of clopidogrel, drug-drug interactions and platelet receptor polymorphisms (Table 1). A mechanism responsible for variability in clopidogrel responsiveness deals with the polymorphisms of the platelet receptor P2Y12. P2Y12 is the target of clopidogrel but the effect of ADP on platelets is mediated by two P2Y receptors and simultaneous activation of both receptors is necessary for optimal aggregation. Fontana et al. [60] found that there is a sequence of variations in the P2Y12 that might explain the interindividual variability of platelet aggregation in response to ADP. Analysis of 98 unrelated male subjects, with platelet aggregation studies, cAMP measurements, genotyping, platelet RNA extraction and reverse transcription, revealed two haplotypes designated H1, comprising 86% of the
Mechanisms Responsible for Individual Responsiveness to Clopidogrel
Genetic Factors
Cellular Factors
Clinical Factors
Polymorphisms of CYP
Accelerated platelet turnover
Failure to prescribe, Poor Compliance
Polymorphisms of GP Ia
Reduced CYP3A metabolic activity
Under- dosing
Polymorphisms of P2Y12
Increased ADP exposure
Poor absorption
Polymorphisms of GP IIIa
UP-regulation of the P2Y12 pathway
Drug-drug interactions involving CYP3A4
UP-regulation of the P2Y1 pathway
Acute coronary syndrome
UP-regulation of the P2Y-independent pathways (collagen, epinephrine, thromboxane A2, thrombin)
Diabetes Mellitus/Insulin resistance
Elevated body mass index
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subjects, and H2, comprising 14% of the subjects. The H2 haplotype was associated with higher maximal aggregation suggesting that clopidogrel may offer less antiplatelet protection in carriers of the H2 allele. Hyperaggregation was not due to specific amino acid substitutions or splice variants but caused by differences in the regulation of cAMP levels. Possibly, an elevated receptor might be associated with platelet responsiveness to ADP. H2 haplotype may be linked to a sequence variation in the promoter region which could increase transcription efficiency. A second source for variability in clopidogrel response is the interaction between clopidogrel and HMG CoA reductase inhibitors (statins). Lipophilic statins (atorvastatin, lovastatin and simvastatin) and clopidogrel require both CYP 3A4 metabolization so the antiplatelet effect of clopidogrel may be decreased by administration of statins. Lau was the first [61] to describe the interaction between clopidogrel and atorvastatin. Neubauer et al. [62], showed the attenuation of the antiplatelet effect of clopidogrel by atorvastatin or simvastatin. However other studies do not support this interaction. Muller et al. [63] studied patients with elective stent placement and demonstrated that there was no attenuation of clopidogrel effect by statin therapy. The results of the MITRA PLUS (Maximal Individual Therapy for Acute Myocardial Infarction) study and the post hoc data from the CREDO trial failed to demonstrate differences in clinical outcomes between patients receiving atorvastatin and those receiving other statins in addition to clopidogrel [64]. Saw et al. [65] demonstrated that there was no drug-drug interaction between clopidogrel and atorvastatin in clinical outcomes because patients treated with and without atorvastatin had the same clinical benefit from clopidogrel. At present, there is no conclusive evidence that statins may interfere with clopidogrel [66]. There is also the interindividual differences in CYP 3A4 metabolic activity. Lau et al. [67] compared patients undergoing elective coronary artery stent implantation with healthy volunteers and demonstrated a significant inverse correlation between CYP 3A4 activity levels and platelet aggregation after clopidogrel. Inhibition of platelet aggregation by clopidogrel was enhanced by rifampin, a CYP 3A4 inducer indicating that agents that induce expression of CYP 3A4 activity decrease clopidogrel resistance. The influence of CYP 3A5 and CYP 2C19 isoenzymes on clopidogrel activation has been demonstrated in a number of studies. Suh et al. [68] showed that clopidogrel responsiveness among subjects with the CYP 3A5 expressor genotype was higher than in subjects without this genotype. Worse outcomes were found in patients with the non-expressor genotype, who underwent stent implantation following treatment with clopidogrel in contrast to patients with the CYP3A5 expressor genotype. The IVS10+12G>A polymorphism of the CYP3A4 gene may be a factor responsible for the variability of clopidogrel response as shown by Angiolillo et al. [69]. The same authors demonstrated in previous studies that there was no association between SNPs of the GP IIIa and P2Y12 receptors and platelet reactivity in patients on chronic clopidogrel therapy [70]. Patients with diabetes mellitus have increased platelet responsiveness to agonists [71, 72]. These data support the concept that insufficient metabolism of clopidogrel is the cause for nonresponsiveness to clopidogrel rather than genetic polymorphisms of platelet receptors or intracellular signaling mechanisms. Aspirin There are many reasons why aspirin might not suppress the production of thromboxane A2 and platelet aggregation resulting in laboratory aspirin resistance (Table 2). In addition there are many other reasons not involving platelet TXA2 production that cause clinical aspirin resistance(Table 3).
Table 2.
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Laboratory Aspirin Resistance: Mechanisms of Reduced Suppression of Thromboxane Production [54]
Impaired suppression of platelet cyclooxygenase-1 Inadequate dose of aspirin Drug interactions (concurrent intake of NSAIDs, preventing access of aspirin to the COX-1 binding site) Increased turnover of platelets (Immature platelets unexposed to aspirin during the 24-h dose interval). Genetic polymorphisms (polymorphisms of COX-1) Bypass of aspirin’s inhibition of platelet cyclooxygenase-1 (aspirin bypass) Alternative sources of thromboxane production (non-platelet sources of thromboxane biosynthesis (eg. monocyte COX-2) Increased turnover of platelets (immature platelets containing measurable levels of COX-2) Genetic polymorphisms (polymorphisms of COX-2)
A classification system of laboratory aspirin resistance was proposed by Weber et al. [73]. Pharmacokinetic resistance (in-vitro but not oral aspirin completely blocks collagen-induced platelet aggregation and thromboxane formation) is different from pharmacodynamic resistance (neither in-vitro nor oral aspirin completely blocks collagen induced aggregation and thromboxane formation) and is different from pseudo-resistance (low dose collagen that induces platelet aggregation despite complete block of thromboxane production). Future work should clarify how valid these definitions are. In 40% of patients with cardiovascular disease the poor compliance of patients with aspirin is responsible for its laboratory and clinical treatment failure [74-77]. In the Physicians Health Study it was found that poorly compliant patients derived less benefit than compliant patients (17% versus 51% reduction in myocardial infarction relative to placebo) [78]. In a study by Ferrari et al. aspirin withdrawal for any reason was associated with hospitalization for an acute coronary syndrome and specifically late stent thrombosis [79]. The reduced enteral absorption of aspirin is another factor contributing to aspirin resistance. Aspirin is absorbed in the stomach and upper intestine with peak blood concentrations achieved in 30 to 40 min after ingestion of soluble aspirin and in 3 to 4 h with enteric –coated formulations [80]. Then, soluble aspirin is rapidly inactivated in the liver and gut and excreted mainly in urine. Platelet exposure to aspirin and COX inhibition occur initially in the portal circulation and complete platelet inhibition has occurred before aspirin enters the systemic circulation. Doses of soluble aspirin as low as 40 mg fully inhibit platelet aggregation in healthy volunteers. Low-dose enteric-coated aspirin does not suppress platelet aggregation completely [81, 82], but there are studies that showed opposite results [83]. Low-dose enteric-coated aspirin preparations are increasingly prescribed to reduce gastrointestinal side effects. Soluble aspirin enters acidic environment of the stomach where it is protected from deacetylation, remains nonionized, lipid-soluble and is rapidly absorbed. But enteric-coated formulations deliver aspirin into the neutral pH of the small intestine where absorption is delayed and bioavailability is reduced [81, 82]. The recommended doses of aspirin are generally based on population studies and not on individual dose-response analysis. The dose-dependent variability has been determined with different biochemical assays. A study by Quinn et al. [84] suggested that response to low dose aspirin varies with anatomic distribution of atherothrombosis. There are numerous epidemiological data and
316 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2008, Vol. 6, No. 4 Table 3.
Aspirin Failure (Possible Causes) [54]
Reduced biovailability of aspirin Inadequate intake of aspirin (poor compliance), Inadequate dose of aspirin, Reduced absorption or increased metabolism of aspirin Altered binding to COX-1 Concurrent intake of certain non-steroidal anti-inflammatory drugs possibly preventing the access of aspirin to COX-1 binding site Other sources of thromboxane production Biosynthesis of thromboxane by pathways that are not blocked by aspirin (e.g. by COX-2 in monocytes and macrophages and vascular endothelial cells) Alternative pathways of platelet activation Platelet activation by pathways that are not blocked by aspirin (e.g. red-cell induced platelet activation, stimulation of collagen, adenosine disphosphate, epinephrine and thrombin receptors on platelets Increased platelet sensitivity to collagen and ADP Increased turnover of platelets Increased production of platelets by the bone marrow in response to stress (e.g. after coronary artery bypass surgery) introducing into bloodstream newly formed platelets unexposed to aspirin during the 24-h dose interval (aspirin is given once daily and has only a 20-min half-life) Genetic polymorphisms Polymorphisms of COX-1, COX-2, thromboxane A2 –synthase or other arachidonate metabolism enzymes Polymorphisms involving platelet glycoprotein Ia/IIa,Ib/V/IX and IIb/IIIa receptors and collagen and von Willebrand factor receptors Factor XIII Val34Leu polymorphism, leading to variable inhibition of factor XIII activation by low dose aspirin Loss of the antiplatelet effect of aspirin with prolonged administration Tachyphylaxis Non - atherothrombotic causes of vascular events Embolism from the heart (red, fibrin thrombi, vegetations, calcium, tumour, prostheses) Arteritis
randomized controlled trials indicating that low-dose aspirin (75 to 150 mg/day) equally is effective as higher doses in preventing cardiovascular disease [85]. Low-dose aspirin suppresses platelet COX-1 in healthy controls and in patients recovering from myocardial infarction [86, 87]. But studies by Patrono et al. [13], Serebruany et al. [88] and the Antithrombotic Trialists Collaboration [5] showed that the estimates of effectiveness of each dose were not precise (the 95% CI were reasonably wide). The long term treatment with aspirin (for months or years) results in loss of suppression of agonist-induced platelet aggregation, a process called tachyphylaxis [89, 90]. This finding might explain the increase in adverse cardiovascular events in prior aspirin users [91, 92]. The exact mechanism is not known but might be explained by progression of atherosclerosis or progressive reduction in compliance over time. Concomitant intake of non-steroidal anti-inflammatory drugs (indomethacin, ibuprofen, naproxen or others) prevents the access of aspirin to the COX-1 substrate binding site and reduces the antiplatelet action of aspirin. Inhibitors of COX-1 such as ibuprofen and naproxen share a common docking site with aspirin and prevent acetylation of aspirin’s target serine residue within the hydrophobic
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pocket of the enzyme [93]. A study by Gislason et al. [94] pointed out that the use of high-dose nonselective nonsteroidal antiinflammatory drugs by patients who took aspirin for secondary prevention was linked to adverse cardiovascular events. However, other studies showed conflicting results [95]. Another possible cause for treatment failure is the fact that there are other sources of thromboxane A2 production except platelets. Monocytes and macrophages produce thromboxane A2 as arachidonic acid is converted to the above substrate in a reaction catalysed by COX-2 and thromboxane synthase. The association between conventional risk factors for cardiovascular disease and enhanced platelet activation might be explained by the following mechanism: the increased biosynthesis of F2 isoprostanes (prostaglandin F2 –like compounds) is associated with aspirin-insensitive thromboxane synthesis. Isoprostane production is augmented by smoking, diabetes, hyperlipidemia, unstable angina and is resistant to the effect of aspirin on platelet activation and altered response of platelets to other agonists [96-98]. These causes may be responsible for the enhanced platelet activation and the risk factors for cardiovascular disease [99-101]. During coronary artery bypass surgery and inflammation there is increased platelet turnover leading to increased proportion of platelets that have not made contact with aspirin and have impaired suppression of COX-1 [102]. Regeneration of COX-1 and COX-2 is associated with enhanced platelet turnover and may overcome the inhibitory response to aspirin [103]. Dennis et al. [104] showed that platelets have the ability to splice endogenous pre-mRNA in response to external signals so they can translate mature mRNAs into biologically active proteins and regenerate COX-1 de novo in response to cellular activation. Evangelista et al. [105] found that TXA2 biosynthesis in response to thrombin and fibrinogen recovered and was stopped by translational inhibitors such as rapamycin. This mechanism may in part explain the loss of platelet inhibition despite chronic aspirin therapy [106]. Even when COX-1 is inhibited, platelets may generate TXA2. Precursors of PGH2 generated by vascular tissue and metabolised by platelet TX synthase may bypass platelet COX inhibition. This transcellular process occurs at sites of atherothrombosis or via platelet-leukocyte aggregates. It is the local release of vascular TX or PG endoperoxides that activates the platelet TP receptor and react synergistically with platelet agonists and subthreshold levels of stronger agonists. [107]. There are single nucleotide polymorphisms in COX-1, COX-2 and other platelet genes that modify the antiplatelet effect of aspirin [108-111]. No conclusive data exist about the exact role of the hundreds of single nucleotide polymorphisms in genes in thromboxane biosynthetic pathways. Genetic variation in platelet glycoprotein receptors and in the ADP receptor gene P2Y1 might be responsible for aspirin resistance [111]. Lim et al. [112] evaluated the dose-related efficacy of aspirin after coronary surgery in patients with PIA2, C807T, A842/C50T polymorphisms and found that polymorphisms in these genes are common. The responses to aspirin were consistently but not significantly impaired in PIA2 and C807T carriers and consistently but not significantly better in C50T carriers. Finally, the response to aspirin in PIA2 polymorphism may be related to dose with significant improvement observed with medium rather than low-dose aspirin. Another possible cause of aspirin failure is the non-atherothromboembolic pathology. The pathologic basis of cardiovascular diseases is the result of many pathways involving inflammation, endothelial function, hemodynamic changes and coagulation cascade. Aspirin cannot be effective in preventing all ischemic events. It is known that only 50% of recurrent ischemic strokes are due to atherothrombotic disease of the large extracranial and intracranial arteries (20% are due to emboli from the heart, 25% to occlusion of the one of the small deep, perforating cerebral arteries
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by lipohyalinosis or microatheroma and 5-10% to vasculitis, infective endocarditis and arterial dissection) [113]. Finally, an interesting issue raised by Lev et al. [114] who evaluated the response to clopidogrel among aspirin-resistant versus aspirin-sensitive patients undergoing percutaneous coronary intervention. They identified a unique group of dual drug-resistant patients who did not achieve adequate antiplatelet effects from either aspirin or clopidogrel. These patients had relatively high incidence of creatine kinase MB (CK-MB) elevation after percutaneous coronary intervention (PCI) so they might be at high risk for thrombotic complications following coronary intervention. The lower response to clopidogrel among aspirin-resistant patients has particular clinical importance because clopidogrel has been suggested as alternative therapy for aspirin-resistant patients. The most likely mechanism that was suggested in their study was a general increase in platelet reactivity. CLINICAL IMPLICATIONS OF CLOPIDOGREL AND ASPIRIN NONRESPONSIVENESS Clopidogrel The concept of incomplete clopidogrel response is an important issue as multiple studies showed the clinical implications (Table 4) [115]. Clinical outcomes that were evaluated were: stent thrombosis, post-stent ischemic events and myocardial infarction. Different groups of patients were examined such as: patients undergoing percutaneous coronary interventions (PCI) electively or not, including patients with non-ST segment elevation (NSTE)- acute coronary syndromes and patients with ST segment elevation myocardial infarction (STEMI). These studies strongly demonstrate that a high platelet reactivity phenotype is a risk factor for ischemia in patients undergoing Table 4.
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PCI. They suggest that insufficient inhibition of P2Y12 receptors by clopidogrel therapy and high post-treatment platelet reactivity are related to the occurrence of stent thrombosis and recurrent ischemic events following PCI. Aspirin Several studies have evaluated the relation between aspirin therapy and platelet reactivity and the risk of vascular events (Table 5) [115]. Aspirin resistance increases the risk for vascular events. These studies had limitations. Adherence to treatment was not assessed by any study. There was heterogeneity in the test results and the samples of patients so conclusions could not be made. MANAGING AN INADEQUATE RESPONSE CLOPIDOGREL When an inadequate response to clopidogrel is examined, a lot of parameters must be evaluated. The compliance of patients with clopidogrel has to be examined. Lipids, glucose and blood pressure levels must be corrected as they might cause platelet dysfunction. A loading dose of 300-mg is considered the standard dose for those patients that are candidates for PCI. Angiolillo et al. [24] and Gurbel et al. [116] concluded that a loading dose of 600 mg leads to a stronger, earlier and more sustained inhibition of platelet function. The benefit of pre-treatment with a loading dose of 600 mg compared with 300 mg in reducing periprocedural myocardial infarction in patients undergoing PCI, without any increase in bleeding hazards, was shown in the ARMYDA–2 (Antiplatelet therapy for Reduction of Myocardial Damage during Angioplasty2) study [117]. Guisset et al. [118] replicated similar results. The issue of the loading dose of 600 mg is a matter of debate and the FDA has not recommended this dose yet. Current recommendations from the American College of Cardiology/American
Clinical Relevance of Clopidogrel Resistance
Study (Year)
Subject (No of patients)
Clopidogrel Dose (mean follow up period)
Clinical Outcome Associated With Clopidogrel Resistance (Assay Results)
Barragan (2003)
Stent thrombosis (
