... 82 / O | 234268$2.7 s / 0. On the Clot-Promoting Activity of Human Platelets in a ... by ADP or collagen can be explained by minor cell lysis accompanying platelet activation. ... had a clotting time of 178 s, Data are shown for one ... result of the action of some platelet agonists ... membrane, the negatively charged phospho-.
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Haemostasis ManagingEditor: H.C. Hemker,Maastricht
Publishers:S. Karger, Basel Reprint (Printed in Switzerland)
Haemostasis 12:268-27a(1982)
@1982S.lgrger AG. Basel 030| 4 | 47/ 82/ O| 234268$2.7s/ 0
On the Clot-PromotingActivity of Human Plateletsin a One-StageProthrombinaseAssay E.M. Bevers,P. Comfurius,H.C. Hemker, R.F.A. Zwaal Biomedical Center, University of Limburg, Department of Biochemistry, Maastricht, The Netherlands
Key Words. Platelets' Coagulation ' Prothrombinasefactor V . Phospholipids . Plateletfactor 3 (PF 3) Abstract.The procoagulantactivity of activated plateletsin a one-stageprothrombinase assayis reevaluated.It is shownthat the apparentprocoagulantactivity ofplatelets activated by ADP or collagencan be explainedby minor cell lysis accompanyingplatelet activation. The reductionin clotting time observedwith thrombin activatedplateletscan be explainedby a combinedeflect of minor cell lysis and releaseand activation of factor V from the platelets. Plateletsstimulatedby ionophore 423187 or by the combinedaction of collagenplus thrombin show a much shorter clotting time than can be accountedfor by minor platelet lysis or releaseand activation of factor V from the platelets. The resultswith this clotting assayessentiallyconfirm previousobservations[Beverset al.: Eur. J. Biochem.122:429436,19821using a spectrophotometricmethod with highly purifred coagulationfagtorsand a chromogenicsubstrateto measurethe rate of thrombin formation with activatedplatelets.
Introduction
cell lysis [3], and to releaseand activation of factor V from the platelets [4]. We have platelets The ability of activated to reduce recently shown, using a chromogenic subthe clotting time in coagulationassaysthat strate assaywith highly purified coagulation are sensitiveto procoagulantphospholipids factors,that plateletsstimulatedby the comhas been well established[-3]. However, bined action of collagen plus thrombin clotting assaysin the presenceofplatelets are stronglyincreasethe rate offactor X and proknown to be sensitiveto low percentagesof thrombin activation,as comparedto nonac-
Bevers/Comfurius/Hemker/Zwaal
270
0 ,i F o^Ez> F o c
+
o UU
25 o "/" tysis
50
75
100
Fig. 1. Clotting time of sonicated platelets as a function of percentagelysis produced by sonication. Clotting was measuredin the absenceof added factor Va (seetext). The longest clotting time shown (114 s) correspondsto 0.4o/oof lysis; non sonicated platelets had a clotting time of 178 s, Data are shown for one representativeplatelet preparation.
Results Figure I shows the relationship between the clotting time (in the absenceof excess added factor Va) and the amount of platelet lysis. Plateletlysis wasinducedby sonication of washed platelet suspensionsfor various time periodsat a minimal output of the sonifier. Lysis, measuredby the activity of lactate dehydrogenasein the supernatant,was expressedas a percentageof this enzyme activity in a completelylysed platelet preparation. Addition of intact platelets to the assaysystemhardly affectsthe clotting time (seealso table I) although this might differ from one plateletpreparationto another,depending on the amount of lysed platelets
alreadypresentin the nonsonicatedpreparation. Trace amountsof lysedplateletsinduce a significantdecreasein clotting time. A nonlinear relationshipbetweenclotting time and platelet lysis is obtained. The decreasein clotting time produced by 10/olysis in the plateletsis more than half of that observed with a completelylysed platelet preparation. A similar nonlinear relationship (with shorter clotting times) was obtained in the presenceof an excessof exogenouslyadded factor Va (comparetable I). The effect of a lO-min activation period with different agonistson the procoagulant activity of intact washedhuman plateletsis given in table I. Platelets contain factor V which can be releasedand activated as a result of the action of some platelet agonists factorVa is a cofactorin the [13-15]. Because prothrombin, this might interactivation of fere with the coagulation assay.Therefore, the assaywas carried out either in the absence or in the presence of exogenously addedexcessof factor Va. As a result of the addition of factor Va, the rate of thrombin formation will be increasedleading to a decreasein clotting time. Also, the blank clotting time with buffer instead of platelets is significantlyloweredin the presenceof added factor Va. Addition of nonstimulated platelets hardly affects the blank clotting time, either with or without addedfactor Va in the assay.Since minor amounts of platelet lysis strongly shorten the clotting time and some plateletlysis accompaniesplateletactivation, it will be clear that for an evaluation of the clot-promoting eflect of activated plateletsa control for the contribution of lysed cells in the assayis an essentialrequirement. When plateletsstimulatedby ADP or collagenare usedin the clotting assay(either in the presenceor absenceofexogenouslyadded
271
Clotting Activity of Platelets
Table I. Percentageoflysis in and clotting time ofsonicated and activated platelets Sonication time, or platelet activator (final concentration)
Buffer Platelets(not activated or sonicated) I s sonication 3 s sonication 6 s sonication l0 s sonication I 2 s sonication 3 min sonication
Lysis o/o
t., 5.1 I 1.9 t9.2 24.9 100
ADP (10 pM) Collagen(10 Uelml) Thrombin (2 nM) Collagen+ thrombin A^23187(3 W)
1.1 1.9 1.7 1.6 2.7
Clotting time, s absenceof excessfactor Va
presenceof excessfactor Va
r93
ll5 98 49 38 3l 27 25 l8
189 79 57 44 38 35 z5
90 76 57 35 27
54 48 49 29 ZJ
Due to minor variations in the amount of lysis, data are given for one representativeplatelet preparation. In total, 17 platelet preparationswere tested that with two exceptionsall showed a similar behavior.
factor Va), the reduction in clotting time to that ofsonicatedprepcloselycorresponds arationsthat show the sameamount of lysis as producedduring the activation procedure. Similar results were obtained with platelets activated with serotonin, epinephrine or kaolin (data not shown).Without addedfactor Va in the clotting assay,thrombin-activated plateletsshow a shorterclotting time than can be accountedfor by lysis, but this discrepancydisappearsin the presenceof an excessofexogenousfactor Va. This strongly suggeststhat in the absenceof added factor Va the reductionin clottingtime is not only causedby minor cell lysis (some l.7olo)but alsoby releaseand activationoffactor Va. It
should be noted here that according to the dataof Chesneyet al. [15], 0.1 ml of platelets (108cells/ml)usedin the assaycancontribute approximately I unit of factor V when this factor is fully releasedand activatedinto factor Va. The small amount of thrombin used to activatethe plateletsdoesnot significantly affectthe clotting time in this assaysystem. In contrast to the platelet activations described above, platelets stimulated by A23187or by the combinedaction of collagen plus thrombin produce a much shorter clotting time (both in the absenceor presence ofaddedfactorVa) than canbe accountedfor by minor cell lysis and/orreleaseand activation offactor V from the platelets.
272
Discussion The clotting time observedin a one-stage prothrombinase assayusing Russell's viper venom will be dependenton the availability of negativelychargedprocoagulantphospholipids for the formation of the prothrombinasecomplex [16]. In the plateletplasma membrane,the negativelychargedphospholipids are almost exclusively present in the inner leafletof the membrane[l7, l8]. This explains why a preparation of intact nonstimulated platelets does not significantly shorten the clotting time. Platelet damage, leadingto the exposureofthe inner surfaceof the plasma membrane, thereby offering a negatively chargedphospholipid surfacefor the formation of the prothrombinase complex, resultsin a decreasein clotting time. Lowering of the clotting time by the addition of activated plateletsin the coagulation assaycan have at least three different explanations:(i) releaseand activation offactor V sincethe plasma from the plateleto,-granules mixture offeredin the coagulationassaynormally doesnot contain an excessof this factor and henceis sensitiveto the addition of factor Va; (ii) exposure of the negatively chargedinner surfaceof the platelet plasma membraneas a result of lysis causedby the activation procedure;(iii) exposureof negatively chargedphospholipids at the platelet outer surfaceas a result of rearrangementsin the plasmamembranecausedby the agonistreceptorinteraction.The data presentedhere showthat the decreasein clotting time caused plateletscan be by ADP or collagen-activated platelet lysis. It should be emby explained phasized,however, that lysis was measured by the activity of the cytoplasmic enzyme in the plateletsupernalactatedehydrogenase tant after activation. Therefore,it cannot be
Bevers/Comfurius/Hemker/Zwaal
excludedthat the presenceof this enzymein the supernatantis a result ofa transientleakage and does not necessarilyreflect platelet damageand exposureof the inner surfaceof the plasma membrane.However, the close correlationin clotting time betweenplatelets lysed by sonication and platelet lysis produced by certain agonists strongly suggests that percentageleakageoflactate dehydrogenaseduring plateletactivation in fact reflects percentageof cell damage. The effectofreleaseand activation offactor V from the platelet granulescan be of importance only in those caseswhere platelets are activatedby thrombin or a combination of another agonistwith thrombin. This can be seenin table I comparing the reduction in clotting time betweenplatelets activated with collagen and platelets activated with thrombin. In the absenceof exogenously addedextra factor Va the assaywill be sensitive to factor Va from the platelets.Although activation by collagenresultsin a similar percentageof lysis as activation by thrombin, the clotting time observed with thrombinactivated platelets is shorter than that observedwith collagen-activatedplatelets.This can be explained by the releaseand activation of factor V from the platelets which occursupon activation with thrombin. (Collagenonly inducesreleasebut not activation of factor V.) When an excessof factor Va is addedto the assay,the differencein clotting time between collagen- and thrombin-activated plateletsdisappears,and equalsthat of sonicated preparations showing the same amount of lysis. Addition of plateletsactivated by a combination of collagenplus thrombin produces an even further reduction in clotting time, which cannotbe explainedby eithercell lysis or releaseand activation of factor V. It has
Clotting Activity of Platelets
been shown previously that this actiyation procedureexposesnegatively chargedphospholipids (particularlyphosphatidylserine)at the cell outer surface,as a result of transbilayer movement of phospholipidsby a processnot involving lysis of the cells [5]. It is suggestedthat this processis in fact mainly for the observedreductionofthe responsible clotting time with plateletsactivatedby collagenplus thrombin. A similar explanationcan be given for plateletsactivatedby ionophore 423187. It should be mentioned that the concentrations of the agonistsused were all suflicient to produceoptimal releaseand aggregation, i.e no increasein o,-granulaor dense body releaseor in the extent of aggregation was observed upon increasing the agonist concentration.Also, the effect of the combined action of collagenplus thrombin could not be evokedby usinghigherthrombin concentrationsto activate the platelets,or combinations of thrombin with other agonists. The data obtainedwith this clotting assay are in close agreementwith those obtained using an assaysystemwith purified coagulation factors and a chromogenicsubstrateto measurethe rate of thrombin formation in the presenceof activated platelets[5]. Although caremust be taken with the interpretation of clottingtimeswith respectto lysisof the plateletsor releaseand activationoffactor V, the assaydescribedhereis suitableto determinewhetheror not negativelycharged phospholipidsbecomeexposedat the platelet outer surfaceas a result of activation by a plateletagonist. References I Spaet,T.H.; Cintron, J.: Studieson plateletfactor3 activity. Br. J. Haemat. l1: 269-2'15(1965).
zl5
2 Hardisty, R.M.; Hutton, R.A.: Platelet aggregation and the availability of platelet factor 3. Br. J. Haemat. I 2: 764-7 76 (1966). 3 Joist, J.H.; Dolezel, G.; Lloyd, J.V.; KinloughRathbone, R.L.; Mustard, J.F.: Platelet factor-3 availability and the platelet release reaction. J. Lab. clin. Med. 1974: 474-4.82. 4 Yan Zfiphen, H.; Bevers, E.M.; Hemker, H.C.; Zwaal, R.F.A.: Contribution of the platelet factor V content to platelet factor 3 activity. Br. J. Haem a t .4 5 : 1 2 1 - 1 3 1( 1 9 8 0 ) . 5 Bevers,E.M.; Comfurius, P.; Rijn, J.L.M.L. van; Hemker, H.C.: Zwaal, R.F.A.: Generation of platelet prothrombin converting activity and the exposure of phosphatidylserine at the platelet outer surface. Eur. J. Biochem. 122: 429436 (1982). 6 Bevers,E.M.; Dieijen, G. van; Rosing,J.; Hornstra, G.; Zwaal, R.F.A.: The effect of prostacyclin on the participation ofplatelets in factor X activation and thrombin formation (Abstract). Thromb. Haemostasis46: 271 (1981). 7 Rosing, J.; Tans, G.; Govers-Riemslag,J.W.P.; Zwaal, R.F.A.; Hemker, H.C.: The role of phospholipids and factor Va in the prothrombinase complex.J. biol. Chem. 255:274-283 (1980). 8 Wroblewski, F.; [,a Due, J.S.: Lactic dehydrogenaseactivity in blood. Proc. Soc.exp. Biol. Med. 90: 210-215 (1955). 9 Tans, G.; Zutphen, H. van; Comfurius,P.; Hemker, H.C.; Zwaal, R.F.A.: Lipid phasetransitions and procoagulant activity. Eur. J. Biochem. 95; 449457 ,1979\. l0 Esmon, C.T.: The subunit structureof thrombin activated factor V. Isolation ofactivated factor V, separation of subunits and reconstitution of biological activity. J. biol. Chem. 254: 964-974 (r9'79). l1 Kappeler, R.; Das Verhalten von Faktor V im Serum unter normalen und pathologischenBedingungen.Z. klin. Med. /JJ: 103-l l3 (1955). l2 Nesheim, M.E.; Myrmel, K.H.; Hibbard, L.; of sinMann, K.G.: Isolationand characterization gle chain bovine factorV. J. biol. Chem.251; 5085t7 /1979\. l3 Breederveld,K.; Giddings, J.C.; Ten Cate, J.W.; Bloom, A.L.: The localizationof factor V within normal human plateletsand the demonstration of a platelet-factor V antigen in congenital factor V deficiency.Br. J. Haemat.29: 405412 (1975).
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14 Osterud, B.; Rapaport, S.I.; Lavine, K.K.: Factor V activity of platelets: Evidence for an activated factor V molecule and for a platelet activator. Blood49:819-834 (1977). 15 Chesney,C.M.;Pifer,D.;Colman,RW.:Subcellular localization and secretion of factor V from human platelets. Proc. natn. Acad. Sci. USA 78; 5 1 8 0 - 5 1 8(41 9 8 1 ) . 16 Zwaal, R.F.A.: Membrane and lipid involvement in blood coagulation.Biochim. biophys. Acta 515r 163-205(1978). 17 Chap, H.J.; Zwaal, R.F.A.; Deenen,L.L.M. van: Action of highly purilied phospholipaseson blood platelets.Evidence for an asymmetric distribution of phospholipids in the surface membrane. Biochim. biophys. Acta 467: 146-164 (1977).
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l8 Perret, B.; Chap, H.J.; Douste-Blazy, L.: Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets.A determination using purified phospholipasesand a rapid method for membrane isolation. Biochim. biophys. Acta 556: 434446 (1979).
Received:IN4arch12, 1982 Acceptedby Editor J. Caen:March 19, 1982 E.M. Bevers,Biomedical Center, University of Limburg, Department of Biochemistry, Maastricht (The Netherlands)
