agionist at platelet

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DOI 10.1378/chest.124.3_suppl.18S

2003;124;18S-25SChestLawrence F. Brass

*Thrombin and Platelet Activation

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Thrombin and Platelet

Activation*

Lawrence F. Brass, MD, PhD

The accumulation of thrombin at sites of vascular

injury provides one of the chief means for recruitingplatelets into a growing hemostatic plug. Studiescompleted over the past 10 years show that plateletresponses to thrombin are mediated by a subset of Gprotein-coupled receptors known as protease-acti-vated receptors. These receptors are activated oncleavage by thrombin, initiating the intracellularsignaling events needed to transform mobile, nonad-hesive platelets into cells that can participate in thegrowth of an immobile hemostatic plug. How this isaccomplished is the subject of this review.

(CHEST 2003; 124:18S25S)

Key words: G proteins; G protein-coupled receptors; phospho-

lipase C; platelet; protease-activated receptors; thrombin

Abbreviations: ADP adenosine diphosphate; cAMP cyclicadenosine monophosphate; GDP guanosine diphosphate;GP glycoprotein; GPCR G protein-coupled receptor;GTP guanosine triphosphate; NO nitric oxide; PAR pro-tease-activated receptor; PGI2 prostaglandin I2; TxA2 thromb-oxane A2; VWFvon Willebrand factor

Human platelets normally circulate in a quiescent state,prevented from premature activation by the presence

of the endothelial cell monolayer, by the signal-inhibitingeffects of prostaglandin I2 (PGI2) and nitric oxide (NO),and by limitations on the local accumulation of plateletagonists. It is only when these barriers are overcome that

platelets can become activated. That can happen afterlocal trauma or in response to the rupture of an athero-sclerotic plaque. Thrombin plays an essential role inactivating platelets, just as it does in the formation of thefibrin clot. When added to human platelets in vitro,thrombin causes platelets to change shape, stick to eachother, and secrete the contents of their storage granules.How this is accomplished is still not fully understood, buta major step forward occurred in 19901,2 with the identi-fication of a G protein-coupled receptor (GPCR) that canbe activated proteolytically by thrombin. Until that recep-tor, now known as protease-activated receptor (PAR)-1,

was identified, there was no clear paradigm for the

initiation of intracellular events by an extracellular pro-tease. In the dozen years since then, a family of protease-responsive receptors has been identified and steady

progress has been made toward understanding how theywork and how thrombin activates platelets. The resultshave provided insights into normal platelet biology andopened an avenue for the development of new antiplateletagentsa promise that has yet to be fulfilled. To place

what is known about the activation of platelets by throm-bin into context, this article begins with an overview of the

events encompassing platelet plug formation and thenfocuses on the role of thrombin.

Stages in the Formation of a Stable

Platelet Plug

Platelet plug formation requires a coordinated series ofevents that can overcome local resistance to plateletactivation long enough for bleeding to stop. This is not atrivial task, particularly if unwarranted platelet activation isto be avoided. The barriers to platelet activation aresubstantial. Part of the barrier is formed by the endothelialcell monolayer, which physically separates platelets fromagonists embedded in the vessel wall, especially collagen

and von Willebrand factor (VWF). VWF is a multimericprotein synthesized by endothelial cells that plays anessential role in the adhesion of platelets to collagen underthe high-flow conditions found in arteries. It is secreted inan ultrahigh-molecular-weight form that is normallycleaved by the metalloprotease, ADAMTS13, preventingit from binding to platelets spontaneously and causing athrombotic microangiopathy.3 The shear stresses pro-duced when blood flows over VWF anchored to collagenexposes platelet-binding sites, allowing the VWF to sup-port platelet/collagen and platelet/platelet interactions(Fig 1). In addition to collagen and VWF, tissue factor isalso present in the vessel wall, on the surfaces of activated

endothelial cells and monocytes, and on circulating mi-crovesicles that stick to activated platelets, so injuries thatalter or remove the endothelial barrier result in the localgeneration of thrombin as well as the exposure of collagen.

In addition to serving as a physical barrier, endothelialcells release PGI2 and NO, whose net effect is to globallydepress the intracellular signaling events needed to sup-port platelet activation by raising cyclic adenosine mono-phosphate (cAMP) and cyclic guanosine monophosphatelevels (Fig 1). The ability of cyclic nucleotides to inhibitplatelet activation has been exploited in the developmentof antiplatelet agents, such as dipyridamole, which work byinhibiting the phosphodiesterases that would otherwise

metabolize cAMP within platelets. The importance ofPGI2 and NO as barriers to platelet activation is indicatednot only by the effectiveness of molecules that mimic PGI2as antiplatelet agents, but also by the prothromboticeffects of deleting the gene encoding the platelet PGI2receptor in mice.4 As a further barrier to platelet activa-tion, some endothelial cells express CD39 on their luminalsurface. CD39 can hydrolyze small quantities of adenosinediphosphate (ADP) released from damaged red cells andactivated platelets, preventing the ADP from activatingadditional platelets.5,6 The use of CD39 as an antithrom-botic is being explored in animal models. Other barriers toplatelet activation include the diluting effects of blood

*From the Departments of Medicine and Pharmacology, and theCenter for Experimental Therapeutics, University of Pennsylva-nia, PA.Supported by grants HL40387 and HL45181 from the NationalInstitutes of Health-National Heart Lung, and Blood Institute.Reproduction of this article is prohibited without written permis-sion from the American College of Chest Physicians (e-mail:[email protected]).Correspondence to: Lawrence F. Brass, MD, PhD, University ofPennsylvania, Room 915 BRB-II, 421 Curie Blvd, Philadelphia,PA 19104; e-mail: [email protected]

18S Thrombin: Physiology and Pathophysiology

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flow, the presence of inhibitors of thrombin, and the shorthalf-life of the platelet-derived agonist, thromboxane A2(TxA2).

Given all of these barriers, platelet activation shouldideally occur only after substantial injuries. If this werealways the case, there would be little need for antiplateletagents in clinical settings. Formation of the platelet plug inresponse to vascular injury can be thought of as occurringin three stages: initiation, extension, and perpetuation(Fig 1). Initiation begins with the tethering, rolling, andarrest of moving platelets on collagen and their subse-quent activation to form a platelet monolayer. Large VWF

multimers are essential to this process, particularly underhigh shear conditions in the arterial circulation, but throm-bin can also help to initiate platelet activation. Extensionrefers to the recruitment of additional platelets throughthe local accumulation of thrombin, ADP and TxA2.Perpetuation refers to the events that stabilize the plateletplug until wound healing can occur, some of which involvemolecules on the platelet surface that are capable ofgenerating intracellular signals only after platelets havecome into sustained contact with each other. The netresult is the formation of a fibrin-anchored platelet plug, astructure in which platelet/platelet interactions are sup-ported by the binding of fibrinogen and fibrin to the

integrin IIb3 (also known as glycoprotein [GP] IIb-IIIa) and by VWF bound to GP Ib and IIb3 (Fig 2).

Initiation

The arrest and eventual activation of moving plateletsby collagen plus VWF requires several receptors on theplatelet surface, including those that can bind directly tocollagen (GP VI and the integrin 21) and those that bindto collagen indirectly via VWF (the GP Ib/IX/V complexand the integrin IIb3). The presence of binding sites forcollagen and VWF on GP VI, GP Ib, 21, and IIb3

allows platelets to stop their forward movement in thearterial circulation long enough to become activated andfully adherent. This can happen rapidly, but only selec-tively. Videomicroscopy of blood vessels following focalinjuries shows that most platelets move by the site of injurytoo quickly to stop.7 In a manner that is very muchreminiscent of how leukocytes escape from the circulation,a small proportion of platelets rolling along the vessel wallis able to react initially to injury and form the nidus for aplatelet plug. This requires platelets to both adhere tocollagen and be activated by it. The binding of collagen toGP VI on the platelet surface causes the clustering of GP

VI and its associated -chain within the plane of the

Figure 1. Stages in platelet plug formation. Prior to vascular injury, platelets are restrained fromactivation by inhibitory factors that include PGI2 and NO released from endothelial cells, the presenceof CD39 on the surface of endothelial cells, and the inability of normal plasma VWF to bindspontaneously to the platelet surface. The development of the platelet plug can be initiated by theexposure of collagen and VWF in the vessel wall, and by the local generation of thrombin, a process thatoccurs more rapidly on the surface of activated platelets. Rolling platelets adhere and spread on thecollagen matrix, forming a monolayer of activated platelets that can act as a surface for subsequentrecruitment of platelets by thrombin, ADP, and TxA2. During the perpetuation stage, close contactsbetween platelets promote the growth and stabilization of the hemostatic plug, in part throughcontact-dependent signaling mechanisms.

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platelet plasma membrane, which leads to the phosphor-ylation of the -chain by tyrosine kinases in the Src family,creating a tandem phosphotyrosine motif that is recog-nized by the tyrosine kinase, Syk, and the activation ofphospholipase C2 (Fig 3).8 PLC2 hydrolyzes PI-4,5-P2to produce 1,4,5-IP3 and diacylglycerol, raising the cyto-solic-free Ca2 concentration within the adherent plate-lets by discharging Ca2 stores from within the densetubular system and activating protein kinase C. Clusteringof a cell-surface receptor (GP VI in this case) followed bysignaling through tyrosine kinases is a theme that occursagain during the perpetuation phase of platelet plugformation.

Extension

The extension phase of platelet plug formation occurswhen activated platelets accumulate on top of the initialmonolayer of platelets bound to collagen (Fig 1). Key tothe extension phase is the presence on the platelet surfaceof receptors that can respond rapidly to soluble agonists,including thrombin, ADP, and TxA2. The local accumula-tion of these agonists allows circulating platelets to berecruited into the growing hemostatic plug even whenthey cannot arrest on collagen. Extension of the plateletplug requires the activation of IIb3 through what iscommonly called inside-out signaling, promoting the

formation of stable platelet/platelet contacts mediated bybridges comprised of fibrinogen and VWF. The receptorsinvolved in these events are typically members of thesuperfamily of GPCRs (Fig 3), which are membraneproteins with an extracellular N-terminus, an intracellularC-terminus, and seven transmembrane domains. Agonistsbind to the surface-accessible domains of GPCRs, causinga conformational change that activates G proteins associ-ated with the intracellular surface of the receptor.9 Gproteins interact with the cytoplasmic domains of thereceptor with specificity determined in part by the se-quence of these domains and in part by the sequence ofthe subunit of the G protein. G proteins are heterotri-

mers comprised of a single , , and subunit. The subunit contains a guanine nucleotide-binding site that isoccupied by guanosine diphosphate (GDP) in the off stateand GTP in the on state. Activation of the receptor causesexchange of guanosine triphosphate (GTP) for GDP, after

which partial dissociation of the GTP-bound G fromG exposes effector interaction sites on both. Fatty

acylation of G and prenylation of G cause them toremain associated with the plasma membrane until hydro-lysis of the GTP bound to G allows the original hetero-trimer to reform.

Mammalian G proteins fall into four families that aretypically referred to by the designation of the subunit.Human platelets express at least one member of the G

s

family and four members of the Gi family (Gi1, Gi2, Gi3,and G

z), which respectively stimulate and inhibit cAMP

formation by adenylyl cyclase, among other functions. Inaddition, platelets express one or more members of the Gqfamily, which stimulate isoforms of phospholipase C,and two members of the G12 family (G12 and G13), whichhelp to regulate the platelet actin cytoskeleton.1014 Basedon evidence from knockout and reconstitution studies, theabundance of G protein types in platelets appears to benecessary to support the actions of multiple dissimilarplatelet agonists.15

The GPCRs that respond to platelet agonists differ intheir potency and their preferences for intracellular effec-tor pathways. Some, such as the receptors for thrombin(PAR-1 and PAR-4), TxA2 (TP), and ADP (P2Y12), causephosphoinositide hydrolysis and raise the cytosolic Ca2

concentration by activating Gq (Fig 3).12 Others, such asthe P2Y12 receptor for ADP and the 2A-adrenergicreceptor for epinephrine, are coupled by Gi2 or Gz to theinhibition of adenylyl cyclase and to the activation of PI

3-kinase and the Ras family member, Rap1.1518 Optimalplatelet activation via GPCRs is thought to require activa-tion of both a Gq-coupled receptor and a Gi-coupledreceptor.19 The ability of the Gi family members inplatelets to inhibit cAMP formation by adenylyl cyclase ismost relevant when PGI2 secreted by endothelial cells hasinhibited platelet activation. In the absence of PGI2, otherGi effectors are more relevant.15 The essential role of theGi2-coupled P2Y12 receptor for ADP is suggested by thephenotypes of the Gi2 and P2Y12 knockout mice and bythe proven utility of the P2Y12 antagonists ticlopidine andclopidogrel as antiplatelet agents.15,17,2022 Thrombin andTxA2 receptors can also cause the rearrangement of the

actin cytoskeleton that underlies platelet shape change bycoupling to guanine nucleotide exchange factors for Rhovia G12 and G13.10

Perpetuation

The third phase of platelet plug formation, perpetua-tion, occurs at a point when direct interactions betweenplatelets are of sufficient duration to make contact-depen-dent signaling feasible. Ultimately, these late events arethought to stabilize the platelet plug, helping to preventpremature disaggregation and regulating retraction of theclot. A number of recent events have helped to definesome of the signaling events that are involved. The

Figure 2. Anatomy of a platelet plug: an enlarged view of theassembled platelet plug, highlighting platelet/platelet interactionsmediated by the binding of fibrinogen, fibrin, and VWF toactivated GP IIb-IIIa (IIb3) and the binding of VWF toGP Ib.




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best-described of these events23 involves outside-in signal-ing through integrins. Other examples include the bindingof Eph kinases to ephrins,24 and the binding of CD40ligand (CD40L) to IIb3 and perhaps CD40.25 Each ofthese depends on one or more molecules expressed on theplatelet surface, but the details differ in essential ways.Eph kinases and ephrins can engage with each other

whenever platelets are in sufficiently close contact for along enough time. Interactions with IIb3 require inside-out signaling before the integrin can bind to its ligands,particularly soluble ligands. CD40L is not expressed onthe surface of resting platelets, but appears there afterplatelets have been activated. Once on the surface,CD40L can bind to activated IIb3 and (perhaps) toCD40. Evidence exists for a role for each of thesecontact-depending signaling mechanisms, but their rela-tive contributions are still being studied.

Thrombin Receptor Structure

and Function

For years after thrombin was shown to activate plate-lets, little was known about how this might be accom-plished. A variety of approaches established that the

proteolytic activity of thrombin was required, and bio-chemical studies showed that G proteins are activated bythrombin, but there was no precedent for G proteinactivation by a protease. Binding studies26,27,48 identifiedhigh-affinity interactions with several sites on the plateletsurface, including GP Ib, but efforts to show that any ofthese constituted a receptor in the signaling sense werenot entirely successful. Substrates for thrombin wereidentified on the platelet surface, including GP V. How-ever, cleavage of GP V did not appear to be required for

platelet activation by thrombin. Before discussing thereceptors that have been identified, it is worth brieflyconsidering criteria that proved useful for establishing aprotein as a true signaling receptor for thrombin. Suchcriteria included the following: (1) demonstrating thepresence of the candidate receptor on the surface ofresting platelets, (2) showing that it is a substrate forthrombin or closely associated with a substrate for throm-bin, (3) demonstrating a link to intracellular signalingcascades, (4) showing that expression of the candidatereceptor could render a cell that was otherwise unrespon-sive to thrombin capable of responding, and (5) showingthat blocking, dismantling, or otherwise removing the

Figure 3. Overview of platelet activation. Most platelet agonists activate platelets via G protein-coupled receptors on the platelet surface. Critical responses include Gq-mediated activation ofphospholipase C isoforms to allow an increase in cytosolic Ca2, activation of phospholipase A2 andprotein kinase C, and G12-mediated activation of Rho family members to support rearrangement of theplatelet cytoskeleton (shape change). The increase in cytosolic Ca2 is initially caused by theIP3-triggered release of Ca

2 from within the dense tubular system of the platelet, which in turntriggers Ca2 influx across the platelet plasma membrane. Activated PGI2 receptors (not shown)stimulate adenylyl cyclase, raising platelet cAMP levels and causing a generalized inhibition of plateletresponses to agonists. Gi family members support the suppression of adenylyl cyclase by plateletagonists and also couple receptors to other critical effector pathways. TP thromboxane receptor.




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candidate receptor would reduce platelet responses tothrombin. So far, PAR family members are the onlyreceptors that meet all of these criteria. However, itremains possible that other proteins on the platelet surfacealso play a role, either by initiating signaling themselves orby facilitating the activation of PARs.

Four members of the PAR family have been identifiedto date. Three (PAR-1, PAR-3, and PAR-4) are thrombinreceptors. The fourth, PAR-2, is activated by serine pro-teases other than thrombin. All four of the PAR familymembers have a structure similar to other GPCRs, includ-ing an exposed N-terminus (Fig 4). Studies1 on PAR-1established the paradigm that applies (with certain excep-tions) to the other three family members. In each case,receptor activation begins when thrombin cleaves theN-terminus of the receptor, exposing a new N-terminusthat serves as a tethered ligand. Given sufficient opportu-nity, proteases other than thrombin can also activatePAR-1 or render the receptor unresponsive to thrombinby cleaving the N-terminus in the wrong place. Thebinding site for the tethered ligand has been mapped to

the extracellular loops of the receptor. Because the ligandis not free to diffuse away, it presents a highly effectivelocal concentration at the receptor. As is the case for otherGPCRs, contact between PAR ligands and the receptor isthought to initiate signaling because of an induced con-formational change in the receptor that is transmittedacross the plane of the plasma membrane to promoteexchange of GTP for GDP on associated G proteins. In the

case of PAR family members, the activation paradigm thatwas initially established for PAR-1 includes the ability torespond to peptides based on the sequence of the tetheredligand. The one exception to the rule is PAR-3, for whichno activating peptide agonist has been identified.

What about the other three members of the PARfamily? PAR-2 is expressed by a number of tissues,

including endothelial cells, but not by platelets. PAR-2 canbe cleaved and activated by trypsin and tryptase, but notby thrombin.28,29 It can also be activated by the tissuefactor/VIIa complex and factor Xa, which may be partic-ularly relevant for endothelial cells.3032 PAR-3 was iden-tified after a gene ablation study33 showed that plateletsfrom mice lacking PAR-1 were still responsive to throm-bin. PAR-3 is a major regulator of thrombin responses inrodent platelets,34 but little else is known about it. Whenoverexpressed, human PAR-3 can respond to thrombin.However, on murine platelets PAR-3 serves solely tofacilitate cleavage of PAR-4 by thrombin.35 The fourthfamily member, PAR-4, was identified by databasesearches using conserved domains of the other threefamily members.36,37 PAR-4 is expressed on human andmouse platelets and accounts for the continued ability ofplatelets from PAR-3 knockout mice to respond to throm-bin.36,37 Simultaneous inhibition of human PAR-1 andPAR-4 with blocking antibodies or a small-molecule an-tagonist completely abolishes platelet responses to throm-bin,38 as does deletion of the gene encoding PAR-4 inmice.39

Thus, the four PAR family members have some featuresin common, but also have differences. Of the three thatcan be activated by thrombin, two (PAR-1 and PAR-3)have similar dose/response curves. The third, PAR-4,requires 10- to 100-fold higher concentrations of throm-

bin, apparently because it lacks the hirudin-like sequencesthat can interact with the anion-binding exosite andfacilitate receptor cleavage of thrombin.3537,40 This dis-tinction is important for understanding the role of PAR-4in human and mouse platelets.

Platelet Activation by Thrombin

Putting all of this together, current evidence suggeststhat thrombin activates human platelets by cleaving andactivating PAR-1 and PAR-4 (Fig 5). In turn, thesereceptors activate Gq, G12, and perhaps Gi family mem-bers, leading to the activation of PLC, PI 3-kinase, andthe monomeric G proteins, Rho, Rac, and Rap1, and also

causing an increase in the cytosolic Ca2

concentrationand inhibiting cAMP formation. This process is supportedby released ADP and TxA2, which bind in turn to theirown GPCRs on the platelet surface (Figs 3, 4). Cleavage ofhuman PAR-4 requires a higher concentration of throm-bin than does cleavage of PAR-1, and it is likely thatPAR-1 is the predominant signaling receptor at lowthrombin concentrations, but PAR-4 activation may bemore sustained.41,42 Mouse platelets provide an interestingcontrast to human platelets. Where human platelets ex-press two functional PAR family members (PAR-1 andPAR-4), mouse platelets express PAR-3 and PAR-4, butsignaling appears to be mediated entirely by PAR-4, with

Figure 4. Structure and features of PAR-1. Cleavage of PAR-1by thrombin between arginine 41 and serine 42 exposes a newN-terminus that serves as a tethered ligand. Activation of PAR-1is followed by a rapid burst of signaling before the receptor isdesensitized and, in some cases, cleared from the cell surface. 49




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PAR-3 serving solely to facilitate the cleavage of PAR-4 atlow thrombin concentrations.35,39

One issue that remains unresolved is the contribution ofother thrombin receptors on platelets, particularly themembers of the GP Ib/IX/V complex. GP Ib is a het-erodimer comprised of an and a subunit, which aredisulfide linked to each other. The heterodimer forms acomplex with GP IX and GP V that serves as both abinding site for VWF and an anchor for the plateletcytoskeleton.43 A high-affinity binding site for thrombinlocated at approximately residues 268287 on GP Ib isthought to interact with domains other than the active

site.44 Deletion of the extracellular domain of GP Ib orblockade of the thrombin-binding site decreases plateletresponses to thrombin.4548 In theory, the binding ofthrombin to GP Ib could facilitate the cleavage of a PARfamily member on human platelets, much as the bindingof thrombin to PAR-3 is thought to facilitate cleavage ofPAR-4 on mouse platelets (Fig 5).48 Thus, although it is

very clear that PAR-1 and PAR-4 provide the primaryresponse elements for thrombin on human platelets, itremains possible that interactions with one or more mem-bers of the GP Ib/IX/V complex may facilitate cleavage/activation of PAR-1 or otherwise regulate platelet activa-tion by thrombin.

Conclusion

In summary, there is no simple answer to the questionhow does thrombin activate human platelets? At leasttwo GPCRs, several heterotrimeric G proteins, and a longlist of intracellular signaling molecules are involved. Othersurface molecules, including GP Ib, may play an acces-sory role. The identification of PAR-1 and the recognitionof its novel mechanism of action suggested that it might bepossible to develop small-molecule antagonists of plateletactivation by thrombin. A number of efforts to do so havebeen launched over the past 10 years. As might be

expected (at least in hindsight), such efforts have met withqualified success. Antagonists for PAR-1 have been iden-tified and tested in vitro and in animal trials. Despite thecompetitive advantage of having a tethered ligand, it hasproved possible to block PAR-1 activation by agonistpeptides and, in some cases, thrombin as well. Oneproblem is that platelets also express functional PAR-4 andfull blockade of thrombin responsiveness requires inhibi-tion of both receptors. One might imagine administrationof a combination of PAR-1 and PAR-4 antagonists.

Whether such a combination will be more useful asantiplatelet therapy than aspirin or an ADP receptorantagonist awaits demonstration. Orally active inhibitors of

Figure 5. Receptor-mediated platelet activation by thrombin. Platelet responses to thrombin aremediated largely by members of the PAR family. Human platelets express PAR-1 and PAR-4, whichcollectively are coupled to Gq-, G12-, and Gi-mediated effector pathways. Secretion of ADP acts as afurther activator of Gi-mediated pathways via the receptor, P2Y12. Cleavage of PAR-1 by thrombinappears to be facilitated by the binding of thrombin to GP Ib in the GP Ib/IX/V complex.




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thrombin are currently under investigation as potentialsubstitutes for warfarin in patients needing long-termanticoagulation. Whether they offer an advantage based inpart on their ability to block platelet activation via PAR-1and PAR-4 remains to be seen.

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DOI 10.1378/chest.124.3_suppl.18S2003;124; 18S-25SChest

Lawrence F. Brass

*Thrombin and Platelet Activation

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