Burger's Medicinal Chemistry and Drug Discovery || Antiplatelet Agents

ANTIPLATELET AGENTS

SAMUEL CHACKALAMANNIL

Schering-PloughResearch Institute,Kenilworth, NJ

1. INTRODUCTION

Platelets are the guardians of vascular integ-rity in the circulatory system where theirprimary function is to stem bleeding at thesite of injury and promote vessel wall re-pair [1–3]. Produced from bonemarrowmega-karyocytes, platelets have an approximatelife-span of 10 days; they lack genomic DNA,but contain messenger RNA and the transla-tional machinery needed for protein synth-esis [4–6]. In the absence of bleeding, plateletscirculate freely in the blood in the restingdiscoid state with minimal interaction witheach other, other blood components, and thevessel wall. Upon endothelial injury, plateletsare stimulated to express their diverse ana-tomic and functional capacities; they undergoshape change (Fig. 1), become adhesive,aggregatory, and spread on the denudedendothelium to form an initial hemostaticplug. With recruitment of additional plateletsand the incorporation of fibrin meshwork, thehemostatic plug grows into a thrombus, whichunder normal physiologic conditions is regu-lated and eventually undergoes fibrinolytic

dissolution once its hemostatic function is ful-filled [7].However,underpathologicconditionssuch as rupture of an atherosclerotic plaque,the very same life-sustaininghemostasis turnsto an aberrant, amplified process leading to anocclusive thrombus that can result in fatal con-ditions such as an acute myocardial infarctionand ischemic stroke [8–11].

2. A BRIEF REVIEW OF THROMBOSIS

2.1. Acute Coronary Syndrome

Platelets play a central role in the pathogen-esis of coronary artery disease (CAD) thatleads to acute myocardial infarction andunstable angina, and cerebrovascular diseasesuch as ischemic stroke, and peripheralvascular disease such as peripheral arterydisease (PAD) [12–15]. Coronary artery dis-ease is the primary cause of cardiovasculardeath with an estimated worldwide mortalityof 7.8 million by 2010 [16,17]. In the UnitedStates alone, over half a million people dieannually of CAD [18]. The underlying etiologyfor CAD is atherosclerosis—a chronic, pro-gressive, inflammatory, and proliferative re-sponse to cholesterol infiltration into arterialwall resulting in progressive luminal narrow-ing [19,20]. The clinicalmanifestations ofCADare triggered by the rupture of an athero-sclerotic plaque that marks the sudden tran-sition from a stable, clinically silent disease to

Figure 1. Visible changes that occur to platelets upon activation. Unactivated platelets have a symmetricaldiscoid shape (left).Uponactivation,platelets lose theirdiscoid shape, becomesomewhatsphericalandextendlong, spiky pseudopods. Taken from Ref. [352]. Copyright Elsevier. Reproduced with permission.

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Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh Edition,edited by Donald J. Abraham and David P. RotellaCopyright � 2010 John Wiley & Sons, Inc.

a symptomatic life-threatening condition. [21]When a plaque ruptures in a coronary artery,blood comes into contact with the vasoactivecomponents of the endothelial matrix trigger-ing activation of platelets and blood coagula-tion to give rise to an occlusive throm-bus [22,23]. The obstruction of blood flowand the myocardial underperfusion thatresults leads to ischemic disorders, presentinga spectrum of clinical conditions known asacute coronary syndrome (ACS) that includesQ-wave myocardial infarction, non-Q-wave myocardial infarction, and unstableangina [24–26].

2.2. The Role of Platelets in ACS

In order to assign theproper role of platelets inACS, a brief review of the mechanism ofthrombosis is in order. The interplay of plate-let aggregation and blood coagulation contri-butes to thrombus formation [27]. When theendothelium is breached due to a pathologiccondition such as rupture of an atheroscleroticplaque or a vascular trauma caused by surgi-cal intervention, blood becomes exposed tosubendothelial collagen and tissue factor [8].Exposure of platelets to collagen initiates theplatelet activation mechanism (Fig. 2). The

Figure 2. The platelet activation mechanisms and activation of the coagulation system synergize inthrombus formation. Upon endothelial injury, platelets are exposed to collagen of subendothelial matrix.Unactivated platelets adhere to collagen via the adhesion protein von Willebrand Factor to form an initialhemostatic plug. At the same time, interaction of platelet surface glycoprotein VI and glycoprotein Ibreceptors with collagen causes activation of platelets. Activated platelets undergo shape change and releaseproaggregatory granular contents such as thromboxane A2 and adenosine diphosphate that further recruitand activate circulating platelet to the site of injury. Concurrent with platelet activation, the coagulationprocess is also triggered by the exposure of tissue factor to blood resulting in the production of thrombin.Thrombin plays a dual role in hemostasis and thrombosis. In its proaggregatory role, thrombin activatesplatelets via protease-activated receptor-1, also known as thrombin receptor. In its procoagulant role,thrombin cleaves soluble fibrinogen to fibrin that cross-links to form an insoluble fibrin meshwork thattraps aggregated platelets and other plasma particles leading to a growing thrombus.

410 ANTIPLATELET AGENTS

initial adhesion of platelets to collagen occursvia vonWillebrand Factor (vWF), an adhesionprotein that has binding sites for both plateletsurface GP Ib receptors and collagen. Theadhesion is followed by platelet activationthat leads to the release of platelet activatingfactors such as thromboxane A2 (TxA2), ser-otonin, adenosine diphosphate (ADP), and soon, which serve to further amplify theactivation process. Activated platelets under-go shape change and display activated glyco-protein IIb/IIIa (GP IIb/IIIa) receptors ontheir surface, which cross-linkwith fibrinogencausing platelet aggregation.

Concurrent with the triggering of plateletactivation mechanism, the plasma coagula-tion system is activated. The tissue factor thatis released from the site of injury activatesfactor VII to VIIa that proteolytically pro-cesses factor X toXa, which, in turn, generatesthrombin from prothrombin.

Thrombin plays a very important dual rolein thrombosis. Thrombin cleaves fibrinogen tofibrin that polymerizes to a meshwork.Thrombin is also the most potent activator ofplatelets via protease-activated receptor-1(PAR-1) activation (see Section 4.4). The poly-merized fibrin meshwork traps aggregatedplatelets, red blood cells, and other plasmaparticles to give rise to a fast growing throm-bus that gains further rigidity andmechanicalstrength by factor XIIIa-mediated cross-link-ing reactions [28].

2.3. Risk Stratification and TherapeuticGuidelines for ACS

Risk stratification criteria have been devel-oped to allow the clinician to make timelydecisions to choose appropriate modality oftreatment for patients presenting signs ofACS [29,30]. Based on electrocardiogram,ACS patients are classified as having eitherunstable angina/non-ST-segment elevationmyocardial infarction (UA/NSTEMI) or STsegment elevation myocardial infarction(STEMI). UA andNSTEMI are closely relatedconditions whose pathogenesis and clinicalpresentations are similar but of differing se-verity. They are caused by reducedmyocardialperfusion resulting from a partially occlusivethrombus that has developed on a disruptedatherosclerotic plaque [31]. UA and NSTEMI

show similar and often unremarkable ECGbut differentiating levels of cardiac biomar-kers. STEMI, on the other hand, signals anacute myocardial infarction due to the suddenthrombotic occlusion of a coronary artery.STEMI usually develops to a final diagnosisof Q-wave MI, and NSTEMI often develops toa non-Q-wave MI.

According to the American College of Car-diology2007guidelines for themanagement ofpatients with STEMI, the use of percutaneouscoronary intervention (PCI) with stent im-plantation is recommended alongwith the useof anticoagulants such as heparin and anti-platelet agents such as aspirin, clopidogrel,and GP IIb/IIIa antagonists [32,33]. If PCI isnot an option, the patients are treated withclot-dissolving fibrinolytics in addition to an-ticoagulants and antiplatelets [34]. For pa-tients presenting with symptoms of UA andNSTEMI, a decision should be made to man-age the patient either using antithromboticagents or using diagnostic angiography; in thelatter case, further decision should bemade totreat the patient with medication, PCI, orcoronary artery bypass graft (CABG). Antipla-telet agents such as aspirin, clopidogrel, andGP IIb/IIIa antagonists are indicated for bothpatients undergoing PCI and those who arebeing medically managed for UA andNSTEMI [35].

2.4. Antiplatelet Agents as a Major Classof Antithrombotic Drugs

Antithrombotic agents are the mainstay ofpharmacological therapy for thromboticdisorders (Table 1) [36–38]. Thrombosis, alocalized clotting of blood, can occur either inthe arterial or in the venous segment of thecirculation system, and depending on the localmilieu thrombus composition and pathophy-siology may vary and need to be treated withdifferent antithrombotic agents [39,40]. Me-chanistically, the function of an antithrombo-tic agent is to prevent the formation of thrombiin the blood vessels or to disrupt the existingones and restore blood flow [41–44]. In gen-eral, arterial thrombi have an abundance ofplatelets and venous thrombi have fibrin asthe major component. Depending on theirfunctional mechanism, the antithromboticagents can be divided into anticoagulants,

A BRIEF REVIEW OF THROMBOSIS 411

antiplatelet agents, and fibrinolytic agents.The anticoagulants either modulate the en-dogenous levels of the key coagulation enzymethrombin or inactivate the enzymatic functionof thrombin [45–47]. They are reviewed else-where in this volume. Antiplatelet agents in-hibit platelet activation and aggregation, akey process of hemostasis and thrombo-sis [48,49]. This topic is the subject of thecurrent review. Fibrinolytic agents, which areenzyme or protein preparations, are intrave-nously administeredunder clinical emergencyto cause lysis of an existing thrombus [50–52].A discussion of these agents is beyond thescope of this chapter.

3. MECHANISM OF PLATELETAGGREGATION

3.1. Functional Anatomy of Platelets

The role that platelets play in hemostasis is afundamental physiologic process [53,54].Normally, platelets circulate freely in bloodvessels without interacting with other plate-lets or the vascular endothelium. In thecontext of endothelial damage, a chain ofevents is triggered leading to aggregation ofplatelets. Depending on the nature of thevascular injury, this may lead to a normalhemostasis or a pathologic condition result-ing in vascular thrombosis, ischemic stroke,and so on. The underlying platelet events

leading to a pathologic condition constitutea complex series of biochemical and cellularprocesses that can be classified into four ca-tegories: adhesion, activation, secretion, andaggregation [55,56].

3.1.1. Adhesion The adhesion of plateletsto denuded endothelium represents the pri-mary hemostatic response to vessel wallinjury [57]. The endothelial cells act as acontinuous barrier between platelets andsubstances within the vessel wall that cancause platelet activation. Platelets do notinteract with normal endothelium, but reactavidly with subendothelial components of adamaged endothelium. When endotheliallayer disruption occurs as a result of avascular trauma, platelets adhere to theexposed endothelium to form a discontinu-ous platelet monolayer. This adhesion ismediated by ligands that are recognized byspecific platelet membrane glycoprotein re-ceptors, which are listed in Table 2. Thesereceptors can be divided into integrins andnonintegrins. Integrins are heterodimericcell-surface molecules composed of a- andb-subunits. Platelets express five a-subu-nits and two b-subunits of integrin, which,in varying combinations, identify distinctsurface receptors [58]. In the initiating stepof adhesion, a circulating, resting plateletmakes “contact” with the damaged vascularwall by interacting with subendothelial

Table 1. Classification of Major Marketed Antithrombotic Agents

Name Mechanism of action Formulation

AnticoagulantsWarfarin Vitamin K antagonist OralHeparin, danaparoid, dalteparin,

tinzaparin, enoxaparin, fondaparinuxIndirect thrombin inhibitors IV or SC

Lepirudin, bivaluridin, argatroban Direct thrombin inhibitors IVAntiplatelet agents

Aspirin COX-1 inhibition (inhibits TxA2 biosynthesis) OralAbciximab, eptifibatide, tirofiban GP IIb/IIIa receptor antagonists IVClopidogrel, ticlopidine, prasugrel P2Y12 (ADP) receptor antagonists OralDipyridamole PDE-V inhibitor Oral

Fibrinolytic agentsStreptokinase, alteplase, tenectaplase,

reteplasePromote plasmin activity IV


collagen [59]. This contact between the un-activated platelet and collagen is accom-plished by interaction between platelet sur-face glycoprotein Ib/IX/V complex and vWF,an accessory molecule that tethers plateletto subendothelial collagen [60]. In additionto vWF mediated binding to collagen, plate-lets also interact with collagen via GP VIreceptor and the integrin a2b1 receptor GPIa/IIa, the latter only after activation of theplatelets, thereby stimulating intracellularproduction and release of TxA2 and ADPinto circulation.

3.1.2. Activation A variety of biochemicalagonists that activate platelets by interactingwithplateletsurfacereceptors(Fig.3)arelistedin Table 3 [61]. Many of these agonists arereleased by platelets themselves after vesselwall adhesion, initiating a biological feedbackmechanism that amplifies the response to theinitialmechanicalorbiochemicalstimulus[62].In the adhesion phase, platelets change shape,spread along the fibrils and release TxA2 andADP into the circulation. The released TxA2

and ADP recruit additional platelets causing

Figure 3. Interaction of collagenwith platelet surface receptors. The initial tethering of platelets to collagenoccurs by the binding of vWF, an adhesive protein, to the platelet GP Ib receptor in the GP Ib/IX/V complex.Subsequent platelet interaction with GP VI and the integrin a2b1 causes release of TxA2 and ADP thatserve to amplify the activation signal.

Table 2. Platelet Surface Glycoproteins Involved in Adhesion

Receptor Ligand Integrins

GP Ia/IIa Collagen a2b1GP Ib/IX Von Willebrand factor —

GP Ic/IIa Fibronectin a5b1GP IV Thrombospondin, Collagen —

GP VI Collagen —

Vitronectin receptor Vitronectin, Thrombospondin Anb3VLA-6 Laminin A6b1

Table 3. Physiologic Agonists for Platelet Activation

Agonist Source Receptor(s)

Thrombin End product of coagulation cascade PAR-1, PAR-4, GP IbaAdenosine diphosphate Platelet dense body P2Y1, P2Y12

Collagen Subendothelium component GP Ia/IIa, GP IIb/IIIa, GP VISerotonin Platelet dense body 5HT2A receptorThromboxane A2 Platelets, other cells PGH2, TxA2 receptorPlatelet activating factor Lipid mediator produced by cells PAF receptor

MECHANISM OF PLATELET AGGREGATION 413

them to aggregate on top of the monolayer.Additionally, thrombin, serotonin, andplateletactivating factor (PAF) also activate plateletsvia their specific receptors (“outside-in sig-naling”) (Fig. 4) [63].

Among the various platelet stimulants,thrombin is themost potent activator of plate-

lets (Fig. 5) [64]. Thrombin is produced at thesite of injury by the activation of coagulationpathways. Vascular injury and inflammationexpose tissue factor resulting in the formationtissue factor/factor VIIa complex that leads tothe local generation of thrombin from pro-thrombin. Platelets also facilitate thrombin

Platelet

ADP

Epinephrine

Serotonin

Collagen

PAR-1

PAR-4

P2Y1

P2Y12

TxA2-R

5HT2A

GGPP IIIIbb

GGPP IIIIIIaa

GP VI

Platelet

GGPP IIIIIIaa

GGPP IIIIbb

Fibrinogen

GP

Thromboxane

Thrombin

Figure 4. Mechanism of platelet aggregation. Platelets have different cell-surface receptors that areactivated by specific agonists. Upon activation, these receptors trigger intracellular signal transductionmechanisms resulting in the activation of integrin receptors such as GP IIb/IIIa. Activated GP IIb/IIIareceptors bind to the RGD motif of fibrinogen thereby cross-linking activated platelets that leads to plateletaggregation that contributes to thrombus formation.

Figure 5. Thrombin plays a dual role in thrombosis by mediating coagulation and platelet activation.Thrombin cleaves soluble fibrinogen to fibrin that polymerizes to form an insoluble fibrin meshwork.Thrombin also is the most potent activator of platelets via PARs. Activated platelets aggregate and gettrapped by fibrin meshwork contributing to thrombus generation.


generation by providing procoagulant phos-pholipid surface that anchor various coagula-tion factors. Thrombin activates platelets atconcentrations in the pM range by interactingwith PARs and GP Iba (see Section 4.4).Thrombin plays a dual role in hemostasis andthrombosis [65]. In addition to activating pla-telets, thrombin also cleaves fibrinogen tofibrin that polymerizes to form a fibrin mesh-work that becomes an integral part of thethrombus.

3.1.3. Secretion Activation of plateletsprompts cytoskeletal rearrangements, mem-brane fusion, exterization, internal synthesisof TxA2 and its passive release acrossplateletmembrane, andexocytosis of granularcontents [66,67]. Platelets contain three typesof granules: (a) dense granules that containplatelet agonists that serve to amplify plateletactivation, (b) a-granules that contain adhe-sive proteins, and (c) lysosomal granules thatcontain glycosidases and proteases. The densegranules contain large amount of purine nu-cleotides (ADP and GDP), divalent cations(Ca2þ , Mg2þ , etc.), serotonin and pyropho-sphates [68]. As discussed above, ADP secre-tion following platelet activation helps recruitadditional platelets to the site of injury. Theplatelet a-granules contain platelet-specificgrowth factors, coagulation factors, and sev-eral glycoproteins [69]. The platelet-derivedgrowth factor (PDGF) promotes smooth mus-cle cell proliferation that occurs following pla-telet interaction with the disrupted vesselwall [70]. Platelet a-granules are a significantsource of coagulation factor V, which is themajor protein secreted following a-thrombinstimulation of platelets [71,72]. Activatedfactor Va, Xa, and Ca2þ are the principalcomponents of the prothrombinase complexthat cleaves prothrombin to a-thrombin [73].Platelets also release fibrinogen in high localconcentrations and plasminogen activatorinhibitor-1 (PAI-1) which inhibits the endo-genous fibrinolysis by tissue plasminogenactivator (tPA) andurokinase-type plasmino-gen activator (uPA) [74–76]. Additionally,platelets secrete the anticoagulant proteinS that is the cofactor for protein C-mediatedinhibition of factors V and VIII, as part of thehemostatic balance. The platelet a-granule

glycoprotein thrombospondin is a modulatorof smooth muscle cell proliferation [77].There is also an internal storage pool of GPIIb/IIIa receptors within the a-granules [78].Following activation, they are expressed onthe platelet surface increasing the total num-ber of surface GP IIb/IIIa receptors [79]. Thelysosomes contain a number of acid hydro-lases (cathepsins) that digest endocytosedmaterial. Lysosome secretion occurs moreslowly than secretion of dense granule anda-granule [80,81].

3.1.4. Aggregation Activation of plateletsand the ensuing intracellular biochemicalevents lead to the activation of surface GPIIb/IIIa receptors. GP IIb/IIIa is a member ofthe integrin family of receptors, composed ofa- and b-subunits (aIIb, b3). The a-subunitconsists of a heavy chain and a light chain.The heavy chain is entirely extracellular,while the light chain spans the platelet mem-brane, ending in a short extracellular do-main [82,83]. In the resting state, plateletGP IIb/IIIa receptors do not bind to fibrino-gen (or bind with a very low affinity) [84].Platelet activation alters the conformation ofGP IIb/IIIa, rendering it capable of binding toextracellularmacromolecular ligands includ-ing fibrinogen and vWF [85]. The arginine–glycine–aspartic acid (RGD) sequence of theadhesive proteins bind to the GP IIb/IIIareceptor [86,87]. Fibrinogen contains twoRGD sequences on its a-chain, one in theN-terminal region and the other in the C-terminal region, rendering itself bivalent inits binding to GP IIb/IIIa receptors, whichallows efficient cross-linking of platelets [88].Although vWF also binds to IIb/IIIa receptorat its various RGD sites, studies in fibrinogenknockout mice have shown that vWF alone isnot sufficient to achieve stable plateletaggregation [89].

In addition to the cross-linking ofplatelets, GP IIb/IIIa binding to fibrinogentriggers outside-in signaling events thatstimulate integrin-associated Src familyand Syk tyrosine kinase activities [90].Studies of platelets from mice lacking thesekinases suggest that these events are re-quired for full, irreversible platelet aggrega-tion (Fig. 6) [91].


3.2. Intracellular Mechanisms of PlateletActivation

Themajor platelet surface receptors thatmed-iateactivationthroughoutside-insignalingareG-protein coupled [63]. GPCRs offer severaladvantages in an efficient platelet activationmechanism. Several of the platelet GPCRsbind their ligands with high affinity. In somecases, each occupied receptor canactivatemul-tiple G-proteins, allowing amplification of asignal thatmight begin with a relatively smallnumber of receptors which range from a fewhundred to a few thousand per platelet. GPCRsignal transduction mechanism also enables

some receptors to signal through more thanone effector pathway (see Table 4) [92–97]. Fi-nally, since several generic mechanisms existfor the abrogation GPCR activation, efficienton-demand inhibitionof platelet activation canbeeffectedunderinappropriatecircumstances.

Table 4 lists the platelet GPCRs alongwiththeir agonists and the major G-protein fa-milies involved in intracellular signaling [84].Activation of platelet GPCRs results in trig-gering two sets of intracellular signal trans-ductionmechanisms. These are phosphoinosi-tide pathway and phospholipase pathway(Fig. 7). The phosphoinositide pathway is in-

Table 4. Main Platelet GPCRs

Agonist Receptor(s) G-protein Effectors Function

ADP P2Y1 Gq PLC "IP3/DAGP2Y12 Gia Adenyl cyclase #cAMP

Thrombin PAR-1 G12 or G13 Rho GEFs Actin cytoskeleton rearrangementGq PLC "IP3/DAGGi Adenylyl cyclase #cAMP

PAR-4 Gq PLC "IP3/DAGG12 Rho CEFs Actin cytoskeleton rearrangement

TxA2 TPa and TPb Gq PLC "IP3/DAGG12 Rho CEFs Actin cytoskeleton rearrangementG13 RhoCEFs Actin cytoskeleton rearrangement

Epinephrine a2A-adrenergic Gzb Adenylyl cyclase #cAMPSerotonin 5HT2A Gq PLC "IP3/DAGPGI2 IP Gs Adenylyl cyclase #cAMP

aThe major Gi protein activated by P2Y12 is Gi2.bGz is a member of Gi family. [103]

Figure 6. Electron micrograph of aggregated platelets trapped by fibrin meshwork in a thrombus. Otherplasma particles such as red blood cells are also shown. From Weisel JW and Veklich Y, University ofPennsylvania. Reproduced with permission.


itiated with the activation of phospholipase C,which cleaves phosphatidylinositol bipho-sphate (PIP2) to form inositol triphosphate(IP3) and diacylglycerol (DAG) [98]. IP3 acti-

vates receptors on calcium storage organellesknown as the dense tubular system, analogousto sarcoplasmic reticulum of muscle, leadingtomobilization of ionized calciumand increas-

Figure7. The two intracellularmechanismsof platelet activation.Thephosphoinositidepathway is initiatedwithactivationofPLCthat cleavesPIP2 to IP3 andDAG. IP3 stimulates [Ca2þ ] from thedense tubular systemthat results in the activation of MLCK, which phosphorylates MLC toMLC-PO4. The other cleavage productDAG stimulates PKC which phspophorylates the intracellular protein P47 to P47-PO4. The arachidonic acidpathway is stimulated by increased intracellular [Ca2þ ] that causes activation of PLA2 to release AA fromphospholipids. AA is converted by CO enzymes to PGG2 and PGH2 that are substrates for thromboxanesynthetase (TS) to produce TxA2 (see also Fig. 8). TxA2 is passively diffused across platelet membrane. MLC-PO4 and P47-PO4 stimulate secretion of dense a-granules and lysosomal granules. The specific intracellularevents that lead to the activation of GP IIb/IIIa receptors are not known with certainty but the early eventsinclude activation of PKC, rise in cytosolic [Ca2þ ] and protein phosphorylations. Alternative effector path-ways mediated by adenyl cyclase and RhoGEFs (Table 4) are not shown. Abbreviations: PLC, phospholipaseC; PIP2, phosphatidylinositol-4,5-biphosphate; DAG, diacylglycerol; IP3, inositol-1,4,5-triphosphate; MLCK,myosin light-chain kinase; MLC, myosin light chain; PLA2, phospholipase A2; CO, cyclooxygenase; AA,arachidonic acid; PGG2, prostaglandin G2, PGH2, prostaglandin H2; TS, thromboxane synthetase;TxA2,thromboxane A2.


ing its cytoplasmic concentration [99]. In-creased cytoplasmicCa2þ concentration leadsto activation of myosin light-chain kinase(MLCK) that phosphorylates myosin lightchain (MLC) to form MLC-PO4. The othercleaved product of PIP2, diacylglycerol, stimu-lates protein kinase C (PKC) which, in turn,phopshorylates the intracellular protein P47to P47-PO4.

ThesecondpathwayinvolvesphospholipaseA2, which is activated by the increased cyto-plasmic Ca2þ concentration. Activated PLA2

produces arachidonic acid (AA) by cleavage ofmembranephospholipidswhich, inturn, iscon-verted by the oxidative cyclooxygenase en-zymes to prostaglandin endoperoxides PGG2

andPGH2 (seeFig. 8). These intermediates areconvertedby thromboxane synthetase toTxA2.TxA2, MLC-PO4, and P47-PO4 together stimu-late secretion of products of the dense a-gran-ules and lysosomal granules.

The intracellular events that link plateletactivation to activation of integrin receptorssuch as GP IIb/IIIa are not known with cer-tainty. In general terms, the early eventsinclude activation of phospholipases result-ing in increase of cytosolic Ca2þ concentra-tion and activation of PKC. Late eventsinclude several cytoskeletal proteins andRap1b and various guanine nucleotide ex-change factors [100,101].

Among the various platelet stimulants,those having the greatest physiological re-levance are the serine protease thrombin,ADP, thromboxane A2, and epinephrine.Epinephrine is the only one among thesethat does not result in platelet shapechange [102].

Activated platelet surface also providesa platform for procoagulant activities byexpressing specific plasma membrane re-ceptors for factors XI, IX, VIII, X, V, andtheir activated forms [104]. These plateletreceptors, functioning in close physicalproximity, enhance the kinetics of activa-tion of factors XIa, IXa, Xa, and finally,thrombin from prothrombin. Thrombin, asmentioned, plays its dual procoagulant andproaggregatory roles in thrombosis by gen-eration of fibrin from fibrinogen and byactivation of platelets via proteolytic activa-tion of PARs.

3.3. Endogenous Regulatory Mechanismfor Controlling Platelet Activation

There are a number of internal factors thatsuppress unwanted platelet activation, con-tributing to a proper hemostatic balance. Inaddition to serving as a physical barrier andproviding vWF, endothelium releases a vari-ety of internal factors such as prostacyclin andNO (originally known as endothelium-derivedrelaxing factor (EDRF)) that have antiaggre-gatory effect on platelets and vasodilatoryeffect on endothelium. These molecules sup-press intracellular signaling in platelets byraising cAMPand cGMPlevels. cGMP inhibitscAMP-phosphodiesterase in platelets contri-buting to cAMP level potentiation. The anti-platelet agent dipyridamole (see Section 4.6.3)inhibits cAMP PDEs increasing the level ofintracellular cAMP. cAMP inhibits plateletactivation by activating protein kinase Awhich phosphorylates multiple platelet pro-teins such as GP Ib b-chain, actin bindingprotein filamin, myosin light chain, vasodila-tor-stimulated phosphoprotein (VSP), and soon.Additionally, endothelial cells release ecto-ADPase3, CD39 on their luminal surface.CD39 can hydrolyze small quantities of ADPthat is released from damaged red cells andactivated platelets, preventing the ADP fromactivating additional platelets. Other barriersto unwanted platelet activation include thediluting effects of continued blood flow, thepresence of endogenous anticoagulants suchas protein C and protein S that limit thrombinformation and the short half-life (<1min) ofthromboxane A2 that helps confine its effectslocally. These endogenous antiplatelet me-chanisms keep the platelet activation processfrom going amuck and maintain a proper bal-ance to hemostasis.

4. ANTIPLATELET AGENTS

4.1. MajorMarketed Antiplatelet Agents andAntiplatelet Agents Under Development

Antiplatelet agents have been themainstay oftreatment for ACS over the past 40 years withwell-established benefit in preventing coron-ary thrombosis and myocardial infarction inpatients with ACS. Results of a meta-analysis


of 145 clinical studies reported a 25% reduc-tion of cardiovascular events in high-risk pa-tients treated with antiplatelet therapy [105].Another meta-analysis of 109 clinical trialshas concluded that antiplatelet medicationswere able to reduce ischemic attacks andstrokes by 22%, reduce coronary artery dis-ease by 29% and reduce peripheral arterydisease by 23% [106]. Table 5 summarizes themajor antiplatelet agents available commer-cially and promising compounds in late stagedevelopment. A detailed discussion of theseagents is provided in this section.

4.1.1. Thromboxane Antagonists During pla-telet activation, the intracellular signalingevents lead to the activation of phopholipaseA2 that cleaves inner membrane phospholi-pids releasing arachidonates (see Section 3.2).Arachidonates are metabolized by cyclooxy-genase-1 enzyme to the prostaglandin endo-peroxide PGH2 that is converted by TxA2

synthase to TxA2 (Fig. 8) [107]. Once synthe-sized, TxA2 diffuses across the plasma mem-brane into circulation causing further plateletactivation by binding to cell-surface TP recep-tors. Like ADP, TxA2 amplifies the initialstimulus for platelet activation and helps re-

cruit additional platelets. As mentioned be-fore, TxA2-mediated effect is exerted only lo-cally, limited by the short half-life (<1min) ofTxA2, which helps confine the spread of plate-let activation to the area of injury.

In human platelets there are two splicevariants of TxA2 receptors (TPa and TPb) thatdiffer in their cytoplasmic tails. TPa and TPb

couple toGqa, G13a, andG12 family of proteins,all of which activate PLC. The TPa form ispredominant in platelets and is not expressedin endothelial cells, whereas TPb is found inendothelial cells [108]. TP receptors have beenalso found in bronchial smooth muscle andmesangial cells of glomeruli. In addition toTxA2, the prostaglandin endoperoxide PGG2

andPGH2also bind to thromboxane receptors.Two stable analogs of PGH2, U44069 andU46619, are known potent agonists of throm-boxane receptors [109].

4.1.2. Aspirin: A Thromboxane A2 BiosynthesisInhibitor Aspirin inactivates the cyclooxy-genase enzymes that convert arachidonate tothe prostaglandin intermediate PGH2. TheCOX-1 isoform, present in all tissues, repre-sents the constitutive form of the enzyme,whereas the COX-2 isoform is expressed in

Table 5. MajorMarketed Antiplatelet Agents andCandidates in Advanced Stage of Development

Generic Name or Code Name Brand Name Formulation Status

Cyclooxygenase inhibitorAcetyl salicylic acid Aspirin Oral LaunchedADP antagonistsTiclopidine Ticlid� Oral LaunchedClopidogrel Plavix� Oral LaunchedPrasugrel Effient� Oral LaunchedCangrelor — IV P-IIITicagrelor (AZD6140) — Oral P-IIIGlycoprotein IIb/IIIa antagonistsAbciximab Reopro� IV LaunchedEptifibatide Integrilin� IV LaunchedTirofiban Aggrastat� IV LaunchedThrombin receptor (PAR-1) antagonistsSCH 530348 — Oral P-IIIE5555 — Oral P-IIPhosphodiesterase-III inhibitorsCilostazol Pletal� Oral LaunchedAdenosine reuptake inhibitorsDipyridamole Persantine�, Aggrenox�

(in combination with aspirin)Oral Launched

ANTIPLATELET AGENTS 419

response to inflammatory conditions [110].PGH2 is a common substrate for a variety ofprostaglandin synthase enzymes (Fig. 8).WhereasPGH2 is produced in platelets almostsolely by COX-1, in endothelial cells bothCOX-1 andCOX-2 producePGH2. In platelets,PGH2 is converted by thomboxane A2

synthase to TxA2 whereas in endothelial cellsand smooth muscle cells, it is converted byprostaglandin I2 synthase to PGI2. Aspirinirreversibly acetylates Ser529 of COX-1 there-by inhibiting the production of TxA2 withinthe platelet [111]. In a similarmanner, aspirinacetylates Ser516 of the COX-2 enzyme, albeitat a 170-fold less potency [112,113]. Since

platelets lack the ability to resynthesize freshquantities of the COX-1 enzyme, its inactiva-tion and the resulting inhibition of the produc-tion of TxA2 persist for the lifetime of platelets(approximately 10 days). Nonsteroidal anti-inflammatory agents (NSAIDs) also inacti-vate COX-1 but without covalently modifyingthe enzyme [114]. It has been demonstratedthat NSAIDs such as ibuprofen prevent theantiplatelet effect of aspirin by stericallyblocking the Ser529 acetylation [115,116].

Theeffect ofaspirin inplatelets functionallycontradicts its effect in endothelial cells.Whereas aspirin is antithrombotic in platelets,it is prothrombotic in endothelial cells due tothe inhibition of COX-1 that produces PGH2

that is converted by prostaglandin I2 synthaseto PGI2 (prostacyclin). PGI2 is a potent endo-genous inhibitor of platelet activation and avasodilator in vascular beds via stimulation ofadenyl cyclase resulting in increased intracel-lular cAMP levels. However, unlike plateletsthat lack transcriptional machinery to gener-ate new enzyme, the endothelial cells recoverCOX-1 activity shortly after exposure to aspir-in. As a result, the antithrombotic effect ofaspirin in platelet persists whereas the pro-thrombotic vascular effect is transient.Aspirin in Acute Coronary Syndrome The useof aspirin for the prevention of coronarythrombosis has been known since1948 [117]. During the past few decades, as-pirin has undergone several landmark clinicalstudies that have established the cardiovas-cular benefit of aspirin that is nearly un-matched by any other pharmaceuticalagent [118]. While several early clinical stu-dies of aspirin in patients with previous myo-cardial infarction had shown trends towardimproved outcome, the first definitive clinicaltrial of aspirin in ACS patients was the Veter-an Administration Cooperative Study whichshowed that treatment with aspirin reducedthe risk of cardiovascular death or MI by51% [119]. Several subsequent placebo-con-trolled studies reinforced the findings of thisinitial study [120,121]. The Second Interna-tional Study of Infarct Survival (ISIS-2), alarge study on ACS patients with ST-segmentelevation myocardial infarction (STEMI),showed a significant 23% relative reductionin vascularmortality relative to placebo [122].

AA

COX-1 orCOX-2

PGH2, PGG2

TxA2

synthasePGI2

synthase

PGE2

synthase

PGF2?synthase

TxA2

PGD2

synthase

PGI2 PGD2PGE2PGF2?

PLA2

Cell membrane

Figure 8. Biosynthesis of thromboxane A2 andother prostanoids. The biosynthesis of TxA2 is in-itiated upon activation of PLA2 following plateletstimulation. PLA2 cleaves membrane phosphogly-cerides to release arachidonate (AA) that is con-verted by COX enzymes (mainly COX-1) to the en-doperoxides prostaglandin H2 (PGH2) and prosta-glandin G2 (PGG2). TxA2 synthase converts theseendoperoxides to TxA2. Once synthesized, TxA2

passively diffuses across platelet membrane intothe surrounding milieu, activating other platelets.PGH2 is a common substrate for a variety of pros-taglandin synthase enzymes in several cell types. Inendothelial cells and smooth muscle cells, prosta-glandin I2 synthase converts PGH2 to PGI2 that actsas anendogenous inhibitor of platelet activationandas a vasodilator in vascular beds.Whereas COX-1 isthe predominant isozyme in platelets, COX-2 ispresent in other cell types such as endothelial cellsand smooth muscle cells.


Aspirin in Secondary Prevention Severalclinical studies have been undertaken toevaluate the benefit of aspirin therapy inpatients who had undergone a previousincidence of MI. The Aspirin MyocardialInfarction Study (AMIS) found no signifi-cant difference in the primary endpoint of3-year mortality in patients who had suf-fered at least one previous episode of MI[123]. However, a combined meta-analysisof 11 placebo-controlled post-MI-trials indi-cated a significant benefit of aspirin in re-ducing nonfatal myocardial infarction [124].Based on this analysis and benefits fromprimary prevention trials, chronic antipla-telet therapy using aspirin is recommendedin patients who have undergone a thrombo-tic coronary event.Aspirin in Primary Prevention A number ofstudies have been conducted to evaluate thebenefit of aspirin in preventing cardiovasculardeath and myocardial infarction in healthyindividuals without a history of prior coronaryevent or stroke. The results of these studieshave been mixed. A British study found nosignificant benefit of aspirin [125]. However, asimilar U.S. trial found that aspirin produceda significant 44% reduction of first heart at-tack, although no change in overall cardiovas-cular mortality was found [126]. The outcomeof theWomen’s Health Study demonstrated alow dose regimen of aspirin had no significanteffect on nonfatal MI or death, but a 17%reduction in the risk of stroke versus placebowas noted [127]. However, further analysis ofthis study indicated that women of 65 years orolder did indeed show a benefit in the reduc-tion of MI, ischemic stroke, and major cardi-ovascular events. In conclusion, the benefit ofaspirin in primary prevention is dependentupon the level of risk of the treatment group,and a broad generalization is difficult. Sincetreatment with aspirin poses serious hemor-rhagic side effects, especially gastrointestinalbleeding, the potential benefit of treatment inprimary prevention should be weighedagainst the risk.Aspirin Resistance The term “aspirin resis-tance” has been often used to define clinicalnonresponsiveness to aspirin treatment[128,129]. However, a more precise definitioninvolves the failure of aspirin to elicit the

desired pharmacological effect of inhibitingthe biosynthesis of TxA2. Laboratory mea-surement of aspirin resistance involves var-ious techniques that measure platelet aggre-gation response to an agonist such as arachi-donic acid, indirect measurement of TxA2 le-vels by measuring its stable metabolites TxB2

and 11-dehydroTxB2 using radioimmunoas-say, and so on [130].

Several mechanisms have been proposedfor aspirin resistance [131]. Drug–drug inter-action with nonsteroid anti-inflammatoryagents is perhaps the best established mole-cular mechanism of laboratory aspirin resis-tance. For example, ibuprofen binds to COX-1enzyme in steric proximity to aspirin bindingsite in the narrowhydrophobic channel, there-by preventing aspirin from accessing its ownbinding site. A COX-1 genetic polymorphismhas been implicated as the genetic cause ofaspirin resistance [132]. Other probablecauses of aspirin resistance involve highplatelet turnover, an unusual recovery of theability of platelets to synthesize TxA2 by denovoCOX-1 synthesis (seeSection4.2), type-IIdiabetes-caused impairment of Ser529 acetyla-tion due to increased glycation of plateletproteins, and so on [133,134].

There is an increasing body of clinicalevidence that positively correlates labora-tory aspirin resistance to adverse cardiovas-cular events. In a meta-analysis of 20 stu-dies that related laboratory aspirin resis-tance to cardiovascular outcome, adversecardiovascular events occurred in 39% ofthe patients in the aspirin-resistant cohortversus 16% of the aspirin-sensitivepatients [135].

The clinical relevance of aspirin resistancehas been assessed inACS and stroke patients.The prevalence of aspirin nonresponsivenessranged from 5% to 65% among various studygroups that were subjected to a meta-analysis(mean 27%) [136,137]. In this study, a signifi-cant association between a laboratory-definedaspirin resistance and occurrence of cardio-vascular events was noted.

In conclusion, based on a large number ofclinical studies and meta-analyses conductedover a period of time, a clear trend seems toemerge that connects impaired aspirin sensi-tivity to cardiovascular risk.


4.1.3. Thromboxane A2 Receptor AntagonistsSeveral potent, orally active thromboxane re-ceptor (TP) antagonists such as ifetroban(BMS 180291), sulotroban (BS 13.177), andGR 32191 have undergone various clinicalstudies. However, despite the preclinical de-monstration of antithrombotic effects inanimal models, clinical studies of these candi-dates have yielded disappointing results, anddevelopment appears to have beendiscontinued [138,139].

The only thromboxane receptor antago-nist that still remains in clinical studiesis terutroban (S18886) (Fig. 9). A TPreceptor antagonist such as terutroban hasthe potential advantage over a conventionalantiplatelet agent by virtue of its dual plate-let antiaggregatory property and anti-in-flammatory effect mediated by endothelialTP receptors. These effects of terutrobanhave been demonstrated in clinicalsettings [140].

In the binding assay, racemate terutroban(S18204) exhibitedhighaffinity for thehumanplatelet TP receptor (Ki¼ 0.82nM). In a plate-let aggregation functional assay, terutrobanshowed poent inhibition of TxA2 agonist-in-duced human platelet aggregation (IC50¼0.23mM) [141]. In a preclinical ex vivo porcineplatelet aggregation inhibition study, terutro-ban at 100mg/kg/day showed more potent in-hibition of ADP-induced platelet aggregationthan clopidogrel at 3mg/kg/day and was aseffective as clopidogrel in inhibiting collagen-induced platelet aggregation [142]. In a por-cine ex vivo arteriovenous (AV) shuntmodel ofthrombosis, terutroban (0.3mg/kg) showedpotent oral antiplatelet effect, comparable tothat of clopidogrel (10mg/kg) plus aspirin

(5mg/kg), with reduced bleeding [143]. Teru-troban also exhibited antithrombotic activityin a dog model of acute platelet-mediatedthrombosis in coronary artery with endothe-lial damage.With regard to its vascular effect,terutroban inhibited atherosclerotic lesion inapoE-deficientmice and induced regression ofatheroscleoris in rabbits [144,145].

In human clinical trials, terutroban causeddose-dependent inhibition of ex vivo plateletaggregation in patients with peripheral arter-ial disease (PAD) and improved endothelialfunction in patients with CAD treated withaspirin [146–148]. In a small clinical studydesigned to measure the anti-inflammatoryeffect of terutroban, treatment with terutro-ban in addition to aspirin for 15 days providedlong-term improvements in endothelial func-tion in patients with severe carotid arteryatherosclerosis [149]. A significant improve-ment in flow-mediated vasodilatation was ob-servedwith all terutroban doses starting fromthe first day of dosing and sustained through-out the treatment period. Terutroban iscurrently undergoing a large, multicenterPhase-III clinical study for the prevention ofcerebrovascular and cardiovascular events ofischemic origin in patients with a historyof ischemic stroke or transient ischemic attack(PERFORM study) [150].

4.1.4. Thromboxane A2 Synthase Inhibitor In-hibition of thromboxane A2 synthase that con-verts PGH2 to TxA2 (Fig. 8) was thought to bean attractive strategy to block TxA2-mediatedplatelet activation. Theoretically, this ap-proach could have a dual effect of causing thebuildupofPGH2and inhibiting theproductionof thromboxane A2 synthase [151]. Elevatedlevels of PGH2 could stimulate the synthesisof PGI2 that is an endogenous antiplateletagent and a vasodilator (see Section 4.2.1).Predicated on this expectation a number ofthromboxaneA2synthaseinhibitorshavebeendeveloped, including ozagrel (OKY-046), pir-magrel (CGS-13080), dazoxiben (UK-37248),isbogrel (CV-4151), furegrelate (U-63557A),dazmagrel, camonagrel, and so on. Numer-ous clinical trials have been conducted fortheir cardiovascular utility; however, onlywith disappointing outcome [152]. One plau-

NH

S

Cl

O

O

COOH

CH3

R

1

Figure 9. Structure of terutroban (S18886).


sible mechanistic explanation for this disap-pointing outcome is that as PGH2 levelsbuild up it also binds to the thromboxaneA2 receptor and produces similar pharmaco-logical effects [153]. Several clinical studieshave also found incomplete inhibition ofTxA2 production [154]. Besides, severalagents had off-target activity inhibitingP450 enzymes and nitric oxide synthase.

4.1.5. Dual Thromboxane A2 Synthase Inhibi-tor/Thromboxane A2 Receptor AntagonistCompounds with dual thromboxane A2

synthase inhibition and thromboxane A2 re-ceptor antagonismwerehoped tomaintain theantiaggregatory property of a TxA2 receptorantagonist and the vasodilatory effect of PGI2that would be produced from PGH2. Based onthis concept, a number of dual thromboxanemodulators have been developed such as pi-cotamide, KDI-792, terbogrel, ridogrel, andBM-573. Picotamide has undergone two largeclinical studies in patients at risk of athero-thrombosis [155]. Although the first study didnot show a significant reduction of cardiovas-cular events by picotamide, a subgroup ana-lysis showed its potential usefulness in pa-tients with diabetes. A subsequent study indiabetic patients showed a significant reduc-tion of overall mortality in the picotamidegroup [156]. KDI-792 increased lower limbblood flow in type II diabetic patients indicat-ing its vasodilatory effect. The pharmacoki-netic and pharmacodynamic evaluation of ter-bogrel in healthy human subjects showed thatat the highest dose tested (150mg) there wasan almost complete inhibition of thromboxanesynthase and thromboxane receptor occu-pancy [157]. This compound is currently underclinical evaluation as an antithromboticagent. However, one study evaluating the useof terbogrel in patients with pulmonary hy-pertensionwas stoppeddue to the side effect ofleg pain [158]. In a comparative clinical studyof ridogrel versus aspirin in patients on strep-tokinase background therapy, ridogrelshowed lower incidence of new ischemicevents (reinfarction, recurrent angina, andischemic stroke) on post hoc analysis, withsimilar incidence of bleeding compared to as-pirin [159]. However, ridogrel was not super-ior to aspirin in enhancing fibrinolytic efficacy.

Another dual agent, BM-573, has demon-strated protective effect in a pig model ofcoronary thrombosis [160].

4.2. Purinergic Receptor Antagonists

The purinergic class of receptors recognizesnucleic acids and their metabolites. The P1purinoreceptors are activated by adenosine.The P2 receptors are activated by adenosinetriphosphate and adenosine diphosphate.There are three subclasses of P2 receptors onthe platelet membrane that are relevant tohemostasis: P2X1, P2Y1, and P2Y12 (Fig. 10).The P2X1 receptor is a ligand-gated ion chan-nel which allows calcium influx upon activa-tion by ATP resulting in fast, reversible plate-let shape change, secretory granule centrali-zation, and pseudopodia formation [161]. TheP2Y1 and P2Y12 are G-protein-coupled recep-tors that are activated by ADP. Activation ofP2Y1 receptor leads to the mobilization ofintracellular calcium stores that result inshape change and aggregation. Activation ofP2Y12 receptors leads to inhibition of intracel-lular adenyl cyclase, initiating a biochemicalsignal cascade that result in platelet activa-tion and aggregation [162].

4.2.1. P2X1 Receptor The role of ATP-stimu-lated P2X1 receptors in human platelets isunclear. They may act alone or in synergywith other pathways to accelerate and en-hance calcium mobilization, shape change,and aggregation [163]. It has been recentlyshown that selective inhibition of P2X1 re-ceptors substantially offsets the intracellularcalcium increase caused by collagen, throm-boxane A2, thrombin, and ADP [164]. Knock-out studies have shown that P2X1

�/� micedisplayed resistance to thrombosis caused bylaser-induced vessel wall injury [165]. On theother hand, overexpression of platelet P2X1

receptor in transgenicmice generated a novelprothrombotic phenotype [166]. P2X1 recep-tor has not received much attention as anantithrombotic drug target. Given the cen-tral role of intracellular calcium in plateletactivation and the rapid release of largeamounts of ATP from damaged cells at sitesof vascular injury, the inhibition of P2X1

receptor represents an untapped possibility


as an adjuvant or a stand-alone antiplatelettherapy [167].

4.2.2. P2Y1 Receptor The P2Y1 receptor wasthe first P2 subtype receptor to be cloned andidentified as a metabotropic adenine nucleo-tide receptor (Fig. 10) [168,169]. Unlike P2Y12

receptor that is expressed only inplatelets andbrain, the P2Y1 receptor is ubiquitously ex-pressed in endothelial cells, smooth muscle,epithelial cells, lungs, platelets, pancreas, andin the central nervous system [170]. The hu-man P2Y1 is a 373-amino acid longGPCR thatis coupled to Gq. Studies in mice lacking Gaq

have revealed that signaling through Gq isessential for platelet shape change [171]. Thisshape change is brought about by calcium-dependent and calcium-independent path-ways. The calcium-dependent pathway istriggered by activation of PLC, and the cal-cium-independent pathway involves activa-tion of RhoA and p160ROCK [172]. ADP ismorepotent at the P2Y1 receptor (EC50� 0.3 uM)than the P2Y12 receptor (EC50� 2uM). Thisdifference in potency explains the distinctADP concentration requirements for shapechange (mediated throughP2Y1) and aggrega-tion (mediated through P2Y12). Activation of

P2Y1 receptor leads to platelet shape changeand rapidly reversible aggregation. Combinedactivation of both P2Y1 and P2Y12 receptors isnecessary for the full ADP-mediated plateletaggregatory response [173].P2Y1 Antagonists The potential of P2Y1 re-ceptor as an antiplatelet target has been ad-dressed recently [174]. Platelets from micethat lack P2Y1 receptor neither change shapenor aggregate in response to ADP, except atvery high concentrations of ADP when partialaggregation occurs (P2Y12-mediated ef-fect?) [175]. It has been shown that P2Y1-deficient mice are resistant to acute throm-boembolism induced by intravenous adminis-tration of ADP [176]. In contrast, transgenicmice that overexpresses P2Y1 receptorsshowed in vitro platelet hyperactivity as evi-denced by increased aggregation response tolower concentration of ADP and collagen thanrequired in wild-type mice [177]. P2Y1 over-expressed platelets showed dense granularsecretion upon treatment with ADP, a phe-nomenon not demonstrated by wild-type pla-telets. Treatment of P2Y1 knockout mice withclopidogrel, a P2Y12 antagonist, showed anenhanced inhibition of thrombus formation,suggesting an additive effect between P2Y1

P2X1 P2Y1 P2Y12

G-Protein

GPCRGq

PLC/IP3

Shape changeTransient aggregation

[Ca2+]

Intrinsicion

channel

Na+/Ca2+

Shape changeaggregation

Molecularstructure

Secondarymessengersystem

Functionalresponse

G-Protein

GPCR

Gi

AC

Sustained aggregationsecretion

[cAMP]

Figure 10. Three subtypes of ADP receptors. ATP activates P2X1, a ligand-gated ion channel, triggeringrapid influx of Ca2þ , leading to platelet shape change and aggregation. P2Y1 and P2Y12 are GPCRs. P2Y1 iscoupled to Gq whose stimulation causes PLC activation and calcium-dependent shape change and transientaggregation. A calcium-independent mechanism leading to platelet shape via RhoA and p16ROCK has alsobeen proposed (not shown). The P2Y12 receptor is coupled to Gi whose activation leads to inhibition of adenylcyclase and a decrease in intracellular cAMP resulting in granular secretion and platelet aggregation.


and P2Y12 receptors. Overall, these resultsindicate that P2Y1 receptor plays an impor-tant role in thrombosis and hemostasis andmay provide a useful pharmacological targetfor intercepting platelet activation [174].

Preclinical development of small-moleculeP2Y1 antagonists has been reported [169].Adenosine 30,50-bisphosphate (A3P5P) andadenosine 20,50-bisphosphate (A2P5P) havebeen known to be competitive P2Y1 antago-nists [178]. Structural modification of thesenucleosides led to the identification of selec-tive P2Y1 antagonists MRS 2179 (Kd¼ 109nM), MRS 2279 (Ki¼ 9nM), and MRS 2500(Ki¼ 0.78nM) [179–181]. These small-mole-cule antagonists inhibited ADP-induced pla-telet aggregation and were efficacious in ar-terial thrombosis models [182]. Administra-tion of MRS 2179 to mice resulted in a sig-nificant decrease in localized thrombusformation in mensenteric arterioles exposedto a solution of FeCl3 [183]. Intravenous ad-ministration of MRS 2500 was effective atreducing arterial thrombosis in a laser-in-duced arterial injury model in mouse [177].The authors also have demonstrated an ad-ditive effect of P2Y1 and P2Y12 antagonism inpreventing thromboembolism in the samethrombosis model. There has been a recentreport of identification of orally active, non-nucleoside P2Y1 antagonists, exemplified bycompound 5 [184].

4.2.3. P2Y12 Receptor The P2Y12 receptoractivation is essential for ADP-induced plate-let aggregation under shear-stress condi-tions [185]. Unlike the P2Y1 receptor, theactivation of P2Y12 receptor by ADP does not

lead to PLC activation, intracellular Ca2þ

mobilization or platelet shape change(Fig. 9) [94,186,187]. The P2Y12 receptor iscoupled to Gi whose activation leads to inhibi-tion of PGE-1 stimulated adenyl cyclase and adecrease in intracellular cAMP resulting ingranular secretion and platelet aggregation.ADP antagonists clopidogrel and ticlopidineabolish ADP-induced adenyl cyclase inhibi-tion and platelet aggregation [188,189]. Plate-lets from P2Y12-null mice do not aggregatenormally to ADP, they fail to inhibit adenylcyclase, and do not respond to treatment withclopidogrel and ticlopidine although they re-tain P2Y1-associated responses such as shapechange and PLC activation [187,190]. In addi-tion, patients with abnormal P2Y12 receptorshave been identified with congenitally defec-tive platelet aggregation [191].P2Y12Antagonists TheP2Y12 antagonists area widely used class of antiplatelet agents,which has had a transformational impact onthe antiplatelet treatment of cardiovasculardisease. The two commercially establishedADP antagonists ticlopidine and clopidogrelbelong to a chemical class known as thieno-pyridine, which require cytochrome P450-mediated hepatic metabolism to active meta-bolites that selectively and irreversibly inac-tivate the P2Y12 receptor by covalent binding.As a result, they have a slow onset of action,which has been a major limitation. Results ofseveral large-scale clinical trials have estab-lished the utility of ticlopidine and clopidogrelas effective antiplatelet drugs for the treat-ment of ischemic cardiovascular and cerebro-vascular conditions, often with additivebenefit when used in combination with other

N

NN

N

NHMe

X

(HO)2OPO

OPO(OH)2

HO

N

N

NHMe

N

N

OPO3Na2Na2O3PO

3 X= Cl MRS 22794 X = I MRS 2500

2 MRS 2179

O HN

HN

O

OCF3

N N

Me

Cl

5

Figure 11. P2Y1 antagonists in preclinical development.


antiplatelet agents such as aspirin. A fasteracting thienopyridine, prasugrel has been cur-rently approved by regulatory agencies. Alsoin clinical trials are a new class of direct-act-ing, reversible P2Y12 antagonists such as can-grelor and ticagrelor that do not requiremeta-bolic activation. This section will briefly ex-amine the pharmacology and clinical trialresults of various ADP antagonists.Irreversible ADP AntagonistsTiclopidine Ticlopidine was the first ADP an-tagonist to be approved for clinical use as avaluable addition to the existing antiplatelettherapy. Ticlopidine is inactive per se againstthe P2Y12 receptor. It is metabolized by cyto-chrome P450 3A4 enzyme to 2-oxo-ticlopidineintermediate 7 that undergoes enzymatic hy-drolysis to the sulfhydryl active metaboliteUR-4501. The sulfhydryl metabolite is postu-lated to form disulfide linkage with cysteineresidues in theactive site, thereby irreversiblyinactivating the receptor. Oxo-ticlopidineand UR-4501 have been isolated and pharma-cologically characterized [192]. UR-4501 pro-duced a concentration-dependent inhibition(3–100uM) of ADP (10uM)-induced humanplatelet aggregation, whereas 2-oxo-ticlopi-dine (3–100uM) did not elicit inhibitoryresponses.

Ticlopidine is highly plasma protein bound(>98%), and its absorption has a favorablefood effect. About 90% of the drug is absorbedand it reaches peak plasma concentrationwithin 1–3h and shows an elimination half-life of 24–36h after a single dose. Due to theslow absorption and the need for metabolicactivation, ticlopidine (Fig. 12) has a slowonset of action and is therefore not the choiceof drug when a quick antiplatelet effect isneeded. A loading dose of ticlopidine has notbeen well studied, presumably because of theavailability of alternative safer therapy using

the second-generation thienopyridine clopido-grel (see below).

Ticlopidine has undergone multiple clini-cal studies in stroke, ACS, PCI, coronary ar-tery bypass surgery, and peripheral arterialdisease. The Ticlopidine Aspirin Stroke Study(TASS) compared ticlopidine with aspirin inpatients with a recent history of minor strokeor transient cerebral ischemia, and demon-strated that ticlopidine was superior to aspir-in in reducing incidence of stroke in 3 years offollow-up [193]. The Canadian American Ti-clopidineStudy (CATS) evaluatedmore thanathousand patients with a history of presumedthromboembolic stroke. The study demon-strated that ticlopidine reduced the combinedendpoint of vascular death, stroke, or myocar-dial infarction compared to placebo [194]. TheAfricanAmericanAntiplatelet StrokePreven-tion Study evaluated the effect of ticlopidineversus aspirin in patients with a recent non-cardioembolic ischemic stroke. In 2 years offollow-up, the incidence of primary combinedendpoint of vascular death,myocardial infarc-tion, or recurrent stroke was similar in ticlo-pidine and aspirin groups [195].

Ticlopidine has shown benefit in ACS pa-tients and in patients undergoing PCI. Addi-tion of ticlopidine to conventional therapywithout aspirin in patients with unstable an-gina resulted in a lower rate of vascular deathor myocardial infarction compared to the con-trol group [196]. The Intracoronary Stentingand Antithrombotic Regimen (ISAR) studywas done in patients undergoing coronaryangioplasty with stent implantation. The pa-tients were randomized to receive ticlopidineplus aspirin versus conventional anticoagu-lants. The 30-day incidence of combined end-point of cardiac death, myocardial infarction,and need for revascularization procedure waslower in the ticlopidine plus aspirin group

N

S Cl

N

S Cl

O450CYP N

HOOCHS Cl

6 Ticlopidine(Prodrug)

8 UR-4501metabolite)active(Ticlopidine

7 2-oxo-Ticlopidine

Figure 12. Metabolic activation of ticlopidine. The active metabolite is formed via CYP450 mediatedoxidation of thienopyridine to 2-oxo-ticlopidine followed by ring opening.


compared to the anticoagulant group [197].The effect of ticlopidine was also evaluated inthe Full Anticoagulation versus Aspirin andTiclopidine (FANTASTIC) trial. In this trial,patients were randomized to receive ticlopi-dine versus warfarin against background as-pirin therapy. The ticlopidine group had alower incidence of subacute stent occlusionwithin 1 week after PCI. However, the rateof early thrombosis within 24h was higher inthe ticlopidine group perhaps due to the slowonset of action of ticlopidine. In the StentAnticoagulation Restenosis Study (STARS),patients undergoing coronary angioplastywith stenting, ticlopidine plus aspirin wascompared with aspirin alone and aspirin pluswarfarin. The primary endpoint of a 30-daycomposite of death, myocardial infarction, thepresence of thrombus on a subsequent angio-gram, or the need for revascularization of thetarget lesion occurred significantly less in theticlopidine plus aspirin group compared to theother groups [198]. Ticlopidine has been alsodemonstrated to reduce the rate of acute oc-clusion of aortocoronary venous bypass graftscompared with placebo [199].

Ticlopidine has shown benefit in peripheralarterial disease also. In the Swedish ticlopi-dine Multicenter Study (STIMS) in patientswith intermittent claudication, a substantiallylower (34%) incidence ofmyocardial infarction,stroke, and transient ischemic attack (TIA)was noted compared to placebo [200]. Therehave also been a number of other clinical stu-dies that corroborate the beneficial effect ofticlopidine in patients with peripheral arterialdisease [201,202]. It is clear that ticlopidineaccords a broad range of benefits in ischemiccardiovascular and cerebrovascular disease.An analysis of the antiplatelet trialist’s colla-boration study demonstrated that ticlopidinegave 10% lowering of risk of cardiovascularevents compared to aspirin [203].

Despite the proven cardiovascular benefitof ticlopidine, it is seldom used today becauseof its hematologic side effects and due to theavailability of a safer second-generation thie-nopyridine ADP antagonist, clopidogrel (seebelow) [204]. The use of ticlopidine has beenassociated with aplastic anemia, neutropenia,thrombocytopenia, and thrombotic thrombo-cytopic purpura (TTP). In approximately 2%of

patients, ticlopidine causes severe neutrope-nia and in 0.02% patients the risk of TTP isseen which is associated with considerablemortality rate [205]. These hematological con-cerns necessitate routine blood monitoring inpatients at risk of these side effects. Due to itsinhibition of P450 2C19 isozyme, ticlopidine isalso known to increase the plasma concentra-tions of drugs that are metabolized by thisenzyme.Clopidogrel Clopidogrel is classified as asecond-generation thienopyridine ADP an-tagonist. Like ticlopidine, clopidogrel belongsto the thienopyridine structural class and it isalso a prodrug that requires cytochorme P450mediated activation. The only structural dif-ference between ticlopidine and clopidogrel isthe presence of a benzylic carbomethoxy groupwith (S)-configuration at the only stereogeniccenter. The active metabolite of clopidogrel(Fig. 13), identified by incubation of clopido-grel with human liver microsomes, maintainsthe original absolute configuration at thebenzylic stereogenic center and has a Z-con-figuration for the unsaturated carboxylicacid [206]. Since the active metabolite ishighly labile, its pharmacokinetic propertieshave not been determined. Approximately50% of clopidogrel is absorbed from the gastro-intestinal tract after an orally administereddose [207]. Clopidogrel is highly plasma pro-tein bound (98% in humans) and food hasno effect on its absorption. The plasma con-centrations of the parent are below the levelof detection after 2 h following an oral doseof 75mg [208]. The main circulating metabo-lite of clopidogrel is an inactive carboxylicacid (SR 26334) that constitutes 85% of thecirculating drug-related compounds in theplasma [207,209].

Dose-dependent inhibition of platelet ag-gregation can be seen 2h after a single oraldose of clopidogrel. After a 75mg oral dose ofclopidogrel, ex vivo antiaggregatory effect isdetected in plasma within two hours inhealthy volunteers [210]. Inhibition of plateletaggregation reaches a level of 40–60% at stea-dy state after 3–7 days of daily administrationof clopidogrel, 75mg. Following an oral dose of14C-labeled clopidogrel in humans, covalentbinding to platelets accounted for 2% of radi-olabel with a half-life of 11 days [211].


The use of a loading dose of clopidogrelreduces the time to achieve maximal inhibi-tory effect on platelet aggregation. In patientsundergoing PCI, a 300mg loading dose ofclopidogrel at the time ofPCIwas significantlymore effective than pretreatment for 2 dayswith 75mg/day at suppressing ADP-inducedplatelet aggregation as well as markers ofplatelet degranulation (P-selectin) and GPIIb/IIIa activation [212]. In patients under-going coronary angiography, a 600mg loadingdose of clopidogrel was associated with signif-icantly greater inhibition of ADP-induced pla-telet aggregation than a 300mg dose, but a900mg loading dose did not achieve greaterinhibition of ADP-induced platelet aggrega-tion [213]. Pharmacokinetic data showed nofurther increase in plasma level of clopidogrelor itsmetabolites at the900mg loadingdose incomparison to the 600mg loading. The 600mgloading dose also gave less interindividualvariability of platelet response [214].

It has been known that the free thiol groupof clopidogrel active metabolite forms a dis-ulfide bridge with extracellular cysteine resi-dues (Cys17 and Cys270) of the P2Y12 receptor,causing its inactivation [215–217]. Corrobor-ating this putative mechanism, the P2Y12 re-ceptor is known to be irreversibly inactivatedby the thiol reagent pCMBS [218]. Due to theirreversible nature of P2Y12 inactivation onplatelets, recovery of platelet function in pa-tients after thewithdrawal of thedrug is about10 days that corresponds to the lifespan of acirculating platelet.

Clinical Studies of Clopidogrel Clopido-grel is indicated for the reduction of athero-thrombotic events in patients with a history ofrecent myocardial infarction (MI), recentstroke, or established peripheral arterial dis-ease and for treatment of non-ST-segmentelevation acute coronary syndrome (unstableangina/non-Q-wave MI) and ST-segment ele-vation acute myocardial infarction [211].The recommended daily dose of clopidogrel is75mg once daily, which should be initiatedwith a loading dose of 300mg for patientswithnon-ST-segment elevation acute coronarysyndrome. As discussed above, higher loadingdoses have been studied to assess faster onsetof antiplatelet effect and reduce interindivi-dual variability.

Results of several large clinical trials haveestablished clopidogrel as an effective antipla-telet agent for the secondary prevention ofischemic events in patients with ACS, stroke,andother cardiovascular conditions [219,220].Since clopidogrel and aspirin exert their anti-platelet effects by complementary mechan-isms, their combination has been clinicallystudied and proven to have additive effect inpreventing ischemic events.

The clinical study that led to the launch ofclopidogrel as a major drug for the treatmentof atherothrombotic disease was Clopidogrelversus Aspirin in Patients at Risk of IschemicEvents (CAPRIE) [221]. It was a large trialthat included 19,185 patients. The patientswere randomized to receive 75mgdaily dose ofclopidogrel versus 325mgdaily dose of aspirin

N

S Cl

COOCH3

N

S Cl

COOCH3

ON

HOOCHS Cl

COOCH3

9 Clopidogrel(Prodrug)

11 activeClopidogrelmetabolite10 2-oxo-Clopdogrel

3A4CYP450S

N

S Cl

COOH

12 26334SRinactiveClopidogrel

metabolite

Esterase

Figure 13. Metabolism of clopidogrel. The labile active metabolite is generated via oxo-clopdiogrel inter-mediate which is formed by CYP450 mediated oxidation of clopidogrel.


for a duration of about 2 years. The primaryendpoint of the CAPRIE trial was a compositeof vascular death, myocardial infarction, andischemic stroke. The annual event rate of theprimary endpoint was lower in patients whoreceived clopidogrel than thosewhowere trea-ted with aspirin (5.32% events in the clopido-grel group versus 5.83% events in the aspiringroup). This corresponded to an 8.7% relativerisk reduction. Most of the risk reduction wasdue to the lower incidence of myocardial in-farction in patients who received clopidogrel.In this trial, clopidogrel was generally welltolerated, and the incidence of bleeding waslower among the patients who received clopi-dogrel than the patients who received aspirin.Thus, the CAPRIE trial demonstrated thatclopidogrel was an effective therapy for thesecondary prevention of ischemic vasculardisease.

TheClopidogrel inUnstable Angina to Pre-vent Recurrent Ischemic Events (CURE) trialwas the first major study that evaluated thebenefit of long-term use of clopidogrel plusaspirin versus aspirin alone in patients withacute coronary syndromes and non-ST-segment elevation MI (NSTEMI) [222]. Pa-tients presenting with UA or NSTEMI re-ceived a 300mg loading of clopidogrel followedby 75mg once daily for 3–12 months. Patientsin both groups received 75–325mg of aspirindaily. Clopidogrel was associated with a 20%relative risk reduction in the primary end-point of a composite of CV death, nonfatal MI,and stroke and 14% relative risk reduction inthe second coprimary endpoint (CV death,nonfatal MI, stoke, or refractory ischemia).For the individual outcome events, the rela-tive risk reduction for clopidogrel was 7% forCV death, 23% for nonfatal myocardial infarc-tion, 14% for stroke, and 7% for refractoryischemia. These findings were confirmed byseveral subsequent retrospective analyses ofCURE data. However, bleeding complicationswere significantly increased in the dual anti-platelet therapy arm that showed 3.7% bleed-ing versus 2.7% in the aspirin only group.

The PCI-CURE was a prospective study ina subgroup of patients in the CURE studywhounderwent PCI [223]. The study was aimed atdetermining whether the addition of clopido-grel to aspirin prior to PCI and then continued

on a long-termbasis after the procedurewouldreduce the incidence of major ischemic events.The primary endpoint was CV death, MI, orurgent target vessel revascularization(UTVR) within 30 days of PCI. Events in theprimary outcome cluster occurred 4.5% in pa-tients in the clopidogrel group compared with6.4% in the placebo group, showing a signifi-cant benefit for clopidogrel treatment. Long-term administration of clopidogrel after PCIwas also associated with a significant reduc-tion of cardiovascular death, MI, or revascu-larization compared to placebo.

In the Clopidogrel as Adjunctive Reperfu-sion Therapy-Thrombolysis in Myocardial In-farction 28 (CLARITY-TIMI 28) trial, patientspresenting within 12h after the onset of ST-segment elevation MI (STEMI) received clo-pidogrel (300mg loading dose followed by75mg daily maintenance) [224]. Essentially,all patients received thrombolytic therapyandaspirin, and most patients received heparinand/or underwent coronary angiography 2–8days after starting the treatment with thestudy drug. In the primary outcome cluster(occurrence of occluded artery on angiogra-phy, or death or recurrent MI before angio-graphy) there was a 36% relative risk reduc-tion in the clopidogrel treated group. In afollow-up evaluation, a composite endpointconsisting of CV death, recurrent MI, or re-current ischemia leading to UTVR was mea-sured. The incidence of this endpoint was11.6% among clopidogrel recipients comparedwith 14.1% among placebo recipients, reflect-ing significantly lower odds in the clopidogrelgroup.

PCI-CLARITY study analyzed patientswho underwent PCI in CLARITY-TIMI 28,with the aim to determine whether pretreat-ment with clopidogrel prior to PCI in patientswith recent STEMI was more effective thanclopidogrel started at the time of PCI in pre-venting major cardiovascular events [225].Results of the analysis showed that pretreat-ment with clopidogrel prior to PCI signifi-cantly reduced the incidence of major cardio-vascular events both before and after PCI,providing support for the early use of clopido-grel in STEMI.

The Clopidogrel Metoprolol MyocardialInfarction Trial/Second Chinese Cardiac


Study (COMMIT/CCS2) was a large studyconducted in patients presenting within24 h of the onset of the symptoms of suspectedMI with supporting ECG abnormalities[226,227]. Patients received 162mg dailydose of aspirin and 75mg clopidogrel for 28days or until discharged from hospital. Thecomparator group received 162mg aspirinper day. Patient groups that received clopi-dogrel showed significantly fewer incidenceof in-hospital death (7.5% compared to 8.1%of placebo recipients). A 9% relative risk re-duction was observed in a coprimary short-term combined endpoint of death, recurrentMI, or stroke, and 9% reduction in the pri-mary endpointwhich constituted a compositeof death, MI, or stroke.

The Clopidogrel for the Reduction of EventDuring Observation (CREDO) study was alarge prospective trial in the setting of electivePCI that evaluated the use of clopidogrel ther-apy prior to PCI and lasted for an extendedperiod of time following the procedure(Table 6) [228]. Patients were randomized toreceive a loading dose of 300mg of clopidogrelor placebo prior to undergoing PCI, and thosewho were on clopidogrel loading dose receiveda daily maintenance dose of 75mg of clopido-grel subsequent to PCI. Patients in bothgroups received aspirin throughout the study.For the 1-year composite endpoint of death,MI, or stroke, clopidogrel administration wasassociated with a significant 26.9% relativereduction in risk compared to the controlgroup. Overall, clopidogrel therapy up to 1year after PCI was associated with a lowerrate of adverse ischemic events. There wasalso a suggestion that administration of a300mg loading dose of clopidogrel prior to PCIwould show improved post-PCI outcome.

Following the favorable outcome of ticlopi-dine in cerebrovascular disease, the MATCHstudy was conducted on clopidogrel in high-risk patients with recent ischemic stroke orTIA to evaluate the effect of clopidogrel versusdual antiplatelet therapywith clopidogrel andaspirin [229]. The patients were randomizedto receive 75mg/day of clopidogrel concomi-tantwithaspirin orplacebo for 18months.Theprimary efficacy analysis indicated a non-significant reduction in ischemic stroke, MI,vascular death, or rehospitalization among

patients who received clopidogrel in additionto aspirin. However, there was an overall in-creased risk of life-threatening bleeding in thegroup that received the dual antiplatelet ther-apy. It appears that clopidogrel alone is super-ior to the combination therapy with similarefficacy, but less bleeding.

The Clopidogrel and Aspirin for Reductionof Emboli in Symptomatic Carotid Stenosis(CARESS) trial was done on a small numberof patients (107)who had symptomatic carotidstenosis and transcranial Doppler evidence ofmicroembolization [230]. All patients receivedaspirin. A repeat examination on day 7 re-vealed that patients on the dual antiplatelettherapy had fewer microemboli compared tothe aspirin monotherapy group.

Anumber of studies are ongoing thatmightprovide further insight into the combination ofclopidogrel and aspirin in the management ofcerebrovascular studies [231]. These includethe Antithrombotic Therapy in Acute Recov-ered cerebral Ischemia (ATARI) trial, AorticArch-Related Cerebral Hazard (ARCH) trial,secondary prevention of Small SubcorticalStrokes (SPS3) trial, the Fast Assessment ofStroke and Transient Ischemic Attack to Pre-vent Early Recurrence (FASTER), and thePrevention Regimen For Effectively avoidingSecond Strokes (PRoFESS) trial.

PrimaryPreventionTrial. The effect of dualantiplatelet therapy with clopidogrel and as-pirin versus aspirin alone was evaluated in abroad population of patients at high risk foratherothrombotic events in theClopidogrel forHigh Atherothrombotic Risk and IschemicStabilization, Management and Avoidance(CHARISMA) trial [232]. A total of 15,603patients either with clinically evident cardio-vascular disease or with multiple athero-thrombotic risk factors were randomized toreceive clopidogrel 75mg/day or placebo, bothwith concomitant aspirin 75–162mg/day.After a median follow-up period of 28 months,the rate of primary endpoint (first occurrenceofMI, stroke, or cardiovascular death) showedno significant benefit for the dual antiplateletregimen. Additionally, the dual therapy wasassociated with an increased risk of moderateto severe bleeding. However, a subgroup ana-lysis for the main secondary endpoint (whichwas a composite of first occurrence of MI,


Table

6.MajorClinicalStu

diesDoneonClopid

ogre

l

Study

Nam

eNumbe

rof

Patients

and

Clinical

Con

dition

Dosean

dDuration

PrimaryEndp

oint

Trial

Outcom

e

Inpa

tien

tswithrecentMI,recentstroke,o

restablished

PAD

CAPRIE

19,185

patien

tswithisch

emic

stroke

,MI,or

PAD

Clop.

75mg/da

yve

rsusas

pirin

325mg/da

y�1–

3ye

ars

Isch

emic

stroke

,MI,or

vascular

death

Relativerisk

redu

ction:8

.7%

Inpa

tien

tswithac

ute

coronar

ysyndromes

(STEMI,UA,o

rNSTEMI)

CURE

12,562

patien

tswithUA

orNSTEMI

Clop.

300mgLD,7

5mg/da

yMD

plusas

pirin7–

325mg/da

yve

rsusaspirin75

–32

5mg/

day�1ye

ar

Com

posite

ofCV

death,n

onfatal

MI,or

stroke

andrefractory

isch

emia

Relativerisk

redu

ction:7

%for

CV

death,2

3%fornon

fatal

MCI,14

%forstroke

,and7%

forrefractory

isch

emia

PCI-CURE

2,65

8pa

tien

tswhounde

rwen

tPCI

Asab

ove

CVde

ath,M

I,or

UTVRwithin

30da

ysof

PCI

Com

posite

ofev

ents:4

.5%

inClop.

versus6.4%

forplaceb

oCLARIT

Y-

TIM

I28

3,49

1pa

tien

tswithSTEMIan

dplan

ned

PCI

Clop.

300mgLD

then

75mg/da

yplusas

pirin

Arterialocclusion

onan

giog

ra-

phy,

orde

athor

recu

rren

tMI

Relativerisk

redu

ction:3

6%

CLARIT

Y-

PCI

1,86

3pa

tien

tswhounde

rwen

tCLARIT

Y-TIM

I28

.Asab

ove

CVde

ath,recurren

tMI,or

stroke

Primaryou

tcom

eoccu

rred

3.6%

inClop.

grou

pve

rsus6.2in

placeb

oCOMMIT

45,852

patien

tswithSTEMI

Clop.

75mg/da

yplusas

pirin

162mg/da

yve

rsusas

pirin

162mg/da

y�28

dayor

discharge

Dea

th,r

einfarction

,strok

eRelativerisk

redu

ction:7

%in

deathan

d9%

inthecompo

site

ofde

ath,M

I,or

stroke

Inpa

tien

tsundergo

ingelective

PCI

CREDO

2,61

1pa

tien

tson

elective

PCI

Clop.

300mgLD

then

75mg/da

yplusas

pirin32

5mg/da

y�1

year

versussa

meregimen

withou

tload

ingdo

se

1-Yea

ren

dpoint:de

ath,M

I,an

dstroke

.28

-day

endp

oint:de

ath,M

I,an

dUTVR

26.9%

relative

redu

ction

(Non

statisticallysign

ifican

t18

.5%

relative

redu

ction)

Inhigh-riskpa

tien

tswithrecentisch

emic

stroke

orTIA

MATCH

7,59

9high-risk(70%

diab

etic)

patien

tswithIS

orTIA

Clop.

75mgplusas

pirin75

mg/

dayve

rsusClop.

75mg/da

y�18

mon

ths

IS,M

I,or

vascularde

ath

Non

statisticallysign

ifican

tde

-crea

sein

IS,M

I,va

scular

death,r

ehospitaliza

tion

.Life-

threaten

ingbleeding

Inabroa

dpo

pulation

ofhigh-riskpa

tien

tswithou

tdocumen

tedeviden

ceof

CVdisea

seCHARIS

MA

15,603

patien

tswithclinically

eviden

tCVdiseas

eor

multiple

risk

factor

Clop.

75mg/da

yplusas

pirin

75–16

2mg/da

yve

rsusas

pirin

75–16

2mg/da

y�28

mon

ths

First

occu

rren

ceof

MI,stroke

,or

CV

death

Nostatisticallysign

ifican

tbe

ne-

fit.In

crea

sedrisk

ofbleeding

andcard

iova

scularde

ath.

431

stroke, and death from cardiovascular causeor hospitalization for unstable angina, TIA, orrevascularization procedure), showed a statis-tical advantage for the dual antiplatelet ther-apy [233]. Although there was a modest re-duction in primary endpoints in this subgroupof patients with documented cardiovasculardisease, in patients with multiple risk factorswho did not have clinically evident athero-thrombosis, the rate of death from cardiovas-cular causeswashigherwith clopidogrel (3.9%versus 2.2%; p¼ 0.01). Although further in-vestigations are warranted, the CHARISMAstudy supports the use of clopidogrel in sec-ondary prevention of cardiovascular events inpatients with a history of established cardio-vascular disease, but not in primary preven-tion of atherothrombosis in patients who donot have a prior history of cardiovasculardisease.Summary of Clopidogrel Clinical TrialsIn summary, long-term administration of clopi-dogrel was associated with a modest, but sta-tistically significant advantage over aspirin inreducing adverse cardiovascular outcomes inpatients with established cardiovascular dis-ease (CAPRIE trial). Dual antiplatelet therapyusing clopidogrel and aspirin improved out-comes in patients with acute coronary syn-drome (CURE, COMMIT, and CLARITY-TIMI28).However,asevident fromtheanalysisof theCHARISMA trial, use of clopidogrel in primaryprevention of atherothrombosis is notsupported.Safety and Tolerability of Clopido-grel The use of antithrombotic agents isvery often associated with hemorrhagic sideeffects. In fact, the current paradigm of an-tithrombotic therapy is “no bleeding, noefficacy.” In major clinical trials the overalltolerability of clopidogrel, including bleedingcomplication, was similar to that of aspirin,although the incidence of hemorrhagic com-plications was generally increased in dualantiplatelet therapy using clopidogrel and as-pirin.There is considerablymorebleeding riskwhen an anticoagulant such as warfarin isadded to the regimen [234]. The severe com-plication of thrombotic thrombocytopenic pur-pura (TTP) is much less commonwith clopido-grel than ticlopidine that it has largely re-placed. Other possible and relatively common

side effects include gastrointestinal distressand abnormalities in liver function [235].Clopidogrel Resistance Patients withclopidogrel resistance show reduced respon-siveness to the drug in blocking ADP-inducedplatelet aggregation, which has been directlycorrelated with reduced clinical response andadverse cardiovascular events [236,237]. Thereported prevalence of nonresponsiveness toclopidogrel therapy among cardiovascular pa-tients varies from 4% to 34% [131]. A numberof mechanisms for clopidogrel resistance havebeen proposed. They include extrinsic me-chanisms such as patient noncompliance, un-der dosing, and reduced intestinal absorptionof the prodrug. Among the intrinsic mechan-isms, drug–drug interactions have beenpointed out as a probable cause. The hepaticenzymes that are involved in the conversion ofclopidogrel to its active metabolites areCYP3A4, CYP2C9, CYP2C19, and CYP1A2.Several widely used drugs such as calciumchannel blockers, ACE inhibitors, statins, andproton pump inhibitors that are metabolizedby these enzymes have been implicated inclopidogrel resistance [238].

Genetic polymorphisms of CYP3A4 andCYP2C19 genes have also been associatedwith lower levels of active metabolites of clo-pidogrel [239,240]. An analysis of the TRI-TON-TIMI 38 trial has revealed that carriersof reduced function CYP2C19 allele had sig-nificantly lower levels of the active metaboliteof clopidogrel, diminished platelet inhibitionand a higher rate of major adverse cardiovas-cular events compared to noncarriers [239]. Asimilar finding has been reported in anotherstudy among young patients who are carriersof loss-of-function CYP2C19 polymorph-ism [241]. Resistance may be also mediatedby variations in P2Y12 receptor density andupregulation of alternative platelet activationmechanisms involving epinephrine, throm-boxane A2, collagen, thrombin, etc.

Clopidogrel resistance may be overcome tosome extent by increasing the dose of thedrug [238]. Recently approved P2Y12 antago-nist prasugrel (see section “Prasugrel”) thatproduces higher plasma level of its activemetabolite at a faster rate and known to havea faster onset of platelet inhibition may be-come an alternative therapy. Other emerging


antiplatelet agents such as thrombin receptorantagonists (see Section 4.3) and reversibleP2Y12 antagonists (see section “ReversibleADP Antagonists”) may also help overcomeresistance by supplementing the ADP antago-nist antiplatelet agents.Prasugrel Prasugrel is a third-generationcommercially launched thienopyridine ADPantagonist that is administered as a racemicmixture, and is faster acting than ticlopidineand clopidogrel [242]. Like ticlopidine andclopidogrel, prasugrel is a prodrug that needsto undergo in vivo metabolism to the activechemical species.Whereas ticlopidine and clo-pidogrel require a CYP-mediated oxidativemetabolism to an intermediate thiolactonemetabolite that then undergoes thiolactonering opening to the active thiol carboxylic acid,prasugrel is a masked thiolactone that pre-sumably undergoes an esterase-mediatedcleavage to the thiolactone and, as the finalstep, ring opening to the sulfhydryl carboxylicacid (Fig. 14) [243]. Additionally, unlike clo-pidogrel, prasugrel active metabolite does notcontain a labile carboxylic ester, making itmore stable. In fact, 85% of clopidogrel israpidly converted to an inactive metabolite byesterase whereas prasugrel does not havesuch a structural disposition for inactivation.These structural characteristics impart a bet-ter pharmacokinetic and pharmacodynamicproperty for prasugrel [244]. Measurement ofthe concentration of the active metabolites ofprasugrel and clopidogrel has confirmed aconsiderably higher plasma level of prasugrelactive metabolite than clopidogrel active me-tabolite in rats, despite the use of a ten timeshigher dose of clopidogrel [245].Pharmacology of Prasugrel The phar-macology of prasugrel was studied in rats.

After a single oral dose (0.3–3.0mg/kg), pra-sugrel produced dose-dependent inhibition ofplatelet aggregation (IPA), being 10 timesmore potent than clopidogrel on a milligramper kilogram basis. [246] The ex vivo plateletinhibitory effect of prasugrel at 1 and10mg/kgdoses in ratwas seen starting at30min, reach-ing a maximum inhibition of 85% and lastingfor more than 3 days, suggesting the irrever-sible nature of platelet inhibition [247]. Pra-sugrel was highly active in a rat arteriovenousshunt thrombosis model, being 10 times morepotent than clopidogrel [244]. The active me-tabolite of prasugrel R-138727 (13) inhibitedADP-induced aggregation of rat and humanplatelets in a concentration-dependentmanner (Fig. 14) [248].Phase-I Clinical Studies of PrasugrelInpharmacodynamicstudiesconductedaspartof the Phase-I clinical trials, prasugrel showeddose-dependent inhibition of platelet aggrega-tion [244]. In a crossover study in healthy as-pirin-free subjects, prasugrel at a loading doseof 50mg demonstrated a robust IPA from30min to 24h, reaching a maximum of about80%. Importantly, individuals who respondedpoorly to clopidogrel achieved robust IPAwhencrossed over to prasugrel. These studies alsoindicated that there was a 60-fold greater ex-posure of the active metabolite of prasugrelthan the active metabolite of clopidogrel, on adose-adjusted basis [249]. In combinationwithaspirin (325mg) prasugrel showed a 60% inhi-bitionofplatelet aggregation2–6haftera load-ing dose of 60mg, whereas a 300mg loadingdose of clopidogrel gave 35% inhibition undersimilar conditions [250].

In a phase Ib study in 101 patients withstable coronary artery disease using clopido-grel as a comparator, prasugrel at a loading

N

S F

O

EsterasesN

S F

O

ON

HS F

OEnzyme-mediated

hydrolsysis HOOC

13 Prasugrel(Prodrug)

15 R-138727active(Prasugrel

metabolite)

2-oxo-Maskedthienorpyridine

14 2-oxo-Prasugrel

O

O

H3C

Figure 14. Metabolic activation of prasugrel. Prasugrel is an enol ester which is presumably cleaved byesterase to produce 2-oxo-prasugurel intermediate which undergoes ring opening to the active metabolite.


dose of 40 or 60mg and a maintenance dose of10 or 15mg maintained stable and dose-de-pendent levels of IPA that were significantlyhigher than that for clopidogrel at 300mgloading dose and 75mg maintenancedose [251]. In these studies, prasugrel showedminor bleeding incidents similar to clopido-grel at a maintenance dose of up to 10mg; at a15mg dose there were nonsignificant in-creases in minor bleeding events compared toclopidogrel.Phase-II Clinical Studies of PrasugrelIn the Joint Utilization of Medications toBlock Platelets Optimally-Thrombolysis InMyocardial Infarction 26 (JUMBO-TIMI 26)dose ranging safety trial, 904 patients whowere undergoing elective or urgent PCI wererandomized to receive either clopidogrel300mg initiation dose, followed by 75mgdailymaintenance dose, or one of the three ascend-ing dose regimens of prasugrel: 40mg loadingdose/7.5mg maintenance dose, 60mg loadingdose/10mgmaintenancedose, and60mg load-ing dose/15mg maintenance dose [252]. Allpatients received 325mg aspirin and the ther-apywas continued for amonth. In the primarysafety endpoint (TIMI major/minor non-CABG-related bleeding), prasugrel showed ahigher rate of bleeding compared to clopido-grel, although it did not reach statistical sig-nificance (1.7% versus 1.2%, p¼ 0.59). TheTIMI major bleeding was similar in patientsreceiving clopidogrel and prasugrel. Overall,prasugrel was well tolerated and patients re-ceiving this drug showed a lower incidence ofthe composite endpoint of major adverse car-diac events and the endpoint of myocardialinfarction and recurrent ischemia.Phase-III Clinical Studies of Prasu-grel The Trial to Assess Improvement inTherapeutic Outcomes byOptimizingPlateletInhibition with Prasugrel-Thrombolysis inMyocardial Infarction 38 (TRITON-TIMI 38)was a large-scale clinical trial that comparedprasugrel with clopidogrel in 13,608 patientswithACSwhowereundergoingPCI [253,254].Patients on a background low-dose aspirinwere randomized to receive either prasugrelin 60mg loading dose, followed by 10mg dailymaintenance dose, or 300mg loading dose ofclopidogrel, followed by 75mg daily mainte-nance dose, for a period of up to 15 months.

The primary objective of the study was to testthe hypothesis that prasugrel is superior toclopidogrel on a background aspirin therapyin patients who undergo PCI. The endpointwas a composite of cardiovascular death, non-fatal myocardial infarction, or nonfatal strokeat a median follow-up of approximately 12months.

In general, patients on prasugrel therapyshowed less variability and hyperresponsive-ness than patients on clopidogrel, as expectedbased on the robust pharmacokinetics andpharmacodynamics of prasugrel discussedabove. The primary endpoint (cardiovasculardeath, MI, or stroke) occurred in 12.1% pa-tients receiving clopidogrel and 9.9% patientsreceiving prasugrel (p< 0.001), thus support-ing the hypothesis that a thienopyridine regi-men that yields higher IPA would result inimproved clinical outcomes.

However, the bleeding episodes were high-er among prasugrel treated patients. TIMImajor bleeding was observed in 2.4% patientsreceiving prasugrel and 1.8% patients receiv-ing clopidogrel (p¼ 0.03). The rate of life-threatening bleeding was greater in the pra-sugrel group than in the clopidogrel group(1.4% versus 0.9%, p¼ 0.01). In general, bothTIMImajor andminor bleeding episodes weremore frequent with prasugrel than with clo-pidogrel. Certain categories of patients weremore responsive to the treatment than others.For example, diabetes patients showed thebest clinical outcome (30% relative risk reduc-tion) without anymajor difference in bleeding.On the other hand, patients with a history ofstroke or transient ischemic attack, elderlypatients and patients with a body weight of<60kg showed less clinical benefit. Prasugrelwas associated with increased intracranialhemorrhage in patients with a history of cere-brovascular disease [255].

In summary, the TRITON-TIMI 38 trialresults reveal that prasugrel significantly re-duced ischemic events compared with clopido-grel, but the at the expense of an increase inmajor (and sometimes fatal) bleeding in ACSpatients. It has been suggested that prasugrelwouldprobably benefit patientswithACSwhoare undergoing PCI and are at high risk ofischemic events and low risk of bleeding,although those with a lower risk for ischemic


events and a high risk of bleeding may bebetter served with clopidogrel [256,257]. In2009, prasugrel received the European andthe U.S. regulatory approvals for the treat-ment of acute coronary syndrome in patientsundergoing PCI.Reversible ADP AntagonistsNonthienopyridineADP antagonists that do not requiremetabolicactivation are on the horizon. Among these,cangrelor (C69931MX), a potent, reversible,short acting intravenous ADP antagonist, ti-cagrelor (AZD6140), a structurally related, or-ally acting agent, and elinogrel (PRT128) areunder clinical investigation [258,259].Cangrelor ATP has been known to be acompetitive antagonist for ADP-induced plate-let aggregation, an observation that led to theinitial characterization ofADPreceptors [260].Since ATP is undesirable as a therapeuticagent due to its lack of stability, structuralmodification of ATP has unveiled a novel classof ATP analogs asADPantagonists. Cangreloremerged from ATP by replacing the distalanhydride oxygen with a dichloromethylenegroup that conferred the molecule higher sta-bility and affinity, and by modifying the ade-nine groupwith nonpolarmoieties and sulfide-linked chains to improve the antagonist activ-ity [261]. Cangrelor (Fig. 15) is a tetrasodiumsalt that is highly soluble inwater,with a shorthalf-life of about 2.6min and a fast plasmaclearance (50L/h) [261, 262].Preclinical Pharmacology of Cangre-lor Cangrelor causes potent inhibition ofADP-induced platelet aggregation in humanwashed platelets. In animalmodels, cangrelorshowed reversible antagonism of P2Y12 recep-

tors, with prevention of both arterial throm-bosis and reocclusion after thrombolytic ther-apy [263]. In a caninemodel of arterial throm-bosis consisting of electrically damaged andpartially occluded carotid artery, intravenousinfusion of cangrelor 15min prior to the in-duction of injury (4.0mg/min, for 6 h) helpedmaintain blood flow in 5 out of 6 treatedanimals for the duration of intravenous infu-sion [264]. Bleeding times were increased inthe drug-treated group, which returned tocontrol values shortly after cessation of drugadministration. The antiplatelet property ofcangrelor was demonstrated in a mechanicalinjury model in anesthetized rabbits and in acaninemodel in conjunctionwith thrombolytictherapy [265,266]. In summary, the preclini-cal pharmacology of cangrelor demonstrates arapid onset of action, potent inhibition ofADP-induced platelet aggregation, and robust an-tithrombotic effects. Dose response relation-ship studies have shown a favorable safetyratio between desired antithrombotic actionand prolongation of bleeding time.Clinical Studies of Cangrelor A Phase-Istudy conducted in healthy volunteers whoreceived four overlapping 1h infusions, can-grelor demonstrated a dose-dependent inhibi-tion of ADP-induced platelet aggregation thatwas completely reversed within 20min of dis-continuation of the drug without the presenceof a rebound effect [267].

In phase II clinical trials designed to assessthe safety and tolerability of cangrelor, thedrug was overall well tolerated as an adjunctto aspirin and heparin. In patients with UAandNSTEMI on background aspirin or hepar-in, the adverse effect of cangrelor was similarto that of placebo, including rates of adversebleeding. Statistically significant lower ratesof angina were reported in patients receivingcangrelor than placebo [268].

In the first arm of a two-part Phase-IIsafety study in patients undergoing PCI,200 patients were randomized to receive 1,2, or 4mg/min of cangrelor in addition to aspir-in andheparin beginning prior to PCI [269]. Inthe second part of the study, 199 patientswererandomized to receive either cangrelor or aGPIIb/IIIa antagonist abciximab. Although notstatistically significant, rates of combinedma-jor and minor bleeding were higher in the

N

N N

N

O

OP

OP

OO

PNa

O–O–O

Na

Cl O–Cl Na O– NaHO OH

SCF3

NHS

CH3

H

16 Cangrelor

Figure 15. Structure of cangrelor that is a stableanalog of ATP.


cangrelor group versus placebo, and compar-able versus those who were randomized toreceive abciximab.

Another small-scale Phase-II clinical studyevaluated the effectiveness of cangrelor as anadjunct to fibrinolytic therapy in patients withacutemyocardial infarction [270].Thepatientswere on background aspirin and heparin ther-apy who received an intravenous infusion ofeither cangrelor alone, full dose of tPAalone, orone of three doses of cangrelor along with half-dose tPA. In this study, the combination ofcangrelor andhalf dose tPA resulted in similar60mincoronaryarterypatencyas full dose tPAaloneandgreaterpatencythancangreloralone.Bleeding and adverse clinical events were si-milar for the study groups. This study supportsthe potential use of cangrelor as an adjunct tofibrinolytics for the treatment of acute MI.

The concurrent effect of clopidogrel andcangrelor on platelet function has been eval-uated in healthy volunteers [271]. Clopidogrel(single dose of 600mg) and cangrelor (30mg/kg,IV, followed by 2h infusion of 4mg/kg/min)alone achieved the expected level of plateletinhibition. However, when both drugs wereadministered simultaneously, clopidogrel wasunable to achieve the anticipated sustainedplatelet inhibition, which suggests a pharma-codynamic interference at the P2Y12 receptorbetween the two drugs. No such effect wasfound when clopidogrel was started upon com-pletion of cangrelor infusion. Presumably, thehigh-affinity binding of cangrelor at the P2Y12

receptor thwarts the irreversiblebinding of theactivemetabolite of clopidogrel.These findingsmay have important implications in the coad-ministration of thienopyridines with direct-acting ADP antagonists in clinical settings.Ticagrelor(AZD6140) Ticagrelor (AZD6140)(Fig. 16) is the first orally active, nonthieno-pyridine reversible P2Y12 receptor antagonist,and it is currently under clinical development.Unlike thienopyridines, it does not requiremetabolic activation. Ticagrelor bears closestructural similarity to cangrelor, but also hassome important differences that contribute toits oral bioavailability [272,273]. Of note, tica-grelor is devoid of the polar phosphate andribofuranose groups of cangrelor, and it haslipophilic substituents such as a difluorophe-nylcyclopropyl group [274].

Preclinical Pharmacology of TicagrelorTicagrelor is a potent inhibitor of the P2Y12

receptor that showed a pIC50 of 7.2 in plateletaggregation assay. In vivo, it produces anactive metabolite (AR-C126910) that is alsoa potent inhibitor of the P2Y12 receptor andcontributes to the overall in vivo potency ofticagrelor [275]. The platelet response to ti-cagrelor was studied inmultiple animalmod-els [272,276]. In diabetic rats, the drug wasabsorbed quickly and inhibited platelet ag-gregation for 24 h. Ticagrelor also reducedthrombus formation in a murine laser injurymodel. In a canine model of arterial throm-bosis, ticagrelor inhibited thrombus forma-tion with less bleeding than clopidogrel.Clinical StudiesofTicagrelor Inphase-Iclinical studies in healthy volunteers, ticagre-lor was rapidly absorbed and showed a dose-dependent pharmacokinetics [267]. After asingle oral dose ranging from 100–400mg,robust platelet inhibition, reaching nearly100% was achieved 2h post dosing.

In a Phase-II dose-selection study (Dose-finding Investigative Study to assess the Phar-macodynamic Effects of AZD6140 in athero-sclerotic disease, DISPERSE) in patients withatherosclerotic disease who were on aspirintherapy, ticagrelor produced more rapid andgreater platelet inhibition (>90%) than clopi-dogrel that produced 60% platelet inhibi-

N

O

HO OH

HO H

17 Ticagrelor

NN

N

N

S

HN

H

F

FH

Figure 16. Structure of ticagrelor that is consider-ably more lipophilic than cangrelor and is devoid ofthe polar phosphate groups, attributes that helpits oral absorption.


tion [275]. There was less interpatient varia-bility inresponse to ticagrelor thanclopidogrel.Thereweredose-relatedminorbleedingeventsanddyspnea; theseadverseeffectswerereport-edly minor. In the second part of this trial(DISPERSE-2), 990 patients with non-ST-seg-ment elevation acute coronary syndromes(NSTE-ACS) were randomly assigned to re-ceive ticagrelor 90 or 180mg, twice daily orclopidogrel75mg,oncedaily forupto12weeks;half the patients receiving ticagrelor were ran-domized to receive a loading dose of 270mg,whereastheotherhalf startedtherapywith themaintenancedose [277].All patients receivedadaily dose of 75–100mg aspirin. The primaryendpoint was a composite of major and minorbleeding. No significant difference in bleedingevents occurred between the ticagrelor andclopidogrel groups. Although treatment withticagrelorwas generallywell tolerated, severaladverse events including dyspnea and brady-cardia were noted. In pharmacodynamic as-says, ticagrelor groups yielded consistentlyhigher levels of IPA than clopidogrel.

Ticagrelor has the potential to overcomesome of the limitation of clopidogrel withsuperior potency, more rapid onset and offsetof activity, and less interpatient variability.Ticagrelor is currently undergoing Phase-IIIclinical studies using clopidogrel as a com-parator (study of PLATelet inhibition andpatient Outcomes or PLATO—A Comparisonof AZD6140 and Clopidogrel in Patients WithAcute Coronary Syndrome). The primary end-point is cardiovascular death, MI, and strokeat a mean follow-up of 11 months. This studywill provide further insight into the clinicalprofile of ticagrelor [278].Elinogrel (PRT128) Another direct-acting,reversible, oral P2Y12 antagonist in develop-ment is elinogrel (PRT060128, PRT128) [279].

Administration of single intravenous doses ofelinogrel, 1–40mg, in healthy volunteersyielded dose-dependent, complete inhibitionof ADP-induced platelet aggregation within20min. When combined with aspirin, elino-grel had a synergistic inhibitory effect on col-lagen-induced platelet aggregation. Elinogrelalso promoted thrombus destabilization and itinhibited thrombus growth starting at a doseof 30mg [280].Elinogrel (Fig. 17) is inPhase-IIclinical trials in patients undergoing nonur-gent PCI [281].

4.3. Thrombin Receptor (Protease-ActivatedReceptor-1) Antagonists

Thrombin, the key enzyme of the coagulationcascade, plays a dual procoagulant and proag-gregatory role in the context of hemostasis(see Section 3.1.2). In its procoagulant role,thrombin proteolytically cleaves fibrinogen tofibrin that polymerizes to form an insolublefibrin meshwork that becomes an integralpart of blood clot. In its proaggregatory role,thrombin activates platelets causing them toaggregate. Aggregated platelets get trappedby the fibrin meshwork to produce a growingthrombus at the site of vascular injury. Thecellular effects of thrombin are mediated byspecific cell-surface receptors known asPARs [282–285]. The prototype of these recep-tors is PAR-1 that is also known as the throm-bin receptor. PAR-1 is a G-protein-coupledreceptor, which is activated by thrombin-spe-cific proteolysis of its extracellular loop trig-gering platelet activation.

Subsequent to the identification of the in-itial protease-activated receptor (PAR-1),three additional protease-activated receptorshave been identified [286]. The current familyof PARs comprises PAR-1, PAR-2 [287],

NH

NF

NH

MeO

O

HN

HN

O

S SCl

O

O

18 Elinogrel

Figure 17. Elinogrel is an ADP antagonist that is currently undergoing Phase-II clinical trials in patientsundergoing percutaneous coronary intervention.


PAR-3 [288], and PAR-4 [289,290]. Of these,PAR-1, PAR-3, and PAR-4 are activated bythrombin. PAR-2 is activated by trypsin, tryp-tase, and coagulation factors VIIa and Xa, butnot by thrombin [291–295]. It has been estab-lished that the “tethered ligand” activationparadigm described below applies to all thePAR receptors. There is considerable speciesspecificity to the nature of PAR receptors onplatelets. In primates, PAR-1 is the mainplatelet protease-activated receptor. It is alsopresent onother cells suchas endothelial cells,smooth muscle cells, monocytes, fibroblasts,and so on. PAR-4 is the second PAR on humanplatelets. Since PAR-4 has only weak affinityfor thrombin, it is activated only at highthrombin concentrations. It is believed to bea “rescue receptor” that is activated in theevent of a serious vascular lesion and theresultant high thrombin concentration.PAR-3 is found in mouse platelets where it isthe major regulator of thrombin response.PAR-4 receptors are also expressed on mouseplatelets. It has been postulated that certainprotease-activated receptors are devoid of in-herent G-protein activation, but serve to func-tion as cofactors in the activation of anotherprotease-activated receptor. For example,PAR-3 serves to facilitate the activation ofPAR-4 at low thrombin concentrations, butdoes not become independently activated by

thrombin [296]. Since PAR-1 receptors are notpresent in mouse and rat platelets, but arepresent in monkey platelets, nonhuman pri-mate models have been used to study the invivo antithrombotic effects of thrombin recep-tor antagonists (see below). However, due tothe presence of PAR-1 receptors on rodentcoronary artery smooth muscle cells, rodentmodels have been used to study the effect ofthrombin receptor antagonists on restenosisand neointimal proliferation [297].

4.3.1. Mechanism of Thrombin Receptor Acti-vation Although a thrombin-specific receptoron platelets that mediates platelet activationhas been known for some time, the exact me-chanism of thrombin-specific cellular activa-tion was unknown [298]. In 1991, expressioncloning of functional thrombin receptor wasachieved by two groups, which unveiled theintriguing mechanistic details of thrombin’scellular activation [299–302]. The amino acidsequence deduced from the mRNA encodingthe thrombin receptor revealed a new G-pro-tein-coupled seven-transmembrane domainreceptor with a large extracellular do-main [303]. The authors postulated thatthrombin binds to the cellular receptorthrough its anion binding exo-site and subse-quently cleaves the extracellular domain atArg41-Ser42 (Fig. 18). The newly unmasked

LDPRSFLLRNP

LDPRNRLLFS

Signal

Figure 18. The tethered ligand mechanism of thrombin receptor activation. Thrombin binds to the extra-cellular domain of the receptor and cleaves it at Arg41-Ser42. The newly generated N-terminus bindsintramolecularly to the second loop of the receptor, triggering cellular activation.


amino terminus acts as a “tethered ligand”binding intramolecularly to the proximal hep-tahelical segment eliciting G-protein-coupledtransmembrane signaling [304–307]. Peptideshaving the sequence SFLLRN that mimic thenew amino terminus of the activated receptor(known as thrombin receptor activatingpeptides or TRAPs) function as agonistsproducingfunctionalresponsessuchasplateletaggregation and mitogenesis [308]. Uncleava-ble mutant thrombin receptors failed to re-spond to thrombin, but were responsive toTRAPs.Intracellular Signaling Once activated, PAR-1and PAR-4 promote guanine nucleotide ex-change on Gq, G12, and perhaps Gi familiesleading to the activation of RhoGEFs (guaninenucleotide exchange factors which activatesmall G-proteins such as Rho), providing apathway to Rho-dependent cytoskeletal rear-rangement leading to platelet shape change.Gaq activates phospholipase Cb, triggeringphopshoinositide hydrolysis which results incalciummobilization and activation of proteinkinase C leading to cellular responses rangingfrom granule secretion and integrin activa-

tion. Gai inhibits adenylate cyclase, therebynegatively regulating cAMP levels. Gbg sub-units can activate phosphoinositide-3 kinase(PI3-K), whichmodifies plasmamembranes toprovide attachment sites for a host of signal-ing proteins (Fig. 19) [309].

4.3.2. Preclinical Pharmacology of ThrombinReceptor Antagonists As mentioned before,two distinct but interrelated mechanisms areoperative in the formation of a thrombus—namely, the activation of the coagulation cas-cadeandactivationof platelets. Thrombin, theendproduct of the coagulation cascade, plays adual role in thrombosis by activating both ofthese mechanisms. As a procoagulant, throm-bin cleaves fibrinogen to fibrin leading to fi-brin meshwork; as a promoter of platelet ag-gregation, thrombin activates platelets viaPARs. Aggregated platelets get trapped in thefibrin meshwork, which further traps otherplasma particles to a rapidly growing throm-bus. A thrombus can be either fibrin-rich orplatelet-rich depending on the part of cardio-vascular system it is formed.Arterial thrombi,which are formed under high shear force of

12/13 q i

PAR-1

RhoGEFs

Rho

Rho-activatedkinases

MLC phosphatase, etc.

Phospholipase

IP3

Ca2+

DAG

PKC

Ca2+-regulated kinases

RasGEFs, MAP kinase, etc.

PI3K

PLC?

K+ channels

G-protein coupledreceptor kinases

Nonreceptortyrosine kinase

Cell shape SecretionIntegrinactivation

Transcriptionalresponse, cell mobilityin endothelial cells

Adenylcyclase

Figure 19. Thrombin receptor signaling. PAR-1 is known to be coupled to G12/13, Gq, and Gi families. The a-subunits of G12 and G13 bind RhoGEFs-stimulating Rho-dependent cytoskeletal responses such as shapechange and migration (for endothelial cells). Gaq activates phospholipase Cb, triggering phosphoinositidehydrolysis. The ensuing cascade of events (see above) leads to granular secretion and integrin activation. Gai

inhibits adenylate cyclase lowering intracellular cAMP levels. Activation of Gbg subunits leads to a host oftranscriptional responses and cell mobility in a number of cells.


blood flow, are abundant in aggregated plate-lets whereas venous thrombi that are formedunder low shear, stasis conditions are abun-dant in polymerized fibrin. PAR-1 activation isthemost potent platelet activationmechanismand various lines of evidence exist suggestingits important role in atherothrombosis.

Since thrombin activates human plateletsvia PAR-1 and PAR-4, it is important to askwhether PAR-1 antagonism by itself is suffi-cient to produce substantial antithromboticeffects [310,311]. Several preclinical experi-ments pointed to the fact that PAR-1 antag-onism can indeed engender strong antithrom-botic effects without the attendant bleedingthat is common to most anticoagulants andantiplatelet agents. PAR-1 antagonists showpotent functional antiplatelet activity in ago-nist-induced platelet aggregation measure-ments, Ca2þ transients, and thymidine incor-poration assays [312,313]. In vivo studies car-ried out using synthetic PAR-1 antagonistpeptides showed inhibition of platelet-richthrombus formation in a baboon thrombosismodel and in a guinea pig thrombosis model,suggesting the promise of a PAR-1 antagonistfor arterial thrombosis [314,315]. In anotherimportant study, an antibody to the PAR-1 N-terminushasbeen reported to inhibitmechan-ical injury-induced thrombosis in a babooncarotid artery model without affecting bleed-ing time and coagulation parameters [316]. Asdescribed below, more recent studies usingpotent PAR-1 antagonists in nonhuman pri-mate antithrombosis models have corrobo-rated the outcome of these earlier studies andestablished the therapeutic potential ofPAR-1antagonists as promising antithromboticagents [317,318]. By selectively inhibitingPAR-1-mediated platelet activation, a throm-bin receptor antagonist should exhibit strongantiplatelet action under conditions in whichthrombin-stimulated platelet activation is im-portant [148]. Since a PAR-1 receptor antago-nist is specific for the cellular actions of throm-binanddoesnot interferewith the coagulationcascade and platelet activation by other ago-nists needed for normal hemostasis, it is likelyto confer an added safety margin with regardto hemorrhagic side effects, which is a compli-cating factor for the currently available an-tithrombotic therapy.

4.3.3. Knockout Animal Experiments Studiesconducted in PAR-deficient mice provide com-pelling evidence for the potential antithrom-botic utility of a PAR-1 antagonist [319]. Cor-responding to the human PAR-1 and PAR-4receptors, mouse platelets contain a high-af-finity PAR-3 receptor and a low affinity PAR-4receptor. However, these receptors work inmechanistically different ways in human pla-telets and mouse platelets. Contrary to thehuman platelets where PAR-1 is the primarymediator of platelet activation, PAR-3 doesnot independently activate mouse platelets.Instead, it acts as a cofactor for PAR-4-mediated platelet activation [320]. Gene dele-tion experiments have showed that PAR-4�/�

mice were totally unresponsive to even micro-molar concentration of thrombin [321]. On theother hand, platelets in PAR-3�/� mice couldbe activated by thrombin, but only at highthrombin concentrations [290]. PAR-4 defi-cient mice were protected against thrombo-plastin-induced pulmonary embolism, ferricchloride-induced thrombosis of mesenteric ar-terioles, and laser-injury-induced thrombosisof cremasteric microvessels [297,322,323].PAR-4-deficient mice were healthy andshowed no evidence for anemia or sponta-neous bleeding. Platelets in PAR-4�/� micewere normal in number and morphology andPAR-4�/� femalemice were found to be able tosupport pregnancy. However, as one wouldexpect, PAR-4-deficient mice showed pro-longed bleedingwhen challenge to hemostasiswas strong.

Studies done using PAR-3�/� mice havedemonstrated that complete ablation ofthrombin signaling is not required for protec-tion against thrombosis. In fact, PAR-3�/�

mice, which have the low affinity PAR-4 re-ceptors still intact, showeda level of protectionagainst thrombosis that was similar to thatseen in PAR-4 deficient mice that showedcomplete ablation of thrombin-mediated pla-telet activation [324]. Moreover, PAR-3�/�

mice showed no spontaneous bleeding. Thefact that PAR-3 deficient mice were protectedagainst thrombosis suggests that even a par-tial attenuation of thrombin signaling in pla-telets might produce a therapeutically usefulantithrombotic effect. Furthermore, these stu-dies challenge the often-raised skepticism


that a dual PAR-1 and PAR-4 antagonism inhumansmight be necessary to engender effec-tive antiplatelet effect.

The role of thrombin in platelet activationand fibrin deposition has been recently stu-died in PAR-4�/� mice using real-time digitalfluorescence microscopy [323]. During laser-induced thrombus formation, juxtamural pla-telet accumulation immediately after laserinjury was not different between wild-typeand PAR4�/� mice. However, subsequentgrowth of platelet thrombi was markedly di-minished in PAR4�/� mice. At the time ofmaximal thrombus size in wild type, plateletaccumulation wasmore than 10-fold higher inwild-type than inPAR4�/�mice. In contrast toplatelet activation and accumulation, the rateand amount of fibrin deposition were indis-tinguishable between PAR4�/� and wild-typemice. These results suggest that platelet acti-vation by thrombin is necessary for thrombusgrowth, but not for the formation of hemo-static plug. Additionally, attenuation of plate-let activationbyPAR-4 deletiondoesnot affectfibrin. This is important because it has beenknown that activated platelets support theassembly of tenase and prothrombinase com-plexes [325]. These studies in PAR4�/� miceare in agreement with the antithrombotic ef-fect and lack of bleeding forPAR-1antagonistsin preclinical nonhuman primate models andPhase-II clinical studies discussed below.

In summary, the results of functional as-says and in vivo antithrombotic data of PAR-1

antagonists taken together with the gene de-letion experiment data provided strong pre-clinical support for the antithrombotic poten-tial for a PAR-1 antagonist. Studies carriedout using nonpeptide, small-molecule throm-bin receptor antagonists in nonhuman pri-mate thrombosis models and Phase-II clinicalstudies in ACS patients have validated thehypothesis that a thrombin receptor antago-nist can exert potent antiplatelet effect with-out attendant bleeding side effect.

4.3.4. Peptidomimetic Thrombin Receptor An-tagonists There has been considerable pro-gress in the discovery of orally active small-molecule thrombin receptor antagonists [326].Two thrombin receptor antagonists arecurrently undergoing clinical trials for acutecoronary syndrome (see 4.3.5). The earlythrombin receptor antagonists were modifiedpeptides such as 19 (Fig. 20) based on thesequence of the tethered ligand of theactivated PAR-1 receptor [327,328]. Furtherresearch in this area led to the identification ofpeptidomimetic analogs such as 20. Whilethese compounds lacked oral bioavailability,they turned out to be excellent tools for theearly validation of the antithrombotic activityof a thrombin receptor antagonist.

Due to the absence of PAR-1 receptors inmouse and rat platelets, nonhuman primatemodelswereused to test the antiplatelet effectof PAR-1 antagonism. The peptidomimeticthrombin receptor (PAR-1) antagonist 20 was

Ph HN N

H

HN N

HO

F

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H2N NH

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NH2

NH

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O19

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HNBnHN

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20 (RWJ 58259)

PAR-1 binding IC50 = 4 nM

Platelet aggregation IC50: 40 nM (TRAP)

PAR-1 binding IC50 = 150 nM

Platelet aggregation IC50: 110 nM (TRAP); 370 nM

(α-thrombin)

Figure 20. Peptidomimetic thrombin receptor antagonists.


tested in a cynomolgus monkey vascular in-jurymodel [318]. The compoundwas adminis-tered intravenously and the degree of vesselocclusion caused by electrolytic injury-in-duced thrombus in each carotid artery wascharacterized. Compound 20 significantly re-duced occlusion in the vessels of all animalsthat completely occluded under experimentalconditions in the absence of drug. Althoughthe plasma level of the drug was relativelyhigh (12mM), ex vivoplatelet aggregationmea-surements indicated complete PAR-1 inhibi-tion under these conditions. In the drug trea-ted group, not only the thrombus size wasreduced but also the composition of the throm-bus indicated a switch from platelet-richthrombi to platelet-depleted thrombi, demon-strating the antiplatelet property of a PAR-1antagonist.

4.3.5. Small-Molecule Thrombin Receptor An-tagonists Efforts in the PAR-1 antagonistarea based on a lead generated from the nat-ural product himbacine (21) (Fig. 21) haveculminated in the identification of SCH530348which is currently in Phase-III clinicaltrials for acute coronary syndrome and sec-ondary prevention of cardiovascular events inpatients who have undergone ischemicevents [329,330]. Optimization of the initialracemic lead 22 that had a structure thatreplaced the piperidine motif of himbacinewith the corresponding pyridine, led to thediscovery of 23 which was a benchmark com-pound in the series [331,332].

Thrombin receptor antagonist 23 had a Ki

of 2.7 nM against the PAR-1 receptor in aradioligand binding assay. It inhibited throm-bin and high-affinity thrombin receptor acti-vating peptide (TRAP)-induced aggregation ofhuman platelets with an IC50 of 44 and 24nM,respectively. It was highly active in thethrombin-mediated calcium transient assay(Kd¼ 2.6 nM) and the proliferation assay(Ki¼ 13.0 nM) in human coronary arterysmooth muscle cells. The compound wastested in an ex vivomodel of platelet aggrega-tion in chair-trained conscious cynomolgusmonkeys. In this model, the compound wasdosed orally, and blood sampleswere collectedat baseline and at several time points post-dosing. These whole-blood samples were sub-jected aggregation induced by TRAP, and theantiaggregatory effect of the PAR-1 antago-nist quantified and correlatedwith theplasmalevels of the drug. In this assay, compound 23showed complete and sustained inhibition ofplatelet aggregation at 3mg/kg after oral ad-ministration. The compound was also testedinanarteriovenous shuntmodel of thrombosisin baboons. In this model, an extracorporalshunt was placed between the femoral arteryand femoral vein catheters. A thrombogenicsurface such as a stent or a segment of a bloodvessel placed in the shunt was used to initiatethrombosis. Compound 23 showed potent in-hibition of platelet deposition on thrombo-genic surfaces in this model after oral admin-istration at 10mg/kg [333]. More recently,another himbacine-derived PAR-1 thrombin

21 (+)-Himbacine(PAR-1 inactive)

22 Initial PAR-1 lead(racemic)

PAR-1 IC50 = 150 nM

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IC50 = 11 nM (K i = 2.7 nM)

CF3

O

O

Me

H

H

H

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H

SCH 530348

F

NHCO2Et

24 (Absolute chirality)23 (Absolute chirality)

Figure 21. Himbacine-based thrombin receptor antagonists.


receptor antagonist was evaluated in a Foltsmodel of thrombosis in anaesthetized cyno-molgus monkeys [334]. Formation of a throm-bus induced by mechanical injury in a carotidartery of the test animal was monitored bymeasuring blood flow. In this model also thehimbacine-derived PAR-1 antagonist inhib-ited thrombosis in a dose-dependent mannerand the effect was additive to the ADP antago-nist, cangrelor.Thrombin Receptor Antagonist SCH530348 SCH 530348 is an exceptionally po-tent thrombin receptor antagonist that sharesits tricyclic structural motif with the naturalproduct himbacine [329]. It is a competitiveinhibitor of the thrombin receptor with a Ki of8.1 nM and with potent activity in a series ofcellular and functional assays. In human pla-telet-rich plasma, SCH 530348 inhibitedthrombin and TRAP-induced platelet aggre-gation with IC50 values of 47 and 25nM, re-spectively, without affecting platelet aggrega-tion induced by other agonists such as ADP,thromboxane A2mimetic U46619, or collagen.SCH 530348 had no effect on coagulationparameters (prothrombin time and activatedpartial thromboplastin time), suggesting thatthe potential for bleeding events may not beincreased. In the ex vivo platelet aggregationassay in cynomolgus monkey after oraladministration, this compound showed an un-precedented level of activity giving completesuppression of TRAP agonist-induced plateletactivation for longer than 24h at 0.1mg/kg,orally. Importantly, SCH 530348, either aloneor in combination with aspirin plus clopido-grel, did not increase surgical blood loss orbleeding time versus placebo and aspirin plusclopidogrel, respectively [335]. The compoundshowed excellent pharmacokinetic propertiesin rat and monkey models. After successfullycompleting preclinical long-term toxicologicalevaluations, SCH 530348 was advanced toclinical studies. In Phase-I clinical studies,the compound demonstrated excellent safetyand tolerability. Inpharmacodynamicplateletaggregation studies, the compound showed arobust>90%inhibition of platelet aggregationfor a sustained period of time after a singleoral dose ranging from 5 to 40 mg [336]. Thetarget level of platelet aggregation throughoutthe 28-day treatment period was maintained

by a 2.5mg once-daily maintenancedose [337].

In a Phase-II randomized double-blind pla-cebo-controlled clinical study, SCH 530348was tested in patients who underwent none-mergent PCI in a trial known as ThrombinReceptor Antagonist for CardiovascularEvent Reduction in Percutaneous CoronaryInterventions (TRA-PCI) [338–343]. The pri-mary endpoint was the bleeding risk asso-ciated with SCH 530348, and the secondaryendpoint was a composite of death or majoradverse cardiac events (myocardial infarction,urgent revascularization, and ischemia re-quiring hospitalization) in PCI patients whoreceived a loading dose followed by a 60-daymaintenance dose of the drug. Patients alsoreceived other standard therapies such asaspirin, clopidogrel, and anticoagulants. Forthe primary safety endpoint, SCH530348wasnot associatedwith increasedTIMImajor plusminor bleeding when compared with placebo.For the secondary outcomeendpoint, althoughthe studywas not powered to detect statisticalsignificance, SCH 530348was associated witha numerical reduction in periprocedural myo-cardial infarction. Overall, treatment withSCH 530348 reduced arterial thromboticevents without an increase in bleedingrisk [339]. A key substudy to evaluate theinhibition of platelet aggregation showed thatthe drug achieved a sustained, potent (>80%)inhibition of TRAP-induced platelet aggrega-tion. Two additional Phase-II studies con-ducted in Japanese patients (one in patientswith acute coronary syndromes and the otherin patients with ischemic stroke) have con-firmed the favorable safety profile of SCH530348. In addition, the study performed inJapanese patients with acute coronary syn-dromes demonstrated a reduction in peripro-cedural myocardial infarctions, similar to thefindings reported in the TRA-PCI study [341].SCH530348 is currently undergoing two largePhase-III studies in patients with acute cor-onary syndrome (TRA-CER) and in patientswho are at risk of atherothrombotic events(TRA-2P) [344].Thrombin Receptor Antagonist E5555 E5555(Fig. 22) is an orally active thrombin receptorantagonist that is currently undergoingPhase-II clinical trials in patients with acute


coronary syndrome [345,346]. The studycalled LANCELOT (Lessons from Antagoniz-ing the Cellular Effects of Thrombin) willevaluate the safety and tolerability of thisdrug in along with its effect on major adversecardiovascular events in patients with acutecoronary syndrome [347]. Additionally, thestudy will evaluate the effect of the drug onex vivo inhibition of platelet aggregation andpresence of intravascular inflammatory mar-kers such as soluble CD40L, interleukin 6 andexpression of P selectin. In the early clinicalstudies, E5555 inhibited release of inflamma-tory markers in platelet-rich plasma fromhealthy volunteers [348,349].

E5555 is an iminoisoindoline with the pre-sumed structure 25 [345]. In the radioligandbinding assay, this compound showed an IC50

of 19nM. It inhibited TRAP-induced humanand guinea pig platelet-rich plasma (PRP)aggregations with IC50 values of 31 and97nM, respectively [350,351]. It also inhibitedthrombin-induced human and guinea pig PRPaggregations with IC50 values of 64 and130nM, respectively. It was reported to beactive in a guinea pig thrombosis model at30 and 100mg/kg with no change in bleedingup to 1000mg/kg.

4.4. Epinephrine Receptors

Epinephrine is a weak activator of humanplatelets and appears to serve primarily asa potentiator of platelet activation by otheragonists [352]. The combination of epinephr-

ine with a suboptimal concentration of ADP,thrombin, or TxA2 analog is a stronger stimu-lus for platelet aggregation than any of theseagonists alone. This potentiation has tradi-tionally been attributed to the ability of epi-nephrine to inhibit cAMP formation, but it isclear now that other effector pathways such asthose leading to the activation of Rap1 areinvolved. Epinephrine has no detectable di-rect effect on PLC in platelets and does notcause shape change [353].

Platelet responses to epinephrine aremediated by a2A-adrenergic receptors (seeTable 4) that are coupled to Gza of the Gi

family. Platelets from Gza knockout miceshowed lack of epinephrine-induced potentia-tion of platelet aggregation caused by otheragonists. Additionally, there was a loss of thenormal ability of epinephrine to inhibit PGI2-stimulated cAMP formation and an increasedresistance to fatal thromboembolism follow-ing injection of epinephrine and collagen, butnot ADP and collagen. There have been re-ports of mild congenital bleeding disordersassociated with impairment of epinephrine-induced platelet aggregation.

4.5. Glycoprotein IIb/IIIa Antagonists

The activation of glycoprotein IIb/IIIa recep-tors on the platelet membrane represents theculminating event in receptor-mediated plate-let activation and it is the final common path-way leading to platelet aggregation. It hasbeen known that patients with Glanzmann’sthrombasthenia, whose platelet GP IIb/IIIacomplex is either deficient or abnormal, havea severe bleeding disorder [354,355]. This ob-servation has helped identify the molecularmechanism of GP IIb/IIIa receptor activationand platelet aggregation.

As mentioned above, platelet response totissue injury occurs in four phases, adhesion,activation, secretion, and aggregation. Adhe-sion of unactivated platelets to denuded en-dothelium takes place by the binding to col-lagen via adhesive proteins such as vWF. Thisis followed by collagen-mediated activation ofplatelets, which leads to the release of procoa-gulant contents of the secretary granules, anevent that causes further platelet activation.Activated platelets display an active confir-

OMeO

N

NHEtO

EtO N

25 (E5555)

t-Bu

O

F

PAR-1 binding IC 50 = 19 nM

Platelet aggregation IC50:

Human = 31 nM (TRAP); 64 nM ( -thrombin)

Guinea pig = 97 nM (TRAP); 130 nM ( -thrombin)

Active in guinea pig thrombosis model

(photochemical injury) at 30 mg/kg and 100 mg/kg

No bleeding up to 1000 mg/kg in this model

Figure 22. Structure and pharmacological profileof E5555.


mation of GP IIb/IIIa receptors on their sur-face, thereby facilitating the binding to theRGD motifs of adhesion proteins such as fi-brinogen with high affinity. In contrast, at theresting stageGP IIb/IIIa receptors have only alow affinity for theRGD sequence. TheGP IIb/IIIa receptor is a member of the integrin fa-mily of adhesion molecules that are found onall cell surfaces [356]. There are 5.0–8.0� 104

GP IIb/IIIa receptors on each platelet [357].Fibrinogen has two RGD sequences at its dis-tal ends that can bind to two platelets, actingas an efficient linker between the two plate-lets, and the process repeats itself rapidlyduring the aggregation phase. Platelet inter-nal microenvironment is also influenced byligand binding to the GP IIb/IIIa receptor thatprovokes an array of intracellular sig-nals [358]. Since activation of GP IIb/IIIa re-ceptors represents the final, convergent stepof platelet activation, the inhibition of thisstep constitutes an efficient approach to in-hibiting platelet aggregation [359].

4.5.1. Intravenous GP IIb/IIIa AntagonistsOnly intravenous formulations of GP IIb/IIIaantagonists have been approved for clinicaluse. The three commercially available intra-venousGP IIb/IIIa antagonists are abciximab,eptifibatideand tirofiban [360].Abciximab is amonoclonal antibody, eptifibatide is a cyclicheptapeptide and tirofiban is low molecularnonpeptide entity. These agents have under-gone extensive clinical trials and have beenindicated for use in setting of percutaneouscoronary intervention and acute coronary syn-drome [30]. Asmentioned, GP IIb/IIIa antago-nists are effective antiplatelet agents sincethey inhibit the final common pathway ofplatelet aggregation. By the same token,bleeding side effect is common for theseagents, which is controlled by adjusting thedose. The preclinical pharmacology and theclinical studies of these agents have been ex-tensively reviewed [359].Abciximab Abciximab is derived fromamur-ine monoclonal antibody for the GP IIb/IIIareceptor [361]. In order to reduce immuno-genicity to humans, it has been modified aspart-murine, part-human chimeric Fab frag-ment that contains the heavy- and light-chain regions of the murine antibody

attached to the constant regions of the hu-man antibody [362].

Abciximab interacts with the GP IIb/IIIareceptor at sites distinct from the RGD se-quence and probably exerts its inhibitoryeffects by steric hindrance [363]. Abciximabalso binds to the avb3 integrin (vitronectinreceptor). Abciximab has a short half-life withthe majority of the drug cleared from plasmawithin 25min. Yet platelet associated abcix-imab can be detected for prolonged periods (upto 14 days) in the plasma, perhaps due todissociation and reassociation of the drug atthe receptor. Maximal inhibition of plateletaggregation was observed when�80%GP IIb/IIIa receptors were blocked by abcixi-mab [364]. This level of IPA was achieved innonhuman primates when abciximab was in-jected at a dose of 0.25mg/kg.

Abciximab is indicated as an adjunct topercutaneous coronary intervention for theprevention of cardiac ischemic complications.Abciximab has been studied in four majorPhase-III clinical trials, all of which evaluatedthe effect of the drug in patients undergoingPCI: in patients at high risk for abrupt closureof the treated coronary vessel (EPIC), in abroader group of patients (EPILOG), in un-stable angina patients not responding to con-ventional medical therapy (CAPTURE), andin patients suitable for either conventionalangioplasty/atherectomy or primary stent im-plantation (EPILOG Stent; EPISTENT). Alltrials involved the use of various, concomitantheparin dose regimens and, unless contrain-dicated, aspirin (325mg). The EPIC studydemonstrated 35% reduction in the 30-dayprimary endpoint of death, MI, or repeatedprocedures [365]. The EPILOG study demon-strated that the positive results seen in high-risk patients could be reproduced in lower riskischemic complications fromurgent or electivePCI [366]. There was no increase in majorbleeding, although minor bleeding incidentswere more frequent. The TARGET trial was adirect comparison of abciximab with tirofiban(below) in patients undergoing PCI [367]. Thetrial showed tirofiban to be less effective thanabciximab. The ISAR-REACT trial evaluatedthe effect of abciximab in PCI patients whowere pretreated with 600mg of clopido-grel [368]. Abciximab was not of additional


benefit in the combined endpoint of death,MI,and urgent target vessel revascularization inthis patient population studied.

In a clinical study evaluating the benefit ofabciximab in non-ST segment elevating ACSpatients (GUSTO 4-ACS), where PCI was notplanned, abciximabwas found not to yield anybenefit [369]. In ST-segment elevation myo-cardial infarction, abciximab was beneficialwithprimaryangioplastyat a cost of increasedmajor bleeding episodes [370]. Other clinicaltrials have examined the timing of abciximabadministration and a trend toward reductionin mortality with early administration of thedrug has been found [371].Eptifibatide Eptifibatide (Fig. 23) is a lowmolecularweight (MW¼ 832) cyclic heptapep-tide, modeled on the structure of barbourin, a73-amino aid peptide found in the snake ve-nomof Southeasternpygmy rattlesnake [372].Eptifibatide reversibly inhibits platelet aggre-gation bypreventing the binding of fibrinogen,von Willebrand factor, and other adhesiveligands to GP IIb/IIIa. When administeredintravenously, eptifibatide inhibits ex vivoplatelet aggregation in a dose- and concentra-tion-dependent manner. Platelet aggregationinhibition is reversible following cessation ofthe eptifibatide infusion. Plasma elimination

half-life of eptifibatide is approximately 2.5 hin humans [373].

Infusion of eptifibatide intobaboons causeda dose-dependent inhibition of ex vivo plateletaggregation, with complete inhibition of ag-gregation achieved at infusion rates greaterthan 5.0mg/kg/min. In a baboon model ofthrombosis that is refractory to aspirin andheparin, doses of eptifibatide that inhibitedplatelet aggregation prevented acute throm-bosis with only a modest prolongation (two- tothreefold) of the bleeding time. Platelet aggre-gation in dogs was also inhibited by infusionsof eptifibatide, with complete inhibition at2.0mg/kg/min. This infusion dose completelyinhibited canine coronary thrombosis inducedby coronary artery injury (Folts model).

Eptifibatide had undergone three placebo-controlled, randomized studies at the time ofits registration [374]. The PURSUIT studyevaluated patients with acute coronary syn-dromes [375]. Compared to placebo, eptifiba-tide administered as a 180mg/kg bolus fol-lowed by a 2.0mg/kg/min infusion significantly(p¼ 0.042) reduced the incidence of combinedendpoint of death or myocardial infarction.The reduction in the incidence of endpointevents in patients receiving eptifibatide wasevident early during treatment, and this re-duction was maintained through at least 30days. The IMPACT II study was conducted inpatients undergoing PCI [376]. The primaryendpoint was the composite of death, MI, orurgent revascularization, analyzed at 30 daysafter randomization in all patients who re-ceived at least one dose of study drug. In thisstudy, eptifibatide regimen reduced the rate ofdeath, MI, or urgent intervention, although at30 days, this finding was statistically signifi-cant only in the lower dose eptifibatide group.As in the PURSUIT study, the effects of epti-fibatide were seen early and persistedthroughout the 30-day period.

The ESPRIT study was conducted in pa-tients undergoing elective or urgent PCI withintended intracoronary stent placement [377].In this study, the incidence of the primaryendpoint (of the composite of death, MI, ur-gent target vessel revascularization) and se-lected secondary endpoints were significantlyreduced in patients who received eptifibatide.A treatment benefit in patients who received

HN

O

NH

O

OH

O

HN

NH2HN

NH

S

S

NH

N

O

NH

OO

O

NH

26 Eptifibatide

H2NO

Figure 23. Eptifibatide has a cyclic hexapeptidestructure that is derived from barbourin, a compo-nent of Southeastern pigmy rattlesnake venom.


eptifibatide was seen by 48h and at the end ofthe 30-day observation period. The treatmenteffect of eptifibatide seen at 48h and 30 daysappeared preserved at 6months and 1 year. Inthe IMPACT II, PURSUIT, and ESPRIT stu-dies of eptifibatide, most patients receivedheparin and aspirin. Additional studies eval-uating eptifibatide in high-risk patients withNSTE-ACS, in ST-elevation myocardial in-farction, and in ischemic stroke have beenundertaken [378,379].Tirofiban Tirofiban (Fig. 24) is a low molecu-lar weight (MW¼ 440), reversible GP IIb/IIIaantagonist that has a modified tyrosine struc-ture [380–382]. When administered intrave-nously, tirofiban inhibits ex vivo platelet ag-gregation in a dose- and concentration-depen-dent manner. When given according to therecommended regimen, more than 90% inhi-bition is attained by the end of the 30mininfusion. Tirofiban has a half-life of approxi-mately 2h.

Three large-scale clinical trials were con-ducted to study the efficacy and safety oftirofiban in the management of patients withAcute Coronary Syndrome. PRISM-PLUS(Platelet Receptor Inhibition for IschemicSyndrome Management–Patients Limited byUnstable Signs and Symptoms) trial evalu-ated the use of tirofiban in combination withheparin inpatientswithdocumentedunstableangina/non-Q-wave myocardial infarctionwithin 12h of entry into the study and initia-tion of treatment [383]. The primary endpointof the study was a composite of refractoryischemia, new myocardial infarction anddeath at 7 days after initiation of tirofibanand heparin. At the primary endpoint, therewas a 32% risk reduction in the overallcomposite.

PRISM (Platelet Receptor Inhibition forIschemic Syndrome Management) studycompared tirofiban to heparin in patientswith unstable angina/non-Q-wave myocar-dial infarction. In this study, the drug wasstarted within 24 h of the time the patientexperienced chest pain. In this study benefitwith the use of tirofiban was confined topatients who had elevated troponinlevels [384].

RESTORE (Randomized Efficacy Study ofTirofiban for Outcomes and Restenosis)study compared tirofiban to heparin in pa-tients undergoing PCI or atherectomywithin72 h of presentation with unstable angina oracute myocardial infarction. This studyshowed that tirofiban when added to heparinreduces ischemic complications in patientswith unstable angina/NSTEMI, whethertreated conservatively or intervention-ally [385]. The benefits seem to be greatestin patients with highest risk for ischemiccomplications.Summary of GP IIb/IIIa Antagonists Gener-ally, there is considerable heterogeneity in theclinical data of GP IIb/IIIa antagonists. Tim-ing and dosing seem to be critically important.Preclinical animal data and dose-finding clin-ical studies have shown that the critical levelof receptor blockage is �80% [386,387]. Low,subtherapeutic levels of synthetic antagonistsactivate quiescent GP IIb/IIIa receptors andcause expression of inflammatory markers,indicating a pro-aggregatory state [388]. Clin-ical studies have demonstrated that the levelof platelet inhibition with GP IIb/IIIa antago-nists is an independent predictor of death andischemic complications after PCI [389]. Thetiming of the use of GP IIb/IIIa antagonists inthe clinical setting is also important for effi-cacy and safety. It is best to use the agent atthe time of maximum platelet aggregationthat occurs as a result of plaque disruption atthe time of balloon angioplasty or intracoron-ary stent-implantation. Use of GP IIb/IIIaantagonists is recommended when risk stra-tification indicates high level of troponin orCD40 levels. Higher troponin levels are asso-ciated with an unstablemilieu at the coronaryplaques and soluble CD40 ligand is an inflam-matory marker that is associated with highermortality in ACS [390].

HNO

O

OH

S

O

O

27 Tirofiban

HN

Figure 24. Tirofiban has amodified, lowmolecularweight tyrosine structure.


4.5.2. Oral GP IIb/IIIa Antagonists Severalorally active GP IIb/IIIa antagonists havebeen reported (Fig. 25) [391–397]. However,in the Phase-III clinical trials, these agentsfailed to reproduce the success of intravenousGP IIb/IIIa antagonists [398]. In fact, oral IIb/IIIa antagonists showed increased mortalityin Phase-III clinical trials [399]. There was adose-dependent increase in bleeding, espe-cially in trials where aspirin was coadminis-tered and a general trend toward increasedincidence of myocardial infarction in longerterm follow-up [400].

The EXCITE (Evaluation of Oral Xemilo-fiban in Controlling Thrombotic Events) trialcompared xemilofiban with placebo in pa-tients undergoing PCI [401]. At 30 days, therewas a statistically significant increase in mor-tality in the xemilofiban group. The OPUSTIMI-16 (Orbofiban in PatientswithUnstableCoronary Syndromes) trial studied the effector orbofiban in>10,000 patients with ACS for6 months [402]. The study was terminatedprematurely due to an excess 30-daymortalityin the drug-treated group. Similarly, the

SYMPHONY trials that studied sibrafiban inACS patients failed to demonstrate a reduc-tion in ischemic endpoints. There was an ex-cess of mortality in the sibrafiban group in thesecond SYMPHONY trial. The BRAVO (TheBlockade of Glycoprotein IIb/IIIa Receptor toAvoidVascularOcclusion) trial that examinedthe combined effect of lotrafibanandaspirin invarious coronary, cerebrovascular and periph-eral vascular disease had to be terminatedprematurely due to an excess mortality in thetreatment group [403].

Several arguments have been put forth toexplain the deleterious effect of long-termtreatment with oral IIb/IIIa antagonists. Apharmacokinetic explanation is that the ad-justed dose-levels to prevent bleeding asso-ciated with long-term use of oral IIb/IIIa an-tagonists prevented reaching sustained plas-ma levels of the drugs necessary to achieve thedesired pharmacodynamic effect. Continuedlowsubtherapeutic exposure of theGPIIb/IIIaantagonists have been in fact shown to have“paradoxic” prothrombotic effect, includingenhanced fibrinogen binding and release of

O

O

HN

OO

NH

HN

H2N

28 Xemilofiban

HN

O

O

O

HNN

O

HN

H2N

29 Orbofiban

OO

O

N

O

NHO

NHO

H2N

30 Sibrafiban

NH

O O

O

OHN

O

O

NH

33 Lefradafiban

NH

OO

O

OHN

OONHN

H2N

32 Roxifiban

NH

N

O

OH

O

O

N

HN

31 Lotrafiban

Figure 25. Examples of orally active GP IIb/IIIa antagonists.


markers of platelet activation [388, 404, 405].Orbofiban has been associated with increasedCD63 expression and in vitro platelet aggre-gation and thromboxane production [406]. Inpatients treated with orbofiban increased pla-telet fibrinogen binding andP-selectin expres-sion have been reported as well as sustainedlevels of neutrophil adhesion mole-cules [407,408]. Increased P-selectin expres-sion may underscore a proinflammatory rolefor IIb/IIIa antagonists. There has been alsothe suggestion that IIb/IIIa antagonist mayrelease soluble CD40 ligand that can lead tostimulation of macrophages to release pro-teins that degrade the fibrous cap of athero-sclerosis andproduction of tissue factor,whichare detrimental events in plaque rupture andacute coronary thrombosis [409]. Also manycells have RGD sequences that can becomeunintended targets of IIb/IIIa antagonists,with deleterious consequences in the longterm [410]. For example, xemilofiban and or-bofiban cause time-dependent increases in theactivity of capsase-3, a protein that is impor-tant in normal cardiac morphogen-esis [411,412]. Despite the speculative natureof many of these mechanistic explanations forthe failure of oral IIb/IIIa antagonists, it iscertain that long-term inhibition of plateletaggregation without inhibiting the earlierstages of platelet activation is undesirable.

4.6. PDE-III Inhibitors

Cyclic AMP is an important secondmessengerin regulating intracellular events that med-iate platelet activation following stimulation

of cell-surface receptors [413–416]. Cellularlevels of cAMP are tightly controlled by itsrate of synthesis by adenylate cyclase and itsrate of hydrolysis by phosphodiesterase en-zymes. The major cAMP phosphodiesterasein platelets is PDE-IIIA, which is inhibited byanother cyclic monophosphate, cGMP [417].Therefore, cAMP levels can be potentiatedeither by directly inhibiting PDE-III enzymesor by elevating cGMP levels.

In addition to platelets, cAMP is also pre-sent in cardiacmuscle and endothelial smoothmuscle [418,419]. Increased cAMP levels inheart muscles increase contractility (ionotro-py), heart rate (chronotropy), and conductionvelocity (dromotropy). Potentiation of cAMPlevels in endothelial cells lead to vasodilata-tion. The overall hemodynamic effect of PDE-III inhibition in the cardiovascular system, inaddition to the platelet effects, is increasedcardiac output and reduced systemic vascularresistance [420]. Because cardiac contractilityleads to increased arterial pressure and vaso-dilatation leads to reduced arterial pressure,the net effect of PDE-III inhibition on arterialpressuredepends on its relative effect onheartversus vascular smooth muscle.

4.6.1. Cilostazol Cilostazol is a PDE-III in-hibitor that has been approved by FDA for thetreatment of intermittent claudication for pa-tients with PAD [421]. The antiplatelet, vaso-dilatory, and cardiostimulative properties of aPDE-III inhibitor can have a beneficial effectfor this condition with limited treatment op-tions (Fig. 26) [422]. In a clinical study con-ducted in Japanese patients, cilostazol was

N

NN

N

N

N

N

N

OH

OH

OH

OH

36 Dipyridamole

HN

NN

O

35 Milrinone

NH

O

ON

N

NN

34 Cilostazol

Figure 26. Antiplatelet agents that work by PDE inhibition.


shown to prevent the recurrence of cerebralinfarction. In another study, cilostazol demon-strated inhibition of intimal proliferation andrestenosis. Additionally, the drug has beenknown to increase HDL level and decreasestriglyceride, by unknown mechanisms [423].Cilostazol is contraindicated (black box warn-ing) in patients with a history of heart failureor compromised ejection fraction. Chronic useof milrinone, another PDE-III inhibitor, inpatients with cardiomyopathy has been asso-ciated with increased mortality. Cilostazolhas shown improved cardiac safety in compar-ison to milrinone. In a meta-analysis of eightrandomized, double-blind, placebo-controlledclinical trials, cilostazol increased pain-freewalking by 67% and was better than pentox-ifylline, the only other drug approved for PADthat works by different mechanisms [424].

4.6.2. Milrinone Milrinone is a PDE-III inhi-bitor, which produces positive inotropic effectsand vasodilation independent of b1-adrenergicreceptor stimulation, with little chronotropicactivity [425,426]. It is used in the manage-ment of heart failure, but only when conven-tional treatment with vasodilators and diure-tics has proven insufficient. This is due to thepotentially fatal adverse effects of milrinone,including ventricular arrhythmias. The rela-tively longer half-life of milrinone (2.5 h) alsomakes it difficult to withdraw the patient fromthe drug, if needed. The acute administrationof intravenousmilrinonehasbeenevaluated inmore than 1600 patients with chronic heartfailure.This studyhasshowed improvement inthe outcome of total death over 48h. However,longer term oral treatment with milrinone didnot show improvement in symptoms, and wasassociated with an increased risk of hospitali-zation and death [427].

4.6.3. Dipyridamole Dipyridamole is a PDE-V inhibitorwithweak inhibition against PDE-III. Dipyridamole is also known to inhibit theuptake of adenosine into platelets, endothelialcells, and erythrocytes in vitro and in vivo in adose-dependent manner at therapeutic con-centrations [428]. This inhibition of the cellu-lar uptake of adenosine results in an increasein local concentrations of adenosine that actson the platelet A2-receptor, thereby stimulat-

ing platelet adenylate cyclase and increasingplatelet cAMP levels. Via this mechanism,platelet aggregation is inhibited in responseto various stimuli [429].

Dipyridamole is indicated as an adjunct tocoumarin anticoagulants in the prevention ofpostoperative thromboembolic complicationsof cardiac valve replacement. It is believedthat platelet reactivity and interaction withprosthetic cardiac valve surfaces, resulting inabnormally shortened platelet survival time,is a significant factor in thromboembolic com-plications occurring in connection with pros-thetic heart valve replacement. Dipyridamolehas been found to lengthen abnormally shor-tened platelet survival time in a dose-depen-dent manner.

In three randomized controlled clinicaltrials involving 854 patients who had under-gone surgical placement of a prosthetic heartvalve, dipyridamole, in combinationwithwar-farin, decreased the incidence of postoperativethromboembolic events by 62–91% comparedto warfarin treatment alone. The incidence ofthromboembolic events in patients receivingthe combination of dipyridamole andwarfarinranged from 1.2 to 1.8%. In three additionalstudies involving 392 patients taking dipyri-damole and coumarin-like anticoagulants, theincidence of thromboembolic events rangedfrom 2.3 to 6.9% [430,431].

4.6.4. Aggrenox Aggrenox is a combinationof dipyridamole in an extended-release formand aspirin that is indicated to reduce the riskof stroke in patients who have had transientischemia of the brain or completed ischemicstroke due to thrombosis [432]. In a double-blind, placebo-controlled, 24-month study(European Stroke Prevention Study 2,ESPS2) in which 6602 patients had an is-chemic stroke (76%) or transient ischemic at-tack (24%) within 3 months prior to entry, thecombination therapy reduced the risk ofstroke by 22.1% compared to aspirin 50mg/day alone (p¼ 0.008) and 36.8% compared toplacebo (p< 0.001).

4.7. EP3 Receptor Antagonists

EP3 is aG-protein-coupled platelet prostanoidreceptor that is activated by PGE2 (see


Fig. 8) [433,434]. The EP3 receptor is coupledto the inhibitory G-protein (Gi) and functionsas a coagonist receptor for other platelet sur-face receptors that are coupled to Gi, such ascollagen, TxA2 and ADP. EP3 stimulation in-dependently does not lead to platelet activa-tion since it has no effect on platelet Ca2þ

mobilization; however, it serves to potentiateplatelet activation by other Gi-coupled recep-tors mentioned above which mobilize plateletCa2þ .

PGE2 is produced by arterial wall that isalso the source for the endogenousantiplateletfactor PGI2 (see Section 3.3) [435]. Whereashealthy arterial wall produces very littlePGE2, arterial wall subjected to inflammatorystimuli under atherosclerosis condition pro-duces substantial amounts of PGE2. Studiesin gene-deleted mice have shown that theproaggregatory effect of PGE2 is mediated bytheEP3 receptor. For example, inmice lackingplatelet EP3 receptor, PGE2-promoted arter-ial thrombosis was impaired.

DG-041 (Fig. 27) is a potent EP3 antagonistthat is currently undergoing Phase-II clinicaltrials for peripheral arterial and occlusivedisease [436]. It is a noncompetitive antago-nistwith an IC50 of 4.6 nMagainst humanEP3

receptor [437]. In the humanplatelet aggrega-tion assay, DG-041 inhibited PGE2 (1mM)stimulated human platelet aggregation withan IC50 of 130nM, and aggregation induced by

sulprostone (0.1mM), another EP3 agonist,with an IC50 of 218nM. In an ex vivo plateletaggregation model in rat, DG-041 completelyinhibited platelet aggregation at a dose of10mg/kg or higher. No increase in bleedingtime post calibrated surgical incision wasnoted up to 100mg/kg, consistent with thelack of bleeding noted in EP3 null ice [3].DG-041 has shown potentiation of the anti-platelet effect of clopidogrel in a rat modelwith no further increase in bleeding, suggest-ing that a lower dose of clopidogrel combinedwith an EP3 antagonist could give improvedsafety margin.

5. PERSPECTIVES ON ANTIPLATELETTHERAPY

Because of the central role of platelets in thepathogenesis and progression of cardiovascu-lar disease, antiplatelet agents have becomethe cornerstone of pharmacotherapy for thetreatment of acute coronary syndromes, is-chemic stroke, peripheral artery disease, andin the settings of PCI. In acute settings, anti-platelet agents are usually coadministeredwith anticoagulants and thrombolytic agents.The current gold standard of antiplatelet ther-apy comprises aspirin, clopidogrel, and GPIIb/IIIa antagonists such as abciximab andeptifibatide. Of these, aspirin and clopidogrelare oral formulations and the GP IIb/IIIaantagonists are intravenous formulations.

The development of newer antiplateletagents has been both challenging and reward-ing. The introduction of ADP antagonists andGP IIb/IIIa antagonists has transformed theantiplatelet field. Therehavebeendisappoint-ments too. For example, efforts to develop anorally active GP IIb/IIIa antagonist have uni-formly failed in the clinic. In an effort toreproduce the success of the intravenous GPIIb/IIIa antagonist for oral formulations,three candidates were taken to clinical stu-dies, but failed to produce a beneficial out-come. In fact, in several clinical studies, car-diovascular mortality was higher in the treat-ment groups than placebo that resulted in thepremature termination of these studies.Whileseveral explanationshavebeen offered for thisfailure (see Section 4.5.2), it is now quite clear

NO O

SS

HN

O

Cl

ClF

Me

Cl

Cl

37

DG-041 (EP3 receptor antagonist)

hEP3 IC50 = 4.6 nM

mEP3 IC50 = 5.3 nM

Inhibition of human platelet aggregation

(agonist: PGE2, 1 µM) IC50 = 130 nM

Figure 27. EP3 antagonist DG-041.

PERSPECTIVES ON ANTIPLATELET THERAPY 451

that sustained, long-term antagonism of theGP IIb/IIIa receptor produces a paradoxic pro-thrombotic effect.

Clopidogrel, the second-generation thieno-pyridine, is awidelyusedADPantagonist thathas effectively replaced its predecessor, ticlo-pidine that had often fatal side effects such asthrombocytopic purpura (TTP) and neutrope-nia. The thienopyridines such as clopidogrelrequire a CYP 450-mediated activation to athiol metabolite that is the active form of thedrug. About 80% of clopidogrel metabolitesare, however, inactive which accounts for itsslow onset of action and amodest inhibition ofplatelet aggregation. This shortcoming is ad-dressed by prasugrel, a third-generation thie-nopyridine that undergoes a highly efficientmetabolic activation (see section “Prasugrel”).As a result, prasugrel is more potent, it showsa robust ex vivo inhibition of platelet aggrega-tion after oral dosing, has a faster onset ofaction, and less interindividual variability.However, in clinical trials, prasugrel hasshown higher rates of major and minor bleed-ing that somewhat offset these favorabletraits. Prasugrel (Effient�) has recently re-ceived regulatory approval for the reduction ofthrombotic events in patients with acute cor-onary syndromes who are managed with per-cutaneous coronary intervention (ACS-PCI) [438].

Ticagrelor (AZD6140) represents the firstorally active, reversible ADP antagonist thatdoes not require metabolic activation (see sec-tion “Ticagrelor”). Phase-II clinical study re-sults suggest potent antiplatelet effect, withbleeding episodes similar to that of clopidogrelat its therapeutic dose. However, there havebeen incidences of dyspnea and bradycardiathat need to be fully assessed in larger trials.

Thrombin receptor antagonists are a newclass of antiplatelet agents. Unlike directthrombin inhibitor (DTI) anticoagulants,thrombin receptor antagonist antiplateletagents do not inhibit the enzymatic activityof thrombin, thereby sparing its role in fibringeneration. Rather, they inhibit thrombin-mediated activation of platelet protease-acti-vated receptor-1 (PAR-1), which is the mostpotent cell-surface inducer of human plateletactivation. Two thrombin receptor antago-nists have advanced to clinical trials (see

Section 4.3.5). Of these, SCH 530348 is apotent, competitive antagonist of plateletthrombin receptor (Ki¼ 8.1 nM)with excellentactivity in multiple functional assays [329].SCH 530348 has demonstrated potent ex vivoantiplatelet effect in nonhuman primates andsimilar compounds from the same structuralseries have demonstrated dose-dependent in-hibition of thrombus formation in nonhumanprimate thrombosis models. As discussedabove, Phase-II clinical trials of SCH 530348showedapromising safetymarginwith regardto TIMI-major or TIMI-minor bleeding epi-sodes andanumerical reduction in thromboticevents in ACS patients during a 2-month fol-low-up. Due to these encouraging results,SCH 530348 has been advanced to Phase-IIIclinical trails to evaluate its efficacy andsafetyin patients with acute coronary syndrome(TRA-CER; n¼ 10,000) and for secondary pre-vention of ischemic events (TRA-2P TIMI 50;n¼ 19,500) [344].

Another orally active thrombin receptorantagonist E5555 is currently undergoingPhase-II clinical trials in patients with acutecoronary syndrome. The trialwill evaluate thesafety and tolerability of E5555 and its effecton major adverse cardiovascular events inpatients with acute coronary syndrome. Inpreclinical pharmacology, this drug demon-strated potent antiplatelet effect in a guineapig thrombosis model without an increase inbleeding.

Bleeding is the main side effect for allantithrombotic therapies, including antipla-telet therapy [439]. In fact, the current para-digm of antithrombotic therapy is “no bleed-ing, no efficacy.” Therefore, clinicians have tostrike a balance between bleeding and out-come by careful selection of dose and adjuncttherapies. This scenario is often made morecomplex by the frail nature of patients andcompromising situations such as advancedage and other underlying pathologic condi-tions. Therefore, it will be very desirable toachieve improved safetymargin in a new anti-platelet drug. The thrombin receptor antago-nists hold some promise in this area. Based onthe published data for SCH 530348 andE5555, these compounds confer potent anti-platelet effect without attendant bleeding inthe preclinical models and, in the case of SCH


530348, in the publishedPhase-II clinical trialresults [338].

There are plausible explanations for theabsence of significant bleeding that is notedfor the thrombin receptor antagonists in pre-clinical animal models and in the TRA-PCIstudy. Although proteolysis of the thrombinreceptor is the most potent mechanism ofplatelet stimulation, other platelet activationmechanisms that remain intact are perhapssufficient for normal hemostasis. Thrombin-mediated platelet activation therefore mightrepresent a more pathologically relevant pla-telet activation mechanism in the context of amajor vascular injury such as rupture of anatherosclerotic plaque or PCI procedures. In arecent study in PAR-4�/� mice, Furie andCoughlin have demonstrated using real-timedigital fluorescence microscopy that plateletactivation by thrombin is necessary for throm-bus growth, but not for primary hemosta-sis [324]. During laser-induced thrombosis,juxtamural platelet accumulation immedi-ately after laser injury was not affected inPAR-4�/� mice. However, subsequent growthof platelet thrombi was markedly diminishedin PAR-4�/� mice. Also, fibrin generation waspreserved in the knockout mice. The conclu-sion from these studies is that a thrombinreceptor antagonist could produce potent anti-platelet effectwithout increased bleeding. Theongoing clinical studies will further validatethe long-term sustainability of the anticipatedsafety margin for thrombin receptorantagonists.

In addition to the third-generation thieno-pyridines, reversible ADP antagonists, andthrombin receptor antagonists mentionedabove, researchers have been exploring newerfrontiers of antiplatelet targets, often trying toadd a new dimension to an existing antiplate-let therapy. For example, recent publicationshave demonstrated a key role for tachykininsin the positive feedback regulation of plateletaggregation. The prothrombotic effects of ta-chykinins on platelets are mediated throughneurokinin 1 (NK-1)whichmay therefore offera novel antiplatelet target [440]. Serotoninreceptor antagonists have been explored asantiplatelets also. The serotonin receptor sub-type 5HT2A is found on platelets, and its sti-mulation constitutes a platelet activation me-

chanism [441]. The 5-HT2A antagonist sarpo-grelate has demonstrated antiplatelet effectin patients with ischemic stroke. In a recentclinical study, sarpogrelate reduced the inci-dence of cerebral infarctions with fewer bleed-ing episodes than aspirin, although its overallefficacy was somewhat less than that of aspir-in [442]. The antiplatelet effect of 5HT2A in-hibition for the treatment of vascular disor-ders can be additionally benefited by the anti-inflammatoryandantiproliferativepropertiesthat 5-HT2A inhibition is known to engen-der [443,444]. Since 5HT2A receptors are dis-tributed throughout the central nervous sys-tem, the cardiovascular utility of a 5-HT2A

antagonist should be weighed against its po-tential CNS side effects. TNF-a is known toinfluence cardiomyocyte apoptosis andplays arole in the mechanism that contributes tocardiac dysfunction during ischemia-reperfu-sion injury and heart failure [445]. Pentoxifyl-line, a marketed phosphodiesterase inhibitorantiplatelet agent that is indicated for thetreatment of patients with intermittent clau-dication, is also known to reduce TNF-a le-vels [446]. Pentoxifylline has been shown toimprove cardiac function in reperfused heartand it has been known to exert beneficialeffects in heart failure and in cardiopulmon-ary bypass surgery [447]. Since inflammationis a major component of cardiovascular dis-orders, an anti-inflammatorymechanismmayadd further benefit to the antiplateletproperty.

In conclusion, several new ADP antago-nists and GP IIb/IIIa antagonist antiplateletagents have been introduced into the marketduring the past two decades. The introductionof these agents has profoundly impacted thetreatment of atherothrombotic disorders.Newer agents such as reversible ADP antago-nists and thrombin receptor antagonists withpotential for improved safety margin are onthe horizon.

ABBREVIATIONS

AA Arachidonic acidACS Acute coronary

syndromeADP Adenosine diphosphate

ABBREVIATIONS 453

AMIS Aspirin inmyocardial in-farction study

ARCH Aortic arch-related cere-bral hazard

ATARI Antithrombotic therapyin acute recovered cere-bral ischemia

AV shunt Arteriovenous shuntBRAVO Blockade of the GP IIb/

IIIa receptor to avoidvascular occlusion

CAD Coronary artery diseaseCABG Coronary artery bypass

graftcAMP Cyclic adenosine

monophosphatecGMP Cyclic guanosine

monophosphateCAPRIE Clopidogrel versus as-

pirin in patients at riskof ischemic events

CAPTURE c7E3 antiplatelet ther-apy in unstable refrac-tory angina

CARESS Clopidogrel and aspirinfor reduction of emboliin symptomatic carotidstenosis

CATS Canadian american ti-clopidine study

CD CyclodextrinCHARISMA Clopidogrel for high

atherothrombotic riskand ischemic stabiliza-tion, management, andavoidance

CLARITY-TIMI Clopidogrel as adjunc-tive reperfusion ther-apy-thrombolysis inmyocardial infarction

CO, COX CyclooxygenaseCOMMIT Clopidogrel and meto-

prolol in myocardial in-farction trial

CREDO Clopidogrel for the re-duction of events dur-ing observation

CURE Clopidogrel in unstableangina to prevent re-

current ischemicevents

CYP3A4 Cytochrome P450 en-zyme 3A4

DAG DiacylglycerolDISPERSE Dose-finding investiga-

tive study to assess thepharmacodynamic ef-fects of AZD6140 inatherosclerotic disease

ECG ElectrocardiogramEDRF Endothelium-derived

relaxing factorEPIC Evaluation of 7E3 for the

prevention of ischemiccomplications

EPILOG Evaluation in PTCA toimprove long-termoutcome with abcixi-mab GP IIb/IIIablockade

EPISTENT Evaluation of plateletIIb/IIIa inhibitor forstenting trial

ESPRIT Enhanced suppressionof the platelet IIb/IIIareceptor with integrilintherapy

EXCITE Evaluation of oral xemi-lofiban in controllingthrombotic events

FANTASTIC Full anticoagulationver-sus aspirin andticlopidine

FASTER Fast assessment ofstroke and transient is-chemic attack to pre-vent early recurrence

GDP Guanosine diphosphateGUSTO Global utilization of

streptokinase and tPAfor occluded arteries

5HT2A 5-hydroxytryptaminereceptor 2A

GP IIb/IIIa Glycoprotein IIb/IIIaGPCR G-protein-coupled

receptorHDL High-density lipoprotein


IMPACT Integrilin tomanagepla-telet aggregation toprevent coronarythrombosis

IP Prostacyclin receptorIP3 Inositol triphosphateIPA Inhibition of platelet

aggregationISIS-2 Second international

study of infarctsurvival

JUMBO-TIMI 26 Joint utilization of med-ications to block plate-lets optimally-throm-bolysis in myocardialinfarction 26

LANCELOT Lessons from antagoniz-ing the cellular effectsof thrombin

MACE Major adverse cardiacevents

MATCH Management of athero-thrombosis with clopi-dogrel in high-riskpatients

MLC Myosin light chainMLCK Myosin light-

chain kinaseNO Nitric oxideNK-1 Neurokinin-1NSAID Nonsteroidal anti-

inflammatory agentsNSTE-ACS Non-ST-segment

elevation acutecoronary syndromes

NSTEMI Non-ST-segment eleva-tion myocardialinfarction

OPUS Orbofiban in patientswith unstable coronarysyndromes

P2X1 Purinergic receptor-X,subclass 1

P2Y1 Purinergic receptor-Y,subclass 1

P2Y12 Purinergic receptor-Y,subclass 12

PAD Peripheral arterydisease

PAF Platelet activatingfactor

PAI Plasminogen activatorinhibitor

PAR Protease-activatedreceptor

PCI Percutaneous coronaryintervention

PDE PhosphodiesterasePDGF Platelet-derived growth

factorPCI Percutaneous coronary

interventionPGF2a Prostaglandin 2aPGG2 Prostaglandin G2

PGH2 Prostaglandin H2

PGD2 Prostglandin D2

PIP2 Phosphatidylinositol-4,5-biphosphate

(PI3-K) Phosphoinositide-3kinase

PKC Protein kinase CPLC Phospholipase CPLA2 Phospholipase A2PLATO Platelet inhibition and

patient outcomesPRISM-PLUS Platelet receptor inhibi-

tion for ischemic syn-drome management inpatients limited to veryunstable signs andsymptoms

PRP Platelet-rich plasmaPRoFESS Prevention regimen for

effectively avoiding sec-ond strokes

PURSUIT Platelet glycoproteinIIb/IIIa in unstable an-gina: receptor suppres-sionusing Integrilintherapy

REACT Rapid early action forcoronary treatment

RESTORE Randomized efficacystudy of tirofibanfor outcomes andrestenosis

ABBREVIATIONS 455

SPS3 Secondary prevention ofsmall subcorticalstrokes trial

STARS Stent anticoagulationrestenosis study

STEMI ST-segment elevationmyocardial infarction

STIMS Swedish ticlopidinemul-ticenter study

SYMPHONY Sibrafiban Versus aspir-in to yield maximumprotection from is-chemic heart eventspostacute coronarysyndromes

TARGET Do tirofiban and reoprogive similar efficacyoutcome trial

TIA Transient ischemicattack

TIMI Thrombolysis inmyocar-dial infarction

TASS Ticlopidine aspirinstroke study

TNF-a Tumor necrosis factor-aTTP thrombotic thrombocy-

topic purpuraTPa Thromboxane-prosta-

noid receptora-subtype(usually known asthromboxane receptor-a)

TS Thromboxanesynthetase

TxA2 Thromboxane A2

tPA tissue plasminogenactivator

TRAP Thrombin receptor acti-vating peptide

VLA-6 Very late antigen-6TRA 2P-TIMI 50 Trial to assess the effects

of SCH 530348 in pre-venting heart attackand stroke in patientswith atherosclerosis

TRA-CER Trial to assess the effectsof Sch 530348 in pre-venting heart attack

and stroke in patientswith acute coronarysyndrome

TRA-PCI Thrombin receptor an-tagonist for cardiovas-cular event reduction inpercutaneous coronaryinterventions

TRITON-TIMI 38 Trial to assess improve-ment in therapeuticoutcomes by optimizingplatelet inhibition withprasugrel-thrombolysisin myocardial infarc-tion 38

UA Unstable anginauPA Urokinase-type plasmi-

nogen activator (alsoknown as urokinase)

VSP Vasodilator-stimulatedphosphoprotein

vWF von willebrand factor

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