One of the early events in thrombosis is the adhesionand subsequent aggregation of platelets at sites of vas-cular injury. This platelet aggregate provides a surfacefor the assembly of procoagulant enzymes and theircofactors, thus localizing the generation of thrombin.Consequently, this process has become the target forthe development of novel antiplatelet agents. Plateletadhesion and aggregation is mediated to a great extentby a receptor, the platelet glycoprotein (GP) IIb/IIIa,interacting with its principle ligand fibrinogen, anadhesion protein. Several compounds that act as antag-onists of this receptor and so prevent platelet aggrega-tion and to some extent adhesion have been devel-oped as oral preparations for long-term administration.The large phase III trials assessing their efficacy andsafety are currently underway.

The platelet GPIIb/IIIa The platelet GPIIb/IIIa is one of a family of adhesion

receptors called integrins that are heterodimers of anα and a β subunit.1 There are at least eight α and 16 βsubunits, the pairing of which results in a wide range ofreceptors with different tissue and ligand specificities.2

In the case of GPIIb/IIIa, the ligand is primarily fibrino-gen and the receptor is confined almost entirely to theplatelet, where there are 50,000 to 80,000 copies mediat-ing platelet aggregation and adhesion.3 The receptoralso recognizes von Willebrand factor and fibronectinand is expressed in megakaryocytes and melanomacells.4,5 In resting platelets, the receptor is present bothon the surface and in α granules,6 where it is thoughtto be complexed with fibrinogen. Under these restingconditions, the receptor has a low affinity for solublefibrinogen, although it can recognize fibrinogen boundto a surface.7 Upon stimulation of the platelet by agonistsacting through G-protein linked receptors, such asthrombin, adenosine diphosphate, and thromboxane A2,

the platelet GPIIb/IIIa assumes a high affinity for fibrino-gen.8 The mechanism for the change in ligand affinity isunknown but appears to involve intracellular signalsinteracting with the relatively short cytoplasmic tails ofthe receptor’s subunits.9 Elevations in cytoplasmic Ca++

and activation of protein kinase C have both been impli-cated with procedures that directly induce these sig-nals.10 In addition, it is possible to generate a constitu-tively active or inactive receptor by deleting portions ofthe cytoplasmic tails of the α and β subunits, respec-tively.11 The link between signaling and the cytoplasmiccomponents of the receptor is unknown, but there areclues. An area in the αIIb, corresponding to thesequence KVGFFKR, is conserved among the α subunitsand binds the Ca++ regulating protein, calreticulin.12 Thisinteraction stabilizes the receptor in a high affinity state.Curiously, GPIIb/IIIa, when purified with ligands corre-sponding to the binding regions of fibrinogen, stablyexpresses a high affinity for fibrinogen. This suggeststhat within platelets resides a mechanism for maintain-ing the receptor in a low affinity state.

The ultimate effect of receptor activation appears tobe a conformational change in GPIIb/IIIa that can bedetected as the expression of a neoepitope recognizedby monoclonal antibodies.13 One of these, PAC-1, hasan antigen recognition sequence within the hypervari-able region of the antibody that resembles the bindingsequence of the ligand and is critical for its interactionwith the receptor.14 Therefore it appears that the ligandrecognition site is cryptic and becomes exposed on cellactivation. This exposure can be induced also by somemonoclonal antibodies (D3, AP-5, LIBS-6)15,16 by nat-ural and synthetic ligands17 and by the reducing agent,dithiothreitol (D77)18 with either cellular or purifiedreceptor, maneuvers that may directly induce a confor-mational change in the receptor. However, it is unclearhow platelet activation induces this conformation.

Fibrinogen as a ligand for GPIIb/IIIaFibrinogen is a hexamer of three identical pairs of

chains (α, β, and γ). The three chains are separatelybundled and the two bundles are linked at one end bydisulfide bonds so that on electron microscopy

Long-term administration of glycoprotein IIb/IIIaantagonistsMartin Quinn, MB, and Desmond J. Fitzgerald, MD Dublin, Ireland

From the Centre for Cardiovascular Science, Royal College of Surgeons in Ireland.Reprint requests: Desmond Fitzgerald, MD, Centre for Cardiovascular Science, RoyalCollege of Surgeons in Ireland, St. Stephens Green, Dublin 2, Ireland.E-mail: dfitzgerald£rcsi.ieAm Heart J 1998;135:S113-S118.Copyright © 1998 by Mosby, Inc.0002-8703/98/$5.00 + 0 4/0/87660

fibrinogen appears to have a central head with twotails.19 Fibrinogen contains several sites that mayinteract with the receptor, including two RGD (Arg-Gly-Asp) sequences in each of its α chains and adodecapeptide in each γ chain at the distal end of thetails.20 RGD is commonly found in adhesion mole-cules, including collagen and fibronectin, where itmediates interactions with adhesion receptors; forthis reason many of the antagonists developedagainst GPIIb/IIIa are based on this motif. However,it is the dodecapeptide that appears to be the mainsite of interaction because fibrinogen mutants notexpressing this sequence are unable to supportplatelet aggregation.21 Moreover, peptides containingthis sequence or the RGD sequence can preventplatelet aggregation and adhesion. In fact, both acton the same binding pocket because they competefor binding, so that it is possible that the dodecapep-tide and the RGD peptides are assuming a similarconformation.22

Ligand receptor interactionA series of events occur after the binding of fibrino-

gen to the platelet GPIIb/IIIa. The receptor undergoesfurther conformational changes detected as theappearance of several epitopes that can be detectedby monoclonal antibodies.16 These ligand-inducedbinding sites (LIBS) may be functionally relevantbecause antibodies to these sites can provoke plateletaggregation or interfere with subsequent events that areGPIIb/IIIa dependent, such as clot retraction15,16 Alterna-tively, the LIBS antibodies may provoke or interfere withadditional postoccupancy conformations of the receptorrequired for subsequent events. The GPIIb/IIIa is not apassive receptor but transduces signals upon ligandbinding, including the assembly of focal adhesion points(where protein kinases localize and become activated),rearrangement of the cytoskeleton, and late activation ofphospholipases and phosphoinositol phosphatekinase.23,24 There is no evidence that these signalingevents are mediated by antagonists of the receptor andin common with other integrins; signaling probablyrequires clustering of GPIIb/IIIa receptors mediated by apolyvalent ligand such as fibrinogen.25 Interfering withpostoccupancy signaling events prevents platelet aggre-gation, suggesting that this is required for the recruit-ment of additional activated receptors.

As with the receptor, the ligand also undergoes con-formational changes detected as the expression ofneoepitopes, referred to as receptor-induced binding

sites.26 The change in conformation may allow fibrino-gen to recognize unactivated receptor (in much thesame way as unactivated platelets adhere to solid-phase fibrinogen) and so recruit other platelets into thedeveloping aggregate. Binding of fibrinogen, initiallyreversible, becomes irreversible over a few minutesand cannot be displaced by addition of an antagonist.

Antagonists of the platelet GPIIb/IIIaSeveral classes of compounds have been developed

(Table I). The first clinically available compound wasthe monoclonal antibody 7E3.27 Initially a murine anti-body, antigenicity of this compound was attenuated bygenerating a chimeric molecule of human and murineprotein.28 7E3 interacts with or close to the ligandbinding site of the receptor. 7E3 is not specific forGPIIb/IIIa because it also interacts with a structurallysimilar receptor, the GPαvβ3, the receptor for vit-ronectin.29 There are only 300 to 500 copies of thisreceptor on the platelet surface, although recent datasuggest that they may be functionally importantbecause compounds directed at this receptor inhibitthrombosis.30 Vitronectin receptors are also found onendothelial cells, but the outcome of interfering withthese receptors is unclear. Several peptide antagonistsof the platelet GPIIb/IIIa have also been generated,including the compound eptifibatide. In place of anRGD sequence, this cyclic heptapeptide contains aLys-Gly-Asp (KGD) that imparts a higher selectivity forthe GPIIb/IIIa.31 Interaction of this compound withGPIIb/IIIa is modulated by Ca++, so that in citratedplasma inhibition of platelet aggregation is exagger-ated.32 The development of synthetic, nonpeptideGPIIb/IIIa antagonists (Table I) has resulted in severalcompounds with oral bioavailability raising the poten-tial for long-term administration, whereas eptifibatideand 7E3 are administered intravenously.

Pharmacology of GPIIb/IIIaantagonists

In vitro, GPIIb/IIIa antagonists bind to the receptorwith high (low nmol/L) affinity, whether the receptoris in an active or inactive state. In some cases bindingis modulated by divalent ions, possibly as the com-pounds interact close to or at a Ca++ binding site.33

Some antagonists exhibit a higher affinity for the acti-vated receptor,34 which is not surprising because thisinvolves a major change in receptor conformation.GPIIb/IIIa antagonists prevent fibrinogen binding andplatelet aggregation induced by all known platelet

American Heart JournalMay 1998Quinn and FitzgeraldS114

activators, including thrombin and collagen. They alsoprevent platelet aggregation induced by maneuversacting directly at the receptor level, such as thatinduced by DTT and the LIBS monoclonal antibodyD3.35 Thus they act solely at the receptor level and donot prevent other outcomes of platelet activation, suchas shape change, that are independent of the plateletGPIIb/IIIa. Antagonists of the platelet GPIIb/IIIa alsoinhibit platelet adhesion to fibrinogen and extracellularmatrixes.36 To date, there is no evidence that postoc-cupancy signaling can be induced by these small mol-ecules, although several, and perhaps all to someextent, induce LIBS expression, indicating that theyinduce conformational changes in the receptor.

In vivo, GPIIb/IIIa antagonists inhibit plateletaggregation and prolong the bleeding time, althoughthe dose response for both is quite different.37 Theyhave been found effective in models of thrombosisin a variety of animals, including baboons, guineapigs, and dogs, whose GPIIb/IIIa receptors appear tobe homologous with the human receptor.38,39 ThusGPIIb/IIIa antagonists have been shown to preventcyclical coronary flow at a site of critical stenosis, aplatelet-dependent model. They have also been shownto prevent electrically induced coronary thrombosisand to accelerate reperfusion and prevent reocclusionin this model.40 In the baboon model of arteriovenousshunt thrombosis, GPIIb/IIIa antagonists are highlyeffective in preventing platelet deposition and subse-quent thrombus formation.41

Several studies have examined the interactions ofGPIIb/IIIa antagonists with other antithrombotics.These studies show that the response to a GPIIb/IIIaantagonist is limited by ongoing thrombin generationand that a more powerful effect is seen when aGPIIb/IIIa antagonist is combined with a thrombininhibitor.42 One possible explanation is that thrombininduces the secretion of intracellular receptors, whichaccount for 30% to 50% of the platelet GPIIb/IIIa, andthat these receptors are less susceptible to the antago-nist.43 The response to GPIIb/IIIa antagonists is alsoenhanced by coadministration of a thromboxane A2

inhibitor such as aspirin.44 Continued thromboxaneformation might be expected as platelet activation perse is unaffected by a GPIIb/IIIa antagonist.45 However,there is increasing evidence that to some extent throm-boxane generation occurs subsequent to GPIIb/IIIaengagement by fibrinogen. Moreover, one compound,Ro 44-9883, suppressed thromboxane formation invivo in a canine model of coronary thrombolysis.46

Consequently, it remains to be seen if aspirin enhancesthe response to all GPIIb/IIIa antagonists.

Human pharmacologyAs in animal models, GPIIb/IIIa antagonists suppress

platelet aggregation ex vivo and prolong the bleedingtime.47,48 The dose response for platelet aggregation islinearly related to the number of receptors occupied,although more than 20% to 30% of receptors must beoccupied before an effect is seen.45 For bleeding time,up to 80% of receptors must be occupied before anyprolongation occurs.47,49 The relation between recep-tor occupancy or plasma drug concentration and inhi-bition of platelet aggregation or prolongation of bleed-ing time is very steep and is linear rather than loglinear.45 Small increases in the plasma drug level trans-late into a marked change in platelet aggregation sothat titrating the dose to this end point is difficult. Insome cases, the bleeding time may become markedlyprolonged to >30 minutes. Whether such prolongedbleeding times predict the risk of serious bleedingindependently of suppression of platelet aggregationor plasma drug level is uncertain.

The steep dose response relation is particularly rele-vant for the oral preparations. Bioavailability of thesepreparations is low, around 5% to 20%.48 Moreover,bioavailability is variable and may be modified byfood. Consequently, it may be difficult to predict the

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Table I. GPIIb/IIIa antagonists currently available or beingdeveloped

Classification Example

Monoclonal antibodies Reopro(7E3 or abciximab)Cyclic peptides DMP-728

TP-9201SC-54701YM 337

Modified peptides EptifibatidePeptides and nonpeptide Lamifiban (Ro-44-9883)derivatives Aggrestat (tirofiban)

Fradifiban (BIBU-52)Orally active agents Xemilofiban (SC-54684A)

OrbofibanLedrafiban (BIBU-104)Rpr-109891Ro-48-3657GR144053SKB-21-4857SR-121787DMP-754EMD-122 347L-738 167

response to an individual patient because wide varia-tions in bioavailability could result in a twofold tothreefold change in plasma drug level.

The response to GPIIb/IIIa antagonists may also bemodified by coadministration of other antiplateletagents, such as aspirin. Catella-Lawson et al.50 showedthat suppression of platelet aggregation and bleedingtime prolongation at intermediate doses of a GPIIb/IIIaantagonist were enhanced by aspirin. It is an openquestion as to whether aspirin is necessary at allbecause it is likely to increase the risk of gastrointesti-nal bleeding. In a study of GPIIb/IIIa antagonism dur-ing coronary angioplasty, patients receiving fradifibanalone showed a marked increase in thromboxane for-mation, demonstrating continued activation ofplatelets.45 Because thromboxane has additionaleffects, including platelet secretion, vascular smoothmuscle contraction, and mitogenesis, it may be reason-able to combine these treatments. Moreover, thetroughs of antiplatelet activity seen with intermittentadministration of a GPIIb/IIIa antagonist may be atten-uated by coadministration of aspirin.

Clinical studiesA number of phase III clinical trials have demon-

strated the efficacy of GPIIb/IIIa antagonists in thesetting of acute intervention.51,52 However, therisk/benefit ratio during long-term administration maybe quite different. First, there is no easily performedor well-characterized surrogate marker of effect. Inhi-bition of platelet aggregation and prolongation of thebleeding time has been used to assess the adequacyof dosing.53 Marked inhibition of platelet aggregationis required before bleeding time is prolonged, so thatthis is of little benefit in assessing dose. On the otherhand, inhibition of platelet aggregation may be help-ful in dose monitoring. A reasonable target duringacute administration is >80% inhibition of plateletaggregation based on animal models and studiesseen in patients undergoing coronary angioplasty.51

However, in the setting of long-term therapy for sta-ble thromboembolic disease, a lower level of inhibi-tion may provide adequate protection while reducingthe risk of bleeding. Moreover, currently availabledrugs are administered once or twice a day, resultingin peaks and troughs,53 so that a stable effect is notpossible.

A major issue in the long-term use of GPIIb/IIIaantagonists is the interindividual variability in pharma-codynamics that may result in a marked variation in

efficacy and safety profile. The importance of thisissue was illustrated in the study by Simpendorfer etal.53 of the long-term administration of xemilofiban.Thirty patients were enrolled into the study; 20 ofthem received one of two doses of xemilofiban for30 days. Initially the majority of patients respondedwell, with adequate inhibition of platelet aggrega-tion. However, at 30 days only 36% of patients had>80% inhibition of platelet aggregation before theirmorning dose. In part this may reflect interindividualvariation in oral bioavailability, which on averagewas low at 13%. In addition, it is possible that theresponse may vary over time, either as a conse-quence of a change in the target receptor or inplatelet activity.

The coadministration of other agents such asaspirin50 or ticlopidine54 appears to potentiate theeffect of GPIIb/IIIa antagonists on the inhibition ofplatelet aggregation and may allow reduced dosing.Similarly, prior administration of the monoclonalantibody 7E3 may enhance the response to subse-quent administration of an oral GPIIb/IIIa antagonistfor several days.55 Administration of GPIIb/IIIa mayalso reduce the requirement for heparin.56,57

In addition to the risk of bleeding, there have beenseveral reports of severe thrombocytopenia. Thiscomplication has been reported with both intra-venous and oral preparations in <1% of patients andresolves once the drug is discontinued.50,58 Thrombo-cytopenia may occur after a single dose or may bedelayed. In the study by Catella-Lawson et al.,50 inwhich patients received RPR 10981 for up to 4weeks, marked thrombocytopenia developed in twopatients, one acutely after a single dose and one after7 days of treatment. Whether thrombocytopenia mayoccur later in long-term drug administration isunknown. One patient described above developedan antiplatelet antibody. Antibodies directed againstthe platelet GPIIb/IIIa complexed to an antagonisthave been reported in a primate. Curiously, this anti-body was present before the animal was ever exposedto the drug.59 Although these studies implicate autoan-tibodies, it is still not clear if this is the mechanism forthe thrombocytopenia associated with GPIIb/IIIaantagonists. In any event, it is important to monitorplatelet counts in the initial stages of therapy.

In conclusion, oral GPIIb/IIIa antagonists provide anovel approach to long-term antiplatelet therapy.However, there are several unique problems with thepharmacologic action of these drugs. In particular,

American Heart JournalMay 1998Quinn and FitzgeraldS116

accurate dosing will prove difficult and it remains tobe seen what effect these drugs will have on plateletand receptor kinetics when administered long term.

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6. Humbert M, Kunicki TJ, Bihour C, Winckler J, Nurden AT, Nurden P.Visualization of activation-dependent epitopes on glycoprotein IIb-IIIacomplexes of platelets stimulated by thrombin: immunogold staining ofultrathin sections. Sem Thromb Hemost 1996;22:279-88.

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