[Handbook of Experimental Pharmacology] Atherosclerosis: Diet and Drugs Volume 170 || Inhibition of Platelet Activation and Aggregation

HEP (2005) 170:443–462c© Springer-Verlag Berlin Heidelberg 2005

Inhibition of Platelet Activation and Aggregation

I. Ahrens (�) · C. Bode · K. Peter

Abteilung für Innere Medizin III (Kardiologie u. Angiologie), UniversitätsklinikumFreiburg, Medizinische Universitätsklinik und Poliklinik, Hugstetter Strasse 55,79106 Freiburg, [email protected]

1 The Role of Platelet Adhesion and Aggregation in Atherosclerosis . . . . . . . 4441.1 Receptors That Mediate Platelet Adhesion, Stimulation and Aggregation . . . 444

2 Clinically Approved Antiplatelet Drugs . . . . . . . . . . . . . . . . . . . . . 4462.1 Inhibition of Cyclooxygenase with Acetylicsalicylic Acid (Aspirin) . . . . . . . 4462.1.1 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4462.1.2 Clinical Use of Acetylsalicylic Acid as an Antiplatelet Drug . . . . . . . . . . . 4472.2 Inhibition of the Phosphodiesterase . . . . . . . . . . . . . . . . . . . . . . . 4482.2.1 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4482.2.2 Dipyridamole (Persantin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4482.2.3 Cilostazol (Pletal) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4492.3 P2Y12 ADP Receptor Antagonists Ticlopidin and Clopidogrel . . . . . . . . . 4502.3.1 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4502.3.2 Ticlopidin (Ticlid) and Clopidogrel (Plavix/Iscover) . . . . . . . . . . . . . . 4502.4 GP IIb/IIIa Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4512.4.1 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4512.4.2 Abciximab (ReoPro) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4522.4.3 Eptifibatide (Integrilin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4542.4.4 Tirofiban (Aggrastat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4552.4.5 Oral GPIIb/IIIa Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

Abstract It has recently been established that platelets are involved at all stages of atheroscle-rotic disease. A major platelet mediated process is the acute vessel closure at the site ofatherosclerotic plaque rupture and there is emerging evidence for platelet adhesion to en-dothelial cells in the early stage of atherosclerotic disease. This, through engagement of othercells, leads to the development of the atherosclerotic plaque. Beside dietary, cholesterol- andlipid-lowering, and other pharmaceutical approaches antiplatelet therapy plays an impor-tant part in the treatment of atherosclerosis and its multifarious clinical manifestations.Antiplatelet therapy and the currently approved substances for oral (acetylsalicylic acid,dipyridamole, cilostazol, ticlopidin and clopidogrel) and parenteral (acetylsalicylic acid,abciximab, eptifibatide and tirofiban) administration are discussed in the following sec-tion. Attention is given to each single agent and its mechanism of action. Differences inpharmacodynamic and pharmacokinetic properties are elucidated and outlook on futureantiplatelet strategies is discussed.

Keywords Atherosclerosis · Antiplatelet drugs · Acetylsalicylic acid · Clopidogrel ·Dipyridamole · GPIIb/IIIa inhibitors · Platelet activation

444 I. Ahrens et al.

1The Role of Platelet Adhesion and Aggregation in Atherosclerosis

The intact endothelium helps to prevent platelets from adhering to vesselwalls. Both the secretion of inhibitory substances, such as nitric oxide andprostacycline, and the intact mechanical barrier to adhesive substrates of thesubendothelial matrix are crucial to keep circulating platelets in their nonre-active state. It is well known that in the late stage of atherogenesis plateletsadhere to denuded vessel wall areas via exposed matrix proteins. Less wellknown is the fact that already very early in atherogenesis platelets stick toendothelial cells that express an ‘atherogenic’ profile of cell membrane-boundadhesion molecules. The adhering platelets recruit other cells (platelets andwhite blood cells) and deliver growth signals to the neighboring vessel wallcells, such as smooth muscle cells and fibroblasts. Aggregating platelets andother blood cells are incorporated into the growing atherosclerotic plaque.Finally, after acute plaque rupture, the formation of platelet aggregates leadsto acute vessel closure and severe clinical sequelae. Coronary artery diseaseand acute coronary syndromes (ACS) are some of the manifold manifestationsof atherosclerosis. Considering the central role of platelets in the pathogenesisdescribed above, it is clearly justified to administer a therapy aiming at theinhibition of platelet function to treat acute and chronic atherosclerosis andtheir clinical sequelae.

1.1Receptors That Mediate Platelet Adhesion, Stimulation and Aggregation

Disruption of the endothelium in atherosclerosis allows platelets to interactwith the subendothelial matrix. The release of subendothelial von Willebrandfactor and the exposure of collagen lead, at conditions of high shear rates, to theadhesion of unactivated platelets (Turrito et al. 1985, Kehrel et al. 1998). GPIb-IX-V is the platelet receptor binding von Willebrand factor, and glycoprotein VI(GPVI) is the major collagen receptor on the platelet surface. Besides bindingto platelet GPVI, collagen also serves as a binding site for von Willebrandfactor in the subendothelial matrix and therefore contributes to the adhesionof unactivated platelets via GPIb-IX-V (Savage et al 1998). The more or lesspassiveprocessofadhesion is followedbyanactivemetabolicprocessofplateletactivation leading to platelet aggregation by binding the activated integrinαIIbβ3 (the platelet receptor for fibrinogen, also termed GPIIb/IIIa) to solublefibrinogen or von Willebrand factor. Both fibrinogen and von Willebrandfactor, bound to activated GPIIb/IIIa, crosslink platelets, which contributes tothe formation of a thrombus.

Collagen and thrombin are the most potent physiological activators ofplatelets. Thrombin is an agonist for two platelet receptors the protease ac-tivated receptors 1 (PAR1) and 4 (PAR4) (Kahn et al. 1998). These receptors

Inhibition of Platelet Activation and Aggregation 445

belong to the family of the G-protein coupled receptors. PAR1 mediates plateletactivation at low concentrations of thrombin while PAR4 is activated at higherconcentrations. A blockade of PAR4 has little effect on platelet aggregation,whereas a blockade of both PAR1 and PAR4 virtually ablates platelet aggrega-tion even at high concentrations of thrombin (Kahn et al. 1999).

Exposed to ADP, platelets undergo shape changes, activate the fibrinogenreceptor GPIIb/IIIa, aggregate, release the contents of their granules and pro-duce thromboxane (TX) A2. The receptors binding extracellular nucleotidessuch as ADP have been classified as P2 receptors. P2 receptors are subdividedinto P2X intrinsic ion channels and P2Y G-protein-coupled receptors. Threedifferent types of P2 receptors have been identified onplatelets: P2X1, P2Y1 andPTY12. P2X1 has been identified as an ADP-stimulated calcium channel thatenables fast calcium entry into the platelet upon ADP binding (MacKenzie etal. 1996). P2Y1 is a G-protein-coupled seven-transmembrane domain receptorthat changes the platelet shape and mobilizes calcium from intracellular storesby activating phospholipase C (Jin et al. 1998). The P2Y12 receptor inhibitsthe platelet adenylate cyclase and seems to be responsible for a positive feed-back mechanism that amplifies platelet stimulation especially by weak agonists(Hollopeter et al. 2001). Therefore, this receptor plays a central part in the finalstep of platelet aggregation and stabilization of aggregates (Gachet 2001).

Epinephrin and other catecholamines stimulate the platelet α2A-adrenergicreceptor coupled to a G-protein and inhibit adenylate cyclase activity (Keulartset al. 2000), thereby antagonizing the cAMP-elevating effect of agents likeprostacyclin and prostaglandin E1 (Paul et al. 1998). Other G-protein coupledreceptors expressed on the platelet surface are the serotonin receptor 5-HT2a(Hourani and Cusack 1991), the vasopressin receptor V1 (Siess et al. 1986), andthe receptor for platelet activating factor (Chao and Olson 1993).

Active phospholipase A2 mediates the release of arachidonic acid frommembrane phospholipids of activated platelets. The arachidonic acid is thenconverted to the prostaglandin endoperoxide intermediates PGG2 and PGH2by action of cyclooxygenase-1 (COX-1). The prostaglandin endoperoxide in-termediates are subsequently converted to TXA2 by the TX synthase (Smithet al. 1996). TXA2, when released from the platelets, binds to the G-protein-coupled TX receptor TP and functions as an agonist for platelet stimulation(Hirata et al. 1991).

After stimulation, the platelet releases the contents of granules in the plateletcytoplasm. This process has been termed platelet secretion. Platelets containseveral dense granules (δ-granules) and around 50 α-granules. δ-granulesmainly contain ADP, ATP, calcium, pyrophosphate and serotonin. α-granulescontain a variety of plasma proteins including fibrinogen, von Willebrandfactor, Factor V and albumin. Other proteins are synthesized by the megakary-ocyte itself, for example platelet-derived growth factor, thrombospondin, β-thromboglobulin, and platelet factor 4. The α-granule membrane also containsthe platelet integrin αIIbβ3, the von Willebrand receptor GPIb-IX-V, P-selectin,


and osteonectin. These receptors reflect the platelet pool of receptors that, onceactivated, can be translocated to and incorporated into the platelet membraneduring the process of secretion. The newly incorporated receptors may con-tribute to some of the pharmacological effects of antiplatelet drugs such asinconsistence in platelet aggregation inhibition.

2Clinically Approved Antiplatelet Drugs

To date, various orally and intraveneously administered agents have been madeavailable for antiplatelet therapy. Some of them do act synergistically by in-hibiting different steps of platelet adhesion and aggregation. In the followingchapter, the four now clinically approved strategies for platelet function inhi-bition are discussed: (1) inhibition of cyclooxygenase; (2) phosphodiesteraseinhibition; (3) blockade of the P2Y12 ADP receptor; and (4) blockade of the GPIIb/IIIa receptor.

2.1Inhibition of Cyclooxygenase with Acetylicsalicylic Acid (Aspirin)

2.1.1Mechanism of Action

Acetylsalicylic acid inhibits the enzymes COX-1[prostaglandin (PG)H-synth-ase-1] and cyclooxygenase-2 (COX-2, PGH-synthase-2) in their conversion ofarachidonic acid to PGG2 and PGH2 (Patrono 1994). COX-1 is constitutivelyexpressed in most cell types, whereas COX-2 is detectable after induction ininflammatory, endothelial and other cells. It is generally believed that plateletsexpress only COX-1, although in one study COX-2 was found in platelets too(Weber et al. 1999). Aspirin selectively acetylates the serine residue at position529 of COX-1, which results in the steric hindrance of arachidonic acid to accessthe catalytic center and thereby in a permanent loss of cyclooxygenase activity.COX-2 is inhibited by the same mechanism; however, higher doses of aspirinare needed for inhibition. In human platelets PGH2, the product of COX 1, ispredominantly metabolized to TXA2. Through its release and binding to theTXA2-receptor, TXA2 represents an amplification system of the platelets thatis active after stimulation with divergent primary platelet agonists (ADP, col-lagen, thrombin, epinephrin, etc.). Since platelets cannot re-synthesize COX-1,the irreversible blockade of COX-1 results in irreversible platelet inhibition.Thus, although aspirin is detectable in plasma only for a limited time (plasmahalf-life, 15 min), platelet inhibition can be demonstrated for about 7–10 days(Patrono et al. 2001).


2.1.2Clinical Use of Acetylsalicylic Acid as an Antiplatelet Drug

The pivotal role of platelets in arterial thrombosis has been established essen-tially by the beneficial effects of aspirin in patients with myocardial infarction(MI). In the ISIS-2 trial, the administration of aspirin reduced the 5-weekmortality of patients with MI by 23%. The effect of platelet inhibition equaledand added to the effect of thrombolysis by streptokinase (ISIS-2 CollaborativeGroup 1988). In the 10-year-follow-up, the original benefit of aspirin was stillpresent (Baigent et al. 1998). Unstable angina is also associated with plateletactivation (Fitzgerald et al. 1986) and, in fact, aspirin does reduce the rate ofdeath and MI in patients with unstable angina (Lewis et al. 1983; Cairns et al.1985; Theroux et al. 1988). Coronary angioplasty is linked to platelet activation,and aspirin significantly reduces the rate of acute vessel closure (Barnathan etal. 1987; Gawaz et al. 1996). In all these acute clinical situations, the adminis-tration of aspirin is a therapeutic necessity. Nevertheless, aspirin saved mostlives in the secondary prevention of cardiovascular events—and as a singledrug it probably saved the most lives in all. In the Antiplatelet Trialists’ meta-analysis of approximately 70,000 patients with coronary artery disease, TIA,stroke or peripheral arterial vascular disease, aspirin showed a risk reductionof 25% for MI, stroke or vascular death (Antiplatelet Trialists’ Collaboration1994). Just recently, a meta-analysis of 200,000 patients treated with antiplateletdrugs confirmed the benefits and even showed additional advantages of an-tiplatelet therapy for the secondary prevention of cardiovascular complicationsin patients with stable angina pectoris, intermittent claudication, and (if oralanticoagulants are unsuitable) atrial fibrillation (Antiplatelet Trialists’ Collab-oration 2002). In the primary prevention of cardiovascular events, the increaseof hemorrhagic strokes—a severe side effect of aspirin—seems to counteractthe beneficial effects of aspirin on the rate of MI (Peto et al. 1988; SteeringCommittee of the Physicians’ Health Study Research Group 1989; ETDRS In-vestigators 1992). In the Physicians’ Health Study, the rate of stroke increasedby 21% and the rate of MI decreased by 44% (Peto et al. 1988). Nevertheless,a reduction of mortality from all cardiovascular causes was not associated withaspirin (Peto et al. 1988). When extrapolated from the data of aspirin treat-ment in primary and secondary prevention, the cut point at which the benefitin the prevention of ischemic cardiovascular events outweighs the increase ofthe risk of stroke has to be determined with caution. A higher cardiovascularrisk seems to correlate with a higher benefit conferred by aspirin. However,patients with clear cardiovascular risk factors, especially with diabetes mel-litus, may indeed profit from aspirin treatment in the primary prevention ofcardiovascular events (Hansson et al. 1998; The Medical Research Council’sGeneral Practice Research Framework 1998; Avanzini et al. 2000).

For a long time, the dosing of aspirin has been debated controversially.There is no doubt that aspirin-induced gastrointestinal toxicity is dose de-


pendent (Roderick et al. 1993). On the other hand, an increase in dose doesnot correlate with an increase in the antiplatelet effects of aspirin. The resultsof the recent Antithrombotic Trialists’ Collaborations’ meta-analysis convinc-ingly demonstrate that high daily doses of aspirin (500–1500 mg) are not moreeffective than medium doses (160–325 mg) or low doses (75–150 mg). (An-tithrombotic Trialists’ Collaboration 2002). Thus, low-dose aspirin seems toexert the full antiplatelet efficiency with the lowest risk of side effects.

2.2Inhibition of the Phosphodiesterase


The phosphodiesterases (PDE) are a family of enzymes catalyzing the hydroly-sis of cyclic nucleoside monophosphates, namely cyclic adenosine monophos-phate (cAMP) and cyclic guanosine monophosphate (cGMP) (Beavo 1995). Atleast nine different isoforms termed PDE1 through PDE9 have been identi-fied in mammalian tissues (Soderling et al. 1998). Platelets have been foundto express PDE2, PDE3 and PDE5 isoenzymes. Two substances acting by in-hibition of PDE—dipyridamole and cilostazol—are now clinically approved.Dipyridamole primarily inhibits the degradation of cGMP (Ziegler et al. 1995)by PDE5 inhibition, but it also elevates platelet cAMP levels. The increasedplatelet cAMP and cGMP concentrations lead to a reduced platelet reactivityby decreasing the cytoplasmic calcium and inhibiting platelet prostaglandinsynthesis. Another nonplatelet-specific effect of dipyridamole is the elevationof extracellular adenosine levels by reducing their uptake and metabolism(Newsholme 1978). Dipyridamole also enhances the release and prevents themetabolic degradation of endothelial prostaglandin PGI2 (Moncada and Kor-but 1978; Neri et al. 1981), a potent inhibitor of platelet aggregation.

Cilostazolpotently inhibits thePDE3andadenosineuptake, thereby increas-ing platelet and vascular smooth muscle cell cAMP levels, which ultimatelyhelps to inhibit platelet aggregation (Schror 2002). Additional beneficial ef-fects of cilostazol have been observed and cannot be explained solely by theelevation of cAMP-levels. Lipid metabolism seems to be influenced in a pos-itive way, as cilostazol facilitates the removal of triglycerides and increaseshigh-density lipoprotein (HDL) cholesterol levels (Elam et al. 1998; Thompsonet al. 2002).

2.2.2Dipyridamole (Persantin)

Since the first introduction of dipyridamole as an antianginal medicationin 1959, there has been a lot of controversy about the clinical relevance ofdipyridamole as an antithrombotic agent. Two major studies were performed


to elucidate the role of dipyridamole in the secondary prevention of MI: first,the Persantine-Aspirin Reinfarction Study (PARIS I) (The Persantine-AspirinReinfarction Study research group 1980) and second, the PARIS II study (Klimtet al. 1986). PARIS I directly compared aspirin 324 mg three times daily to thesame dose of aspirin plus dipyridamole 75 mg three times daily in a total of2,026 patients. Primary endpoints were total mortality, coronary mortality,and fatal plus nonfatal MI. The average follow-up was 41 months. There was nostatistical significant difference between the two treatment groups. In PARISII, patients were randomized to aspirin plus dipyridamole or placebo. A 24%reduction of coronary adverse events was found in the group treated withaspirin plus dipyridamole. However, a group receiving aspirin only was notincluded in this trial, so that a direct beneficial effect of dipyridamole addedto aspirin could not be proven.

Furthermore, other clinical trials failed to show consistent benefit of dipyri-damole added to aspirin in patients having undergone coronary artery bypasssurgery (Sanz et al. 1990), in patients with peripheral vascular disease (Kohleret al. 1984), or in patients with prosthetic heart valves (Stein et al. 1986).However there is evidence that dipyridamole plays an important part in theprevention of stroke. The European Stroke Prevention Study 2 (ESPS-2) wasa randomized placebo-controlled double-blind trial examining aspirin 50 mgdaily, dipyridamole 400 mg daily, a regimen of both drugs together or placebo.The combined treatment with aspirin 50 mg and dipyridamole 400 mg dailyreduced the relative risk of major vascular events by 22% compared to a treat-ment with aspirin alone (The ESPS-2 Group 1997). The mechanism by whichdipyridamole prevents stroke still needs to be determined. A recent studyfailed to establish a link between dipyridamole and a permanent reduction ofblood pressure. This link would have explained how strokes are prevented (DeSchryver 2003).

2.2.3Cilostazol (Pletal)

The quinolinone derivative cilostazol has been shown to inhibit platelet ac-tivation (Kimura et al. 1985) and increase vasodilation (Tanaka et al. 1988).Cilostazol inhibits the proliferation of vascular smooth muscle cells (Takahashiet al. 1992). After oral administration, cilostazol is extensively metabolizedby hepatic cytochrome P-450 enzymes. Two metabolites of cilostazol are ac-tive and account for the pharmacologic effects observed in patients treatedwith cilostazol 100 mg or 50 mg (when administered together with otherantiplatelet drugs) twice daily. The elimination half-lives of cilostazol and itsactive metabolites amount to 11–13 h. The substance has been extensively stud-ied in patients with peripheral vascular disease and intermittent claudication.A meta-analysis of placebo controlled trials with cilostazol in the treatment ofperipheral artery disease showed that cilostazol therapy (administered for 12–


24 weeks) increased the maximal and pain-free walking distance in patientswith intermittent claudication by 50% and 67%, respectively (Thompson etal. 2002). In addition to the enhancement of quality of life, treatment withcilostazol reduced plasma triglyceride levels by 15.8% and increased HDL by12.8% (Thompson et al. 2002). Cilostazol is primarily used for the treatment ofsymptomatic peripheral artery disease. The role of cilostazol in the treatmentof other diseases secondary to atherosclerosis needs to be carefully investigatedin larger scale placebo-controlled clinical trials.

2.3P2Y12 ADP Receptor Antagonists Ticlopidin and Clopidogrel


The thienonyridines ticlopidin and clopidogrel are prodrugs metabolized inthe liver by cytochrome P450. The short-lived active metabolite of clopidogrelwas recently identified as being a thiol derivate of the parent agent clopidogrel(Savi et al. 2000). The selectivity of thienopyridines for the P2Y12 ADP receptoris thought to be caused by covalent modification of the four cystein residues inthe P2Y12 receptor via the thiol metabolites (Hollopeter et al. 2001; Savi et al.2000).

2.3.2Ticlopidin (Ticlid) and Clopidogrel (Plavix/Iscover)

The inhibition of platelet aggregation by the thienopyridine ticlopidine wasalready reported by Thebault et al. (1975). Rather recently, clopidogrel, alsobelonging to the thienopyridines, replaced ticlopidine in its clinical use, asticlopidine showed significant adverse effects such as skin rash, gastrointesti-nal symptoms and bone marrow toxicity with severe and fatal neutropenia.Treatment with clopidogrel, however, caused none or many fewer of theseproblems (CAPRIE Steering Committee 1996; Bennett et al. 2000; Bertrand etal. 2000; Bhatt et al. 2002). The breakthrough study for the widespread clin-ical use of clopidogrel was the CAPRIE trial (CAPRIE Steering Committee1996). In this study, clopidogrel proved to be slightly more effective than as-pirin for the secondary prevention of thrombembolic complications in patientswith atherosclerotic disease (prior MI, ischemic stroke or peripheral vasculardisease). In addition, safety and tolerability of clopidogrel and aspirin weresimilar. In interventional cardiology the combinational therapy of aspirin andclopidogrel after stent placement has become a widespread standard (Bhattet al. 2002). In particular, the risk of a subacute stent thrombosis after stentplacement could be reduced substantially by the combination of aspirin andthienopyridines (Bhatt et al. 2002; Schomig et al. 1996; Moussa et al. 1999;


Cosmi et al. 2001). Finally, just recently evolving has been the concept of long-term treatment of coronary syndromes with the combination of aspirin andclopidogrel (Yusuf et al. 2001).

The specific abundance of the P2Y12 ADP receptor limited to platelets andpotentially to the brain makes this receptor an interesting pharmacologicaltarget, promising nearly ideal selectivity (Hollopeter et al. 2001). Clopidogrelhas been shown to block all available P2Y12 receptors on the platelet and thusdemonstrates highest efficiency (Gachet 2001). Clopidogrel presents an al-most ideal safety and tolerability profile (CAPRIE Steering Committee 1996;Bertrand et al. 2000; Bhatt et al. 2002). In fact, clopidogrel is a remarkablepharmaceutical agent. Nevertheless, two characteristics of clopidogrel justifythe search for other P2Y12 ADP receptor inhibitors. First, even with a loadingdose of 300 mg at least two, but probably up to 12 h are needed for the fullantiplatelet effect of clopidogrel to develop (Savcic et al. 1999). The data of theISAR REACT study suggest that with a loading dose of 600 mg, a steady statecan be reached in less time than with 300 mg, and that the higher dose maylead to a better outcome in the setting of percutaneous coronary interventions(PCI) (ACC 2003, late breaking trials). However, in acute MI or urgent coronaryinterventions for example, the antiplatelet effect of clopidogrel comes too late.Second, the P2Y12 receptor is irreversibly blocked and thus the effect persistsfor the entire platelet life span (Weber et al. 2001). Since there is no antag-onist available, needed for example when urgent operations are performed,bleeding complications may occur. To overcome these problems, a new classof P2Y12 ADP receptor inhibitors has been developed (Story 2001), based onthe fact that ATP is a competitive antagonist of ADP. Structural homologueswere screened for selectivity against the P2Y12 receptor, and there were indeedseveral agents found, some of them already having been tested in clinical trials.The advantage of these agents consists in their immediate antiplatelet effectsafter intravenous application that, because of their short half-life, are rapidlyreversible (Storey 2001). Some of these agents can be used as intravenous, someas oral drugs (Storey 2001).

2.4GP IIb/IIIa Inhibitors


GPIIb/IIIa, or integrin αIIbβ3, is the platelet receptor for fibrinogen and me-diates the final step in platelet aggregation. As the default state of αIIbβ3 isa nonactivated and resting state, it needs to become activated in order to bindits major ligand, i.e., soluble fibrinogen. This occurs after the activation ofplatelets by physiological agonists (e.g., thrombin, ADP, collagen) stimulateintracellular signal pathways and thereby induce conformational changes ofαIIbβ3. This process is termed ’inside-out’ signaling (Shattli 1999). It switches


the receptor into an activated state with high affinity for fibrinogen and numer-ous other ligands (Phillips et al. 1998). As a result of αIIbβ3-mediated bindingto the bivalent molecule fibrinogen, platelets aggregate and form a throm-bus rich in platelets. To date, two binding sites have been well characterizedin αIIbβ3: an Arg-Gly-Asp (RGD)-binding site and a Lys-Glu-Ala-Gly-Asp-Val (KQAGDV)-binding site (Tcheng 2000). Other binding sites have beendescribed as well, with yet unknown functional properties (Lin et al. 1997;Basani et al 2000). Interestingly, fibrinogen binds via the KQAGDV-bindingsite. Agents that bind within the ligand-binding region of αIIbβ3 and blockthe binding of its natural ligands have been developed and termed GPIIb/IIIainhibitors. Of the three clinically approved parenteral GPIIb/IIIa inhibitors,one (abciximab; ReoPro, Lilly, IN, USA) is based on an antibody structure, andthe two others (eptifibatide; Integrilin, Millennium/Schering-Plough, NJ, USAand tirofiban; Aggrastat, Merck, NJ, USA) are described as small-moleculeGPIIb/IIIa inhibitors. All of them bind to αIIbβ3 either in the resting or acti-vated conformation and inhibit fibrinogen binding and, consequently plateletaggregation. The first substance to be developed was a murine monoclonalantibody (mAb) that blocked αIIbβ3 (Coller et al. 1989). The immunogenicityof this mAb could be reduced by its humanization, i.e., by the exchange of theconstant regions of the mouse antibody with the constant regions of humanimmunoglobulin IgG1 to produce a chimeric Fab fragment called abciximab.The other two GPIIb/IIIa inhibitors belong to the group of small molecules andcan be subdivided into synthetic peptide inhibitors (eptifibatide) and syntheticnonpeptide inhibitors (tirofiban).

2.4.2Abciximab (ReoPro)

The Fab fragment abciximab, with a molecular mass of 48 kDa and a reportedequilibrium dissociation constant (KD) of 5 nM is a high-affinity GPIIb/IIIainhibitor for parenteral use only (Scarborough et al. 1999). The high receptoraffinity and low KD characterize the antagonist–receptor binding as noncom-petitive, rendering a low plasma concentration of unbound abciximab that iseliminated through protein metabolism. Besides binding to αIIbβ3, abciximabhas been reported to bind to with equal affinity to integrin αVβ3 (Tam et al.1998) and with lower affinity (KD, 160 nM) to the activated integrin αMβ2(Mac-1) on monocytes and granulocytes (Coller 1999). The blockade of Mac-1by abciximab inhibits leukocyte adhesion and aggregation and thus directlyattenuates inflammatory reactions (Schwarz et al. 2002). Furthermore, sinceabciximab inhibits the binding of Factor X and its conversion to Factor Xa,it attenuates the cell-based initiation of the coagulation cascade (Schwarz etal. 2002). However, the clinical relevance of the cross-reactivity of abciximabagainst αVβ3 and Mac-1 remains to be determined. Interestingly, abciximabcompetes with heparin for binding to Mac-1 (Peter et al. 1999) and may thereby


prolong the activated clotting time. This has been observed in patients treatedwith unfractionated heparin and abciximab (Moliterno et al. 1995; Ammaret al. 1997). The cross-reactivity of abciximab with the endothelial cell inte-grin αVβ3 and the leukocyte integrin Mac-1 provides a possible explanationfor abciximab-related effects on the coronary microcirculation. Microvascularcoronary resistance decreased after abciximab bolus administration in patientswith unstable refractory angina pectoris and hemodynamic-relevant coronaryartery stenosis (Marzilli et al. 2002). Abciximab is currently approved for theuse in patients undergoing PCI. It has also been studied as first-line medicaltreatment in patients with ACS in the absence of PCI, but for up to 48 h nobenefit of abciximab treatment could be reported in this setting (The GUSTOIV-ACS Investigators 2001). Initial dose-finding trials were performed to iden-tify the dosage leading to over 80% inhibition of platelet aggregation in humansor to less than 20% of baseline ADP-induced platelet aggregation. The findingsled to the current administration as a bolus of 0.25 mg/kg followed by a 12-hinfusion of 0.125 µg/kg/min (Tcheng et al. 1994). This dosing regimen hasbeen kept constant in several clinical trials with a huge number of patients.The trials proved the benefit of abciximab treatment in the setting of acuteMI and PCI. A newer study using the current dosing regimen showed that al-though most of the abciximab-treated patients achieved a platelet inhibition of95% or higher 10 min after bolus administration, as assessed with the UltegraRapid Platelet Function Assay, the level of platelet inhibition 8 h after bolusadministration and during continuous infusion of 0.125µg/kg/min was only90%±11%. Patients showed a significant higher rate of major cardiac adverseevents if platelet inhibition was less than 95% 10 min after bolus administration(Steinhubl et al. 2001). This result may lead to further dose-adjustment trialsmonitoring the platelet function inhibition. The high affinity and the low half-time rate of dissociation from the receptor led to prolonged platelet inhibitionlasting for up to 7 days and distinguished abciximab from the small-moleculeinhibitors with a lower affinity and more rapid dissociation (Mascelli et al.1998; Peter et al. 2000). Due to the rapid plasma clearance of unbound abcix-imab and the high receptor affinity, a dose reduction in patients with renaldisease does not seem to be necessary and platelet inhibition can be reversedrapidly by platelet transfusion. A rare adverse event of abciximab adminis-tration is the development of acute, severe thrombocytopenia with plateletcounts of less than 20,000 platelets/µl. This condition develops within 24 h inapproximately 0.7% of patients (Berkowitz et al. 1997). The pathogenesis of thisthrombocytopenia is unknown. One of the most often discussed theories statesthat pre-existing antibodies against αIIbβ3 conformations induced by abcix-imab or other antagonists can induce thrombocytopenia (Bednar et al. 1999).There was one patient described who presented with severe thrombocytopeniaand in whom abciximab treatment resulted in direct platelet activation. It hasbeen hypothesized that the sequestration of these activated platelets causedthrombocytopenia (Peter et al. 1999).


2.4.3Eptifibatide (Integrilin)

Eptifibatide is a synthetic heptapeptide with a mass of 800 Da, modeled onthe active site of barbourin, a peptide from the disintegrin family found in thevenom of the southeastern pigmy rattlesnake. Unlike other disintegrins thathave an RGD sequence and block all integrins that recognize this sequence,barbourin mediates its high affinity to αIIbβ3 through a Lys-Gly-Asp (KGD)sequence (Phillips and Scarborough 1997). Eptifibatide contains a modifiedKGD sequence and binds with high specificity to αIIbβ3, but with a loweraffinity (KD, 120 nM) than abciximab.

Despite its initially proposed specificity for αIIbβ3, binding of eptifibatideto αV integrins has been reported (Thibault et al. 2001) and inhibition ofαVβ3 on different cell types, including smooth muscle and endothelial cells,has been demonstrated (Lele et al. 2001). The low affinity of the drug accountsfor the high plasma concentration of unbound eptifibatide with only 25% ofeptifibatide bound to plasma proteins. As eptifibatide is eliminated throughthe kidney, dose reductions in patients with severe renal disease or renal fail-ure are necessary. Eptifibatide is currently approved for patients with ACSand patients undergoing PCI. The Integrilin to Minimize Platelet Aggrega-tion and Coronary Thrombosis (IMPACT II) trial (IMPACT-II Investigators1997) showed that eptifibatide, given as a 0.135 µg/kg bolus followed by a0.75-µg/kg/min infusion, reduced the rate of ischemic events during the 24-htreatment period in patients undergoing elective, urgent or emergency PCI.It was found that the calcium-chelating property of the anticoagulant sodiumcitrate used for platelet aggregation assays in the IMPACT II trial led to anoverestimation of the inhibitory effect of eptifibatide (Phillips et al. 1997).The dosing regimen was re-evaluated in the Posicor Reduction of IschemiaDuring Exercise (PRIDE) trial (Tcheng et al. 2001), with the direct thrombininhibitor Phe-Pro-Arg chloromethyl ketone (PPACK) as an anticoagulant forplatelet aggregation assays. In the PRIDE trial, the dose of 180µg/kg bolusfollowed by a 2.0-µg/kg/min infusion proved to be sufficient to achieve andmaintain over 90% inhibition of 20 µM ADP-induced platelet aggregation. Thisdosing regimen was confirmed in the Platelet Glycoprotein IIb/IIIa in Unsta-ble Angina trial [Receptor Suppression Using Integrilin Therapy (PURSUIT)trial]. Compared to a treatment with placebo, patients with unstable angina ornon-ST-segment elevation MI (NSTEMI) had a significantly lower incidenceof death or MI when treated with eptifibatide 180 µg/kg bolus followed by2.0-µg/kg/min infusion (The PURSUIT trial investigators 1998).

The plasma elimination half-life of eptifibatide is approximately 2.5 h. Thelevel of platelet aggregation inhibition 4 h after cessation of the infusion isless than 50% and the bleeding time 6 h after cessation is not remarkablyprolonged. As an early peak level with a small decline within 4–6 h after thebolus administration was observable, a double bolus of 180µg/kg followed by


another 180µg/kg 10 min later was considered safe in patients undergoingPCI in the Enhanced Suppression of the Platelet IIb/IIIa Receptor with Inte-grilin Therapy (ESPRIT) trial (The ESPRIT trial investigators 2000). Data frompatients who underwent PCI suggest that the decline in platelet aggregation in-hibition could be overcome by the double bolus administration of eptifibatide(Gilchrist et al. 2001).

2.4.4Tirofiban (Aggrastat)

The synthetic nonpeptide tirofiban is a small-molecule (500 Da) GPIIb/IIIainhibitor that mimics the RGD sequence (Egbertson et al. 1994). Tirofiban isa competitive antagonist with a receptor affinity higher than eptifibatide butlower than abciximab (KD, 15 mM). To date, no cross-reactivity of tirofibanwith other integrins has been reported. The fraction of unbound tirofibanin human plasma is 35%, the plasma elimination half-life of 2 h is compa-rable with eptifibatide. Normal hemostatic function is restored within 4 h ofcessation of the drug infusion. The renal clearance of tirofiban accounts for39%–69% of the plasma clearance. Patients with creatinine clearance of lessthan 30 ml/min present a significantly decreased plasma clearance (>50%) oftirofiban. For these patients, the manufacturer recommends a weight-adjustedreduction of the dose by 50%. Tirofiban is licensed for the treatment of ACS andin the setting of PCI. The dosing regimen for the use in ACS is a bolus infusionof 0.4 µg/kg/min over 30 min, followed by an infusion of 0.1 µg/kg/min. Inpatients undergoing PCI, a bolus dose of 10 µg/kg given 10 min prior to PCIfollowed by a 0.15-µg/kg/min infusion for 18 h has been used in the Random-ized Efficacy Study of Tirofiban for Outcomes and Restenosis (RESTORE) trial(RESTORE Investigators 1997). Sufficient and consistent platelet aggregationinhibition of more than 80% in the response to 20 µM ADP has been reportedwith either dosing regimen during the time of infusion (Batchelor et al. 2002).However, an inhibition of platelet aggregation exceeding 80% after a bolus of10 µg/kg (RESTORE regimen) was not achieved in all patients at 15 and 30 min,but could be observed after 4 h (Batchelor et al. 2002). The same dosing regi-men for tirofiban was used in the Tirofiban and ReoPro Give Similar EfficacyOutcomes trial (TARGET) (The Target Investigators 2001). This trial, intendedto asses the noninferiority of tirofiban compared to abciximab, was the firstdirect comparison of the two GPIIb/IIIa inhibitors abciximab and tirofiban inthe setting of PCI. The 30-day results demonstrated the superiority of abcix-imab. The primary end point occurred in 7.6% of patients treated with tirofibanversus 6% of patients treated with abciximab. A possible explanation for thisphenomenon is the incomplete inhibition of platelet aggregation by tirofiban.Platelet aggregation measurements were not incorporated in the study proto-col. Therefore, this issue has to be addressed by further investigations.


2.4.5Oral GPIIb/IIIa Inhibitors

The rationale behind the development of oral GPIIb/IIIa antagonist was theassumption that long-term inhibition of αIIbβ3 would provide a new thera-peutic approach for the prevention of recurrent ischemic events in patientswith cardiovascular disease. Four oral GPIIb/IIIa antagonists (lotrafiban, or-bofiban, sibrafiban and xemilofiban), all of which are nonpeptide prodrugs,have been examined in large Phase III clinical trials. None of them fulfilled thehigh expectations. Therapies applying these substances have not proven to besuperior to aspirin therapy. Furthermore, a meta-analysis of four major PhaseIII trials [Evaluation of Oral Xemilofiban in Controlling Thrombotic Events(EXCITE), Orbofiban Post Unstable Coronary Syndromes (OPUS), Sibrafibanversus Aspirin to Yield Maximum Protection from Ischemic Heart Events Post-acute Coronary Syndromes (SYMPHONY) and 2nd SYMPHONY] including33,326 patients showed a statistically significant increase in mortality in pa-tients treated with the oral GPIIb/IIIa antagonists (Chew et al. 2001). The shortplasma half-life of these drugs that causes undulating plasma levels and pro-aggregatory effects especially at low concentrations of the GPIIb/IIIa blockersis discussed as a potential reason for the disappointing outcomes of these largePhase III trials (Peter et al. 1998).

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Part IV

Targets of Future Anti-Atherosclerotic Drug Therapy

Documents

[Handbook of Experimental Pharmacology] Atherosclerosis: Diet and Drugs Volume 170 || Inhibition of Platelet Activation and Aggregation