Anti-platelet therapy: glycoprotein IIb-IIIa antagonists

Glycoprotein (GP) IIb-IIIa antagonists inhibit the aggregation of activated platelets. Three agents are approved for clinical use. In this review, the characteristics of each agent, their pharmacodynamic profile, results in pivotal clinical trials and the associated clinical implications are discussed. GP IIb-IIIa antagonists have greatest benefit when used as adjunctive therapy during percutaneous coronary intervention (PCI) when the patient has intra-coronary thrombosis. These agents appear to provide greatest benefit when used in combination with heparin. The clinical niche for parenteral GP IIb-IIIa antagonists is evolving. The rapid onset and offset of GP IIb-IIIa antagonists plus dosing designed to inhibit extensively platelet aggregation differentiates them from oral agents. The contemporary niche appears to include patients in transition, such as individuals requiring emergent PCI before oral agents are fully active and for unstable patients requiring transport to PCI centres, particularly in patients likely to have intracoronary thrombus. Subsequent studies should evaluate the optimal duration of therapy with GP IIb-IIIa antagonists.     

The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation. Ticlopidine has no direct effect on the GP IIb-IIIa complex. We studied the effects of ticlopidine (500 mg/day for 8 days) in four healthy male volunteers on washed platelet aggregation induced by 5 μM ADP or thrombin (0.1 units/mL) and potentiated by 1 μM adrenaline (Adr), on basal and 1 μM PGE₁-stimulated cAMP levels and on elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). We found that: (i) ticlopidine inhibits aggregation by ADP but not the potentiation by Adr of ADP-induced aggregation; (ii) ADP, Adr or thrombin decreases cAMP levels raised by PGE₁, an effect inhibited by ticlopidine only for ADP and not for Adr or thrombin; and (iii) Ca²⁺ influx and Ca²⁺ mobilization from internal stores were not affected. These results suggested that ticlopidine or a metabolite impairs the coupling mechanism of the ADP aggregation pathway at an unknown level.     

The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GP IIb-IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb-IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb-IIIa complex.     

Overview of clinical trials with glycoprotein IIb-IIIa receptor antagonists in the prevention and management of coronary

Platelet aggregation is mediated by the glycoprotein IIb-IIIa receptor, a member of the integrin superfamily of membrane-bound adhesion molecules. In the activated platelet, binding to the major adhesive proteins, fibrinogen and von Willebrand, occurs due to a conformational change of the glycoprotein IIb-IIIa receptor. Glycoprotein IIb-IIIa receptor antagonists effectively block the binding of these adhesive proteins and thus inhibit platelet aggregation. Large-scale clinical trials have demonstrated the benefits of these agents in patients undergoing percutaneous coronary angioplasty and with acute coronary syndromes compared to conventional antiplatelet therapy. Furthermore, trials are in progress in patients with acute myocardial infarction. The beneficial effects of these agents was first demonstrated with abciximab, a monoclonal antibody to the glycoprotein IIb-IIIa receptor, in patients at risk of coronary arterial thrombosis, and was further illustrated in trials with other IIb-IIIa receptor blocking agents, both with synthetic peptide and non-peptide receptor antagonists. This review focuses on the glycoprotein IIb-IIIa receptor antagonists most advanced in clinical development.     

Optimal use of platelet glycoprotein IIb/IIIa receptor antagonists in patients undergoing percutaneous coronary interventions

Discovery of the central role of platelets in the pathogenesis of acute coronary syndromes (ACS) and ischaemic complications of percutaneous coronary interventions (PCI) has led to the widespread use of oral and parenteral platelet inhibitors to treat these conditions. Glycoprotein (GP) IIb/IIIa (also known as α(IIb)β(3)) receptors on the surface of platelets play an essential role in platelet aggregation and serve as a key mediator in the formation of arterial thrombus. When activated, GP IIb/IIIa receptors bind to fibrinogen, which serves as the 'final common pathway' in platelet aggregation. Of the numerous agents developed for modulating platelet activity, intravenous platelet GP IIb/IIIa receptor antagonists are the most potent. There are four agents in clinical use, including abciximab, eptifibatide, tirofiban and lamifiban, although lamifiban is not approved for use in the US. While all agents block fibrinogen binding to GP IIb/IIIa, they do so by different mechanisms. Abciximab is a humanized form of a murine monoclonal antibody directed against GP IIb/IIIa, eptifibatide is a synthetic, cyclic heptapeptide that contains a lysine-glycine-aspartic acid (KGD) sequence that mimics the arginine-glycine-aspartic acid (RGD) sequence found on GP IIb/IIIa, tirofiban is a non-peptide antagonist derived by optimization of the tyrosine analogue that structurally mimicks the RGD-containing loop of the disintegrin echistatin, and lamifiban is a synthetic, non-cyclic, non-peptide, low-molecular-weight compound. In clinical trials, use of these agents reduces ischaemic adverse cardiovascular events in patients with ACS undergoing PCI, but at a cost of increased bleeding.     

Modulation of the activity and assessment of the receptor selectivity in a series of new RGD-containing peptides

We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related β3-integrins of the vasculature, GPIIb-IIIa (α_IIbβ₃), present in platelets, and the vitronectin receptor (α_vβ₃), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et. al. (1993, J. Biol. Chem. 2 68 , 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the ^N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.     

Low molecular weight, non-peptide fibrinogen receptor antagonists.

The tetrapeptide H-Arg-Gly-Asp-Ser-OH (1) (RGDS), representing a recognition sequence of fibrinogen for its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3), served as lead compound for the development of highly potent and selective fibrinogen receptor antagonists. Replacement of the N-terminal arginine by p-amidinophenylalanine or the Gly moiety by m-aminobenzoic acid led to compounds which are superior to the lead peptide with regard to activity and selectivity for GP IIb-IIIa vs the closely related vitronectin receptor alpha v beta 3. By random screening [(p-amidinobenzenesulfonamido)ethyl]-p-phenoxyacetic acid derivatives have been identified as fibrinogen receptor antagonists. Further structure-activity relationship studies culminated in the preparation of N-[N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl]-3-phenyl-L- alanine (29h, Ro 43-5054) and [[1-[N-(p-amidinobenzoyl)-L-tyrosyl]-4-piperidinyl]oxy]acetic acid (37f, Ro 44-9883), which exhibit very high activity as platelet aggregation inhibitors (IC50s 0.06 and 0.03 microM, respectively, human PRP/ADP) as well as marked selectivity for GP IIb-IIIa vs alpha v beta 3. Since the activity of 37f in dogs declines according to a two-compartment model with an initial phase having a t1/2 of 8 min and a second phase with a t1/2 of 110 min, this compound is a suitable candidate for the development as iv platelet inhibitor.     

血小板膜糖蛋白Ⅱb/Ⅲa受体拮抗剂的研究进展

血小板糖蛋白(GP)Ⅱb/Ⅲa受体是血小板聚集血栓形成的最终共同途径,(GP)Ⅱb/Ⅲa受体拮抗剂通过与受体结合抑制血小板凝集,从而抑制血栓形成,是一类高效和特异的抗血小板药物,越来越广泛地应用于择期经皮冠状动脉介入治疗(PCI)、非ST段抬高的急性冠脉综合征(NSTE-ACS)、ST段抬高心肌梗死(STEMI)等疾病。     

An in vitro investigation of the cardiovascular effects of the 5-HT4 receptor selective agonists,velusetrag and TD-8954

The 5-HT₄ receptor agonists, and gastrointestinal (GI) prokinetic agents, cisapride and tegaserod, lack selectivity for the 5-HT₄ receptor. Cisapride is a potent human ether-à-go-go-related gene (hERG) potassium channel inhibitor while cisapride and tegaserod have significant affinity for 5-HT₁ and 5-HT₂ receptor subtypes. Marketing of both compounds was discontinued due to cardiovascular concerns (cardiac arrhythmias with cisapride and ischemic events with tegaserod). The reported association of tegaserod with ischemia has been postulated to involve coronary artery constriction or augmentation of platelet aggregation. This in vitro study investigated the effects of two of the new generation of highly selective 5-HT₄ receptor agonists, velusetrag and TD-8954, on canine, porcine and human coronary artery tone, human platelet aggregation and hERG potassium channel conductance. No significant off-target actions of velusetrag or TD-8954 were identified in these, and prior, studies. While cisapride inhibited potently the hERG channel currents, tegaserod failed to affect platelet aggregation, and had only a small contractile effect on the canine coronary artery at high concentrations. Tegaserod inhibited the 5-HT-induced contractile response in the porcine coronary artery. New generation 5-HT₄ receptor agonists hold promise for the treatment of patients suffering from GI motility disorders with a reduced cardiovascular risk.     

Evidence for functional 5-HT2 receptor sites on human blood platelets

The aggregation of normal human platelets by 5-hydroxytryptamine (5-HT) is the result of a specific interaction of the monoamine with a platelet receptor since it is not influenced by adrenergic receptor blockade, inhibition of fatty acid cyclo-oxygenase or ADP-scavenging. The 5-HT induced platelet reaction is inhibited in a concentration-dependent wat by various serotonergic antagonists; the potency of these compounds in this respect correlates strongly with their potential to inhibit the specific binding of [³H] ketanserin, a selective label for 5-HT₂ binding sites, to rat prefrontal cortex and striatum and to cat platelet membranes. This study thus provides evidence for a functional role as true receptor initiating a physiological response of the 5-HT₂ receptor on human platelets.     

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation

BACKGROUND AND PURPOSE

Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.

EXPERIMENTAL APPROACH

The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.

KEY RESULTS

When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2-methylthioadenosine 5′-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5′-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.     

Snake venom probes of platelet adhesion receptors and their ligands

Snake venom proteins that modulate platelet adhesive interactions are chiefly from either of two main structural families: the C-type lectin-like family, or the metalloproteinase-disintegrins. Snake venom probes from both families selectively target platelet adhesion receptors, including glycoprotein (GP) Ib-IX-V, GP VI, 2β1 and IIbβ3 (GP IIb-IIIa). These receptors act together to mediate platelet adhesion, activation and aggregation (thrombus formation) under hydrodynamic shear stress in flowing blood. The receptors are members of the leucine-rich repeat family (GP Ib-IX-V), the immunoglobulin superfamily (GP VI), or integrins (2β1, IIbβ3). In addition, adhesive glycoproteins in matrix and/or plasma such as von Willebrand factor (that binds GP Ib and IIbβ3), collagen (that binds GP V, GP VI and 2β1), or fibrinogen (that binds IIbβ3), are also targeted by C-type lectin family or metalloproteinase-disintegrin snake venom proteins. Emerging structural and functional evidence is beginning to explain how interactions between the conserved structural module-domains that make up these mammalian and snake proteins are regulated. Whether homologous adhesion/counter-receptors on platelets and other vascular cells are also potential snake venom targets is as yet largely unexplored.     

Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist

By means of Sephadex G-75 and CM-Sephadex C-50 column chromatography and reverse-phase HPLC, a low molecular weight (Mr = 7500), cysteine-rich peptide, halysin, was purified from Agkistrodon halys (mamushi) snake venom. Halysin is a potent platelet aggregation inhibitor that concentration-dependently inhibited human platelet aggregation stimulated by ADP, thrombin and collagen (IC50 = 0.16 to 0.36 microM) without affecting platelet secretion. It was active in inhibiting platelet aggregation of platelet-rich plasma and whole blood. Halysin had no effect on thromboxane B2 formation of platelets or intracellular Ca2+ mobilization of Quin 2-AM loaded platelets stimulated by thrombin. It inhibited the fibrinogen-induced aggregation of elastase-treated platelets. Halysin concentration-dependently inhibited the 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50 = 0.16 microM). 125I-Halysin bound to resting platelets (Kd = 1.6 x 10(-7) M) and to ADP-stimulated platelets (Kd = 3.4 x 10(-8) M) in a saturable manner. EDTA, the Arg-Gly-Asp (RGD)-containing snake venom peptides trigamin and rhodostomin, Arg-Gly-Asp-Ser (RGDS), and Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val blocked both 125I-fibrinogen binding and 125I-halysin binding to ADP-stimulated platelets. The monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex blocked both 125I-fibrinogen and 125I-halysin binding, whereas 10E5 had no significant effect on halysin binding to ADP-stimulated platelets, indicating that 7E3 and halysin bind to an epitope which is different from that of 10E5. RGDS concentration-dependently inhibited 125I-halysin binding in a competitive manner. We determined the primary structure of halysin which is a single peptide chain of 71 amino acid residues. An RGD sequence appeared in the carboxy-terminal domain of halysin. Halysin showed about an 85% identical sequence with trigamin which is a specific antagonist of fibrinogen receptor associated with glycoprotein IIb-IIIa complex. In conclusion, halysin inhibited platelet aggregation by interfering with fibrinogen binding to the fibrinogen receptor of the activated platelets. The RGD sequence of halysin plays an important role in the expression of its biological activity.     

AoGDW肽抑制血小板聚集的作用机制

目的研究RGD(精氨酸-甘氨酸-天门冬氨酸)肽衍生物-AoGDW(ω-氨基辛酸-甘氨酸-天门冬氨酸-色氨酸)抑制血小板聚集的作用机制。方法采用双色荧光标记以及流式细胞技术检测AoGDW肽对CD41抗体与血小板GPⅡb/Ⅲa受体结合的抑制作用以及对血小板膜上P-选择素表达的影响。结果随着AoGDW肽浓度的增加,CD41抗体与活化血小板GPⅡb/Ⅲa受体结合率呈现下降趋势,相对于阴性对照组,P<0.01。但CD41抗体与静止血小板GPⅡb/Ⅲa受体的结合率以及CD62p抗体与静止血小板P选择素的结合率与阴性对照组相比,P>0.05。结论AoG-DW肽是通过占据血小板上纤维蛋白原的结合位点即活化的GPⅡb/Ⅲa受体而发挥抑制血小板聚集的作用,并且不具有活化血小板的作用,是一种选择性的GPⅡb/Ⅲa受体拮抗剂。     

The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation. Ticlopidine has no direct effect on the GP IIb-IIIa complex. We studied the effects of ticlopidine (500 mg/day for 8 days) in four healthy male volunteers on washed platelet aggregation induced by 5 microM ADP or thrombin (0.1 units/mL) and potentiated by 1 microM adrenaline (Adr), on basal and 1 microM PGE1-stimulated cAMP levels and on elevation of cytosolic free Ca2+ concentration ([Ca2+]i). We found that: (i) ticlopidine inhibits aggregation by ADP but not the potentiation by Adr of ADP-induced aggregation; (ii) ADP, Adr or thrombin decreases cAMP levels raised by PGE1, an effect inhibited by ticlopidine only for ADP and not for Adr or thrombin; and (iii) Ca2+ influx and Ca2+ mobilization from internal stores were not affected. These results suggested that ticlopidine or a metabolite impairs the coupling mechanism of the ADP aggregation pathway at an unknown level.     

Understanding the mechanism of platelet thrombus formation under blood flow conditions and the effect of new antiplatelet agents

Platelet aggregation induced by stirring of a platelet suspension after activation by the exogenous addition of stimulants, such as ADP, epinephrine and thrombin, has long been used as a platelet function test, and for the screening of antiplatelet agents. Since platelet aggregation has been demonstrated to be mediated exclusively by the binding of plasma fibrinogen to the activated GP IIb/IIIa, anti-GP IIb/IIIa agents, which can completely inhibit platelet aggregation, were expected to become among the most potent of antithrombotic agents. The strong preventive effects of anti-GP IIb/IIIa agents against thrombotic complications after coronary intervention, and the weaker preventive effects of the same agents against the onset of coronary thrombosis in unstable angina pectoris patients point to both the efficacy and the limitations of anti-GP IIb/IIIa antithrombotic agents. Recently, many investigators have reported that platelets play a major role in thrombus formation at sites exposed to blood flow. Several platelet-function assay systems have been developed to elucidate the mechanism of thrombus formation under blood flow conditions. Numerous studies have demonstrated that von Willebrand factor (VWF) and its interaction with its receptors on platelets, including GP Ibalpha and GP IIb/IIIa, play essential roles not only in platelet activation, but also in platelet adhesion and possibly platelet cohesion. These findings prompted us to explore whether the VWF-mediated process of thrombus formation could be exploited as a target for potential antiplatelet agents. Moreover, recent studies have demonstrated that multiple synergistic signals, including those mediated by catecholamine receptors, purine nucleotide receptors, as well as some types of collagen receptors are involved in the process of VWF-mediated platelet thrombus formation. We now have new antiplatelet agents on the horizon, targeted against thrombus formation under blood flow conditions.     

替罗非班治疗急性冠脉综合征的临床应用进展

替罗非班是一种非肽类的血小板糖蛋白Ⅱb/Ⅲa受体的可逆性拮抗剂,其作用机制是通过抑制纤维蛋白原和血小板Ⅱb/Ⅲa受体结合,从而抗血小板聚集、激活以及血栓形成,广泛应用于急性冠脉综合征患者。本文对替罗非班在机型冠脉综合征中的应用价值、疗效及作用机制做一综述。     

Prednisolone exerts exquisite inhibitory properties on platelet functions

We have previously reported presence of the glucocorticoid (GC) receptor (GR) alpha on blood platelets, and its ability to modulate platelet aggregation when activated by the synthetic GC prednisolone (Pred). In the present study we investigated the effects of Pred on broader aspects of platelet functions to unveil novel non-genomic actions on this cell type. Using whole blood assay we demonstrated that Pred was the only GC able to inhibit platelet aggregation and platelet–monocyte interactions. This latter effect was due to regulation of platelets, not monocytes. We next examined the effects of Pred on physiological actions of platelets, observing inhibition of platelet adhesion and spreading on collagen under static conditions. Moreover Pred inhibited thrombus formation under flow, suggesting potential important effects in haemostasis and thrombosis. Pred was unable to regulate platelet reactivity under conditions where the effects of platelet-derived ADP and TxA₂ were blocked, suggesting that the GC targeted the activation-dependent component of the adhesion and aggregation response. The effects of Pred were not mediated through cyclic nucleotide signaling, but rather seemed to evolve around selective regulation of P2Y₁₂ ADP receptor signaling, intimating a novel mode of action. This study details the actions of Pred on platelets unveiling novel properties which could be relevant for this GC in controlling unwanted vascular and thrombotic diseases.     

The Role of the Platelet in the Pathogenesis of Atherothrombosis

Platelet adhesion, activation, and aggregation at sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. Platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von Willebrand factor (vWF). Platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (GP) Ib-V-IX receptor to circulating vWF, which binds to exposed collagen, and directly, via interaction with the platelet receptors GP VI and GP Ia/IIb. Platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, ADP, and thromboxane A2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet alpha(IIb)beta3 integrin (GP IIb/IIIa receptor). Platelet aggregation, mediated primarily by interaction between the activated platelet GP IIb/IIIa receptor and its ligands, fibrinogen and vWF, results in the formation of a platelet-rich thrombus. Currently available antiplatelet drugs (aspirin [acetylsalicylic acid], dipyridamole, clopidogrel, ticlopidine, abciximab, eptifibatide, tirofiban) act on specific targets to inhibit platelet activation and aggregation. Elucidation of the multiple mechanisms involved in platelet thrombus formation provides opportunities for selectively inhibiting the pathways most relevant to the pathophysiology of atherothrombosis.     

ADP receptor antagonists as antiplatelet therapeutics

With the cloning of the P2Y12 receptor, the molecular basis for ADP-induced platelet aggregation is seemingly complete. Two platelet-bound ADP receptors, P2Y1 and P2Y12, operate through unique pathways to induce and sustain platelet aggregation via the glycoprotein (GP)IIb-IIIa integrin. P2Y1 operates via a glycoprotein q (Gq) pathway, activates phospholipase C, induces platelet shape change and is responsible for intracellular calcium mobilisation. P2Y12 inhibits adenylyl cyclase through a glycoprotein i (Gi)-dependent pathway, and is the target of the clinically used thienopyridines, ticlopidine (Ticlid, F. Hoffman-La Roche) and clopidogrel (Plavix, Bristol-Myers Squibb/Sanofi-Synthelabo). In addition, the receptor is targeted by the ADP analogue AR-C66096, which is currently in Phase IIb clinical trials, as well as other non-nucleoside-based preclinical leads.