Comparison of the pharmacodynamic effects of the platelet ADP receptor antagonists clopidogrel and AR-C69931MX in patients with ischaemic heart disease

We compared the antiplatelet effects of clopidogrel and the intravenous platelet P2Y 12 receptor antagonist AR-C69931MX, which acts on the same receptor as clopidogrel by a different and reversible mechanism and, unlike clopidogrel, is active in vitro . Thirteen patients with acute coronary syndromes entered into a phase II study of intravenous AR-C69931MX (Group 1) and eight patients undergoing intracoronary stent implantation and treated with clopidogrel (Group 2) were studied using a whole blood single-platelet counting aggregation assay. Group 2 patients were also studied using turbidimetry with ADP and TRAP as agonists and whole blood [ 14 C]5HT release to study dense granule secretion in response to ADP, collagen and TRAP. In Group 2 studies, a therapeutic concentration of AR-C69931MX was added in vitro before and after clopidogrel administration. AR-C69931MX in Group 1 achieved greater inhibition of ADP-induced platelet aggregation than clopidogrel in Group 2 and AR-C69931MX in vitro added to the effects of clopidogrel on ADP-induced aggregation. AR-C69931MX but not clopidogrel inhibited TRAP-induced aggregation and granule secretion and AR-C69931MX had a more consistent inhibitory effect on collagen-induced responses. In conclusion, therapeutic administration of clopidogrel moderately inhibits platelet P2Y 12 receptor activation and substantially greater P2Y 12 receptor blockade can be achieved with AR-C69931MX.     

Comparison of the pharmacodynamic effects of the platelet ADP receptor antagonists clopidogrel and AR-C69931MX in patients with ischaemic heart disease

We compared the antiplatelet effects of clopidogrel and the intravenous platelet P2Y(12) receptor antagonist AR-C69931MX, which acts on the same receptor as clopidogrel by a different and reversible mechanism and, unlike clopidogrel, is active in vitro. Thirteen patients with acute coronary syndromes entered into a phase II study of intravenous AR-C69931MX (Group 1) and eight patients undergoing intracoronary stent implantation and treated with clopidogrel (Group 2) were studied using a whole blood single-platelet counting aggregation assay. Group 2 patients were also studied using turbidimetry with ADP and TRAP as agonists and whole blood [(14)C]5HT release to study dense granule secretion in response to ADP, collagen and TRAP. In Group 2 studies, a therapeutic concentration of AR-C69931MX was added in vitro before and after clopidogrel administration. AR-C69931MX in Group 1 achieved greater inhibition of ADP-induced platelet aggregation than clopidogrel in Group 2 and AR-C69931MX in vitro added to the effects of clopidogrel on ADP-induced aggregation. AR-C69931MX but not clopidogrel inhibited TRAP-induced aggregation and granule secretion and AR-C69931MX had a more consistent inhibitory effect on collagen-induced responses. In conclusion, therapeutic administration of clopidogrel moderately inhibits platelet P2Y(12) receptor activation and substantially greater P2Y(12) receptor blockade can be achieved with AR-C69931MX.     

The P2Y 12 receptor as a therapeutic target in cardiovascular disease

Coronary thrombosis complicating rupture of atherosclerotic plaque is the predominant cause of acute coronary syndromes and platelets play a crucial role in this thrombus formation. Whilst aspirin has been successful in reducing cardiovascular morbidity and mortality, appreciation of its limited antiplatelet effects has stimulated the search for more effective antiplatelet agents. The thienopyridines, ticlopidine and clopidogrel, act, via metabolites, on the platelet ADP receptor subtype now designated P2Y 12 (formerly P 2T , P2T AC , P2Y ADP or P2Y cyc ) and these agents have proven clinical efficacy. Analogues of the natural P2Y 12 receptor antagonist ATP have been developed that act directly on the receptor and have a rapid onset of action. One such antagonist, AR-C69931MX, is being developed for clinical use. AR-C69931MX is a potent antagonist of ADPinduced platelet activation, aggregation and secretion and also antagonises platelet responses, including procoagulant activity, induced by all other agonists in view of the central role of the P2Y 12 receptor in amplifying platelet responses. Phase II studies of intravenous AR-C69931MX in patients with acute coronary syndromes show that this agent has a rapid onset of action, rapidly achieving steady-state inhibition of platelet aggregation, with a half-life of only a few minutes. AR-C69931MX appears to be safe and well tolerated as adjunctive therapy in these patients, and more effective inhibition of platelet function is achieved than with clopidogrel. Orally active ATP analogues are also being developed that may be more effective than clopidogrel. Limitations of platelet glycoprotein IIb/IIIa antagonists leave scope for development of alternative antiplatelet agents.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.     

Inhibitory effects of P2Y12 receptor antagonists on TRAP-induced platelet aggregation, procoagulant activity, microparticle formation and intracellular calcium responses in patients with acute coronary syndromes

Thrombin induces platelet aggregation and membrane rearrangements leading to enhanced procoagulant activity and microparticle production, all of which are thought to contribute to thrombus formation in patients with acute coronary syndromes (ACS). Clopidogrel, an adenosine diphosphate (ADP) receptor antagonist acting at the P2Y(12) receptor, has been shown to provide clinical benefit in ACS. We aimed to investigate the effects of clopidogrel ex vivo and another ADP-antagonist, AR-C69931MX in vitro on thrombin receptor activating peptide (TRAP)-induced platelet aggregation, procoagulant activity, microparticle formation and [Ca(2+)]i responses in patients with ACS. Measurements were performed in platelet-rich plasma using aggregometry and flow cytometry (n = 12). Clopidogrel (300 mg loading dose plus 75 mg daily) significantly inhibited TRAP-induced aggregation, procoagulant activity (annexin V binding) and microparticle production (all P < 0.05) but not as extensively as AR-C69931MX (400 nmol/l). [Ca(2+)]i responses induced by a combination of TRAP and ADP designed to mimic the physiological effects of released ADP showed that clopidogrel partially and AR-C69931MX completely removed the ADP component of the [Ca(2+)]i responses (n = 6). The results provide new information on the mechanisms involved in the beneficial effects of P2Y(12) antagonists in patients with ACS.     

Inhibitory effects of P2Y12 receptor antagonists on TRAP-induced platelet aggregation,procoagulant activity,microparticle formation and intracellular calcium responses in patients with acute coronary syndromes

Thrombin induces platelet aggregation and membrane rearrangements leading to enhanced procoagulant activity and microparticle production, all of which are thought to contribute to thrombus formation in patients with acute coronary syndromes (ACS). Clopidogrel, an adenosine diphosphate (ADP) receptor antagonist acting at the P2Y₁₂ receptor, has been shown to provide clinical benefit in ACS. We aimed to investigate the effects of clopidogrel ex vivo and another ADP-antagonist, AR-C69931MX in vitro on thrombin receptor activating peptide (TRAP)-induced platelet aggregation, procoagulant activity, microparticle formation and [Ca²⁺]_i responses in patients with ACS. Measurements were performed in platelet-rich plasma using aggregometry and flow cytometry (n?=?12). Clopidogrel (300?mg loading dose plus 75?mg daily) significantly inhibited TRAP-induced aggregation, procoagulant activity (annexin V binding) and microparticle production (all P?<?0.05) but not as extensively as AR-C69931MX (400?nmol/l). [Ca²⁺]_i responses induced by a combination of TRAP and ADP designed to mimic the physiological effects of released ADP showed that clopidogrel partially and AR-C69931MX completely removed the ADP component of the [Ca²⁺]_i responses (n?=?6). The results provide new information on the mechanisms involved in the beneficial effects of P2Y₁₂ antagonists in patients with ACS.     

Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling

Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A(2) have been shown to cause platelet aggregation by concomitant signaling through G(q) and G(i) pathways, we investigated whether coactivation of G(q) and G(i) signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (alphaIIbbeta3). A PAR1-activating peptide, SFLLRN, and PAR4-activating peptides GYPGKF and AYPGKF, caused inhibition of stimulated adenylyl cyclase in human platelets but not in the presence of either Ro 31-8220, a protein kinase C selective inhibitor that abolishes secretion, or AR-C66096, a P2Y12 receptor-selective antagonist; alpha-thrombin-induced inhibition of adenylyl cyclase was also blocked by Ro 31-8220 or AR-C66096. In platelets from a P2Y12 receptor-defective patient, alpha-thrombin, SFLLRN, and GYPGKF also failed to inhibit adenylyl cyclase. In platelets from mice lacking the P2Y12 receptor, neither alpha-thrombin nor AYPGKF caused inhibition of adenylyl cyclase. Furthermore, AR-C66096 caused a rightward shift of human platelet aggregation induced by the lower concentrations of alpha-thrombin and AYPGKF but had no effect at higher concentrations. Similar results were obtained with platelets from mice deficient in the P2Y12. We conclude that (1) thrombin- and thrombin receptor-activating peptide-induced inhibition of adenylyl cyclase in platelets depends exclusively on secreted adenosine diphosphate that stimulates G(i) signaling pathways and (2) thrombin and thrombin receptor-activating peptides cause platelet aggregation independently of G(i) signaling.     

Platelet aggregation and adenosine diphosphate/adenosine triphosphate receptors: a historical perspective

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.     

The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.     

Both the ADP receptors P2Y1 and P2Y12, play a role in controlling shape change in human platelets

Two types of ADP receptors, P2Y(1) and P2Y(12) are involved in platelet aggregation. The P2X(1) receptor is also present but its role, in terms of platelet function, is not yet defined. The aim of this study was to establish if the ADP receptors, P2Y(1,) P2Y(12) and P2X(1) play a role in controlling platelet shape change (PSC) in human platelets. PSC is an early phase of platelet activation that precedes aggregation. Using a high-resolution channelyzer, PSC was assessed by measuring the median platelet volume (MPV). The P2Y(1) receptor antagonist MRS 2179 (1.06 - 10.25 micro mol/l) blocked ADP-induced PSC (by 100%). The median IC(50) was 3.16 micro mol/l. MRS 2179 also significantly (P = 0.01) inhibited PSC induced by the combination of ADP + serotonin (5HT). The P2Y(12) receptor antagonist AR-C69931MX significantly inhibited (at 10s, P = 0.009; 15 s, P = 0.001 and 30 s, P = 0.015) ADP-induced PSC. The P2X(1) receptor antagonist TNP-ATP had no significant effect on ADP- or ADP + 5HT-induced PSC. We conclude that the IC(50) of a P2Y(1)-blocker can be derived because of the high-resolution and reproducibility of the channelyzer technique. In addition to the P2Y(1) purinoceptor, the P2Y(12)receptor appears to be involved in ADP-induced PSC since this process was significantly inhibited by AR-C69931MX. The channelyzer technique may be more reliable than optical aggregometry to assess PSC.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca²⁺ influx. Binding studies, using the non- hydrolysable ligand [³³P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (K_d∼ 5 niw). These sites present pharmacological characteristics of a P_2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [³³P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca²⁺ influx and the other a Gi protein-coupled receptor responsible for Ca²⁺ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.     

Effects on blood compatibility in vitro by combining a direct P2Y12 receptor inhibitor and heparin coating of stents

The effect of the direct platelet P2Y12 receptor inhibitor, AR-C69931MX, on activation of blood induced by stents with and without heparin coating was investigated using a whole blood Chandler loop model in vitro. Stents were deployed in Chandler loops. Fresh human blood with heparin and AR-C69931MX was rotated for 1 h at 37 degrees C and used for measurements of platelets, microparticles, thrombin-antithrombin complex (TAT), fibrinogen binding to platelets, P-selectin expression by platelets, CD11b, Prothrombin Fragment F1+2, FXIa-AT, FXIIa-AT, C3a, sC5b-9 and stent score. In the first experiment there were four study groups with unmodified stents: 1a, no AR-C69931MX; 1b, 250 nmol/L; 1c, 750 nmol/L; 1d, 2250 nmol/L of AR-C69931MX. In the second experiment the concentration of AR-C69931MX was 500 nmol/L: 2a; tubings without stent; 2b; tubings with heparin-coated stent; 2c; tubings with unmodified stents. Heparin-coated stents were used in the third experiment: 3a; no AR-C69931MX; 3b; 500 nmol/L of AR-C69931MX. In the first experiment there were significant differences in all parameters analysed except for C3a, and stent score when the group with no AR-C69931MX was compared to all the groups with AR-C69931MX. In the second experiment there were significant differences in platelet count, TAT, FXIa-AT, FXIIa-AT and stent score when unmodified stents were compared to loops with no stents and partly to loops with heparin-coated stents. In the third experiment there was a significant reduction in generation of TAT, stent score and better preservation of platelet number by combining the platelet inhibitor and heparin-coated stents as compared to heparin-coated stents alone. The conclusion is that the direct P2Y12 receptor inhibitor AR-C69931MX reduced the different aspects of activation of blood induced by both unmodified and heparin-coated stents.     

Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses

The role of the G(i)-coupled platelet P2Y(12) receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to alphaIIbbeta3 activation in human platelets has not been delineated. As the P2Y(12) receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y(12) receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y(12)-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G(q)-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y(12) receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y(12) receptor. We conclude that GIRK channels are important functional effectors of the P2Y(12) receptor in human platelets.     

Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G(i)/adenylyl cyclase (P2Y(AC) ADP receptors). Clopidogrel abolishes the inhibitory P2Y(AC) receptor-mediated ADP effects on prostaglandin E(1)-stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y(AC) ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action.     

Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5'-diphosphate receptor stimulation and cyclic calcium signaling.

OBJECTIVES: We sought to evaluate the mechanisms that support the stability of platelet aggregates on a thrombogenic surface exposed to flowing blood. BACKGROUND: Activation of the membrane glycoprotein (GP) IIb/IIIa--mediated in part through the P2Y1 and P2Y12 adenosine 5'-diphosphate (ADP) receptors--is necessary for platelet aggregation. Platelets in growing thrombi exhibit cyclic calcium signal, suggesting that sustained activation may be required for thrombus stability. METHODS: Blood was perfused over type I collagen fibrils at the wall shear rate of 1,500 s(-1). Three-dimensional visualization of platelet thrombi was obtained in real time with confocal microscopy. The intracytoplasmic Ca2+ concentration ([Ca2+]i) was measured in fluo-3AM-loaded platelets. RESULTS: The height of platelet thrombi in control blood was 13.5 +/- 3.3 microm after 6 min, and increased to 16.3 +/- 4.5 microm (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 +/- 2.2 and 3.3 +/- 1.3 microm, respectively (p < 0.01, n = 8), when the blood used in the second 6-min perfusion contained a P2Y1 (MRS2179) or P2Y12 (AR-C69931MX) inhibitor. The [Ca2+]i of platelets within forming thrombi oscillated between 212 +/- 38 nmol/l and 924 +/- 458 nmol/l, with cycles lasting 4.2 +/- 2.8 s that were inhibited completely by AR-C69931MX and partially by MRS2179. Accordingly, thrombi became unstable upon perfusion of blood containing the Ca2+ channel blocker, lanthanum chloride. Flow cytometric studies demonstrated that AR-C69931MX, MRS2179, and lanthanum chloride reduced monoclonal antibody PAC-1 binding to platelets, indicating a decrease of membrane-expressed activated GP IIb/IIIa. CONCLUSIONS: Continuous P2Y1 and P2Y12 stimulation resulting in cyclic [Ca2+]i oscillations is required for maintaining the activation of GP IIb/IIIa needed for thrombus stability in flowing blood.     

P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes

Ninety-eight diabetic patients (type 2) were studied together with 24 healthy normotensive controls. Microaggregates (particle scale, <25 microm) of platelets were detected by a laser scattering system. Microaggregates in the control group showed a time-dependent reversible change; however, they existed continuously in 82 of 98 diabetic patients. When platelets of diabetics were stimulated by a shear stress alone without ADP, 74 also showed spontaneous and irreversible microaggregates even though they were not observed in all control subjects. In control subjects, microaggregates were inhibited by MRS2279 (a P2Y1 antagonist), but not AR-C69931MX (a P2Y12 antagonist). However, AR-C69931MX prevented irreversible microaggregates in diabetic patients. When either aspirin or ticlopidine was administered to diabetic patients with irreversible microaggregates, both drugs significantly decreased microaggregates induced by a low dose of ADP. Ticlopidine additionally reduced the microaggregates induced by shear stress alone. In conclusion, microaggregates of platelets via P2Y12 receptors could play a key role in the hypersensitivity of platelets in diabetic patients, and the measurement of microaggregation could be a useful marker to estimate of thrombogenesis. These findings present a possible new means for patients with diabetes to prevent ischemic events.     

The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T(AC) receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T(AC), in mouse platelets with similar function as in human platelets.     

Preclinical and clinical studies with selective reversible direct P2Y12 antagonists

An important role for adenosine diphosphate (ADP)-induced platelet activation and aggregation was proposed more than 40 years ago. The clinical use of clopidogrel, a prodrug of an irreversible P2Y (12) antagonist, has further proved the relevance of inhibiting signaling via the platelet-specific P2Y (12) ADP receptor in the prevention of cardiovascular events. Pharmacological studies at AstraZeneca R&D Charnwood have identified direct, selective, and competitive P2Y (12) antagonists, including cangrelor (also known as AR-C69931MX), which is suitable for intravenous administration, and AZD6140, which is suitable for oral administration. In preclinical use, these compounds predictably and effectively inhibited platelet aggregation without significant increases in bleeding time. In clinical use, these compounds may have significant advantages over current antiplatelet agents. This article summarizes preclinical and clinical data on cangrelor and AZD6140 and discusses the potential of these compounds as novel antiplatelet therapies.     

The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T AC receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptorselective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T AC , in mouse platelets with similar function as in human platelets.