Mode of action of P2Y₁₂ antagonists as inhibitors of platelet function

Summary

P2Y₁₂ receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y₁₂ receptors. However, some P2Y₁₂ receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y₁₂ antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y₁₂ receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A_2A agonists, which act at G_s-coupled receptors. All three P2Y₁₂ antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A₂ mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y₁₂ antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the G_s-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y₁₂ antagonists acting through PAR1, TP, IP, EP4, A_2A or EP3 receptors. All three P2Y₁₂ antagonists enhanced VASPphosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A_2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y₁₂ receptors and not through another G-protein-coupled receptor.

Keywords

ADP - G_s-coupled receptors - platelet aggregation - P2Y₁₂ antagonists - VASP phosphorylation

• References

• 1 Heemskerk JWM. et al. Platelet activation and blood coagulation. Thromb Haemost 2002; 88: 186-193.
• 2 Munix ICA. et al. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102: 1149-1156.
• 3 Storey RF. et al. The central role of the P2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity. Br J Haematol 2000; 110: 925-934.
• 4 Storey RF. et al. Inhibition of ADP-induced P-selectin expression and platelet-leukocyte conjugate formation by clopidogrel and the P2Y12 receptor antagonist AR-C69931MX but not aspirin. Thromb Haemost 2002; 88: 488-494.
• 5 Dorsam RT, Kunapuli S. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340-344.
• 6 Gachet C. P2 receptors, platelet function and pharmacological implications. Thromb Haemost 2008; 99: 466-472.
• 7 Evans DJ. et al. Platelet P2Y12 receptor influences the vessel wall response to arterial injury and thrombosis. Circulation 2009; 119: 116-122.
• 8 Judge HM. et al. Relationship between degree of P2Y12 receptor blockade and inhibition of P2Y12-mediated platelet function. Thromb Haemost 2010; 103: 1210-1217.
• 9 Cattaneo M. New P2Y12 inhibitors. Circulation 2010; 121: 171-179.
• 10 Becker RC, Gurbel PA. Platelet P2Y12 receptor antagonist pharmacokinetics and pharmacodynamics: a foundation for distinguishing mechanisms of bleeding and anticipated risk for platelet-directed therapies. Thromb Haemost 2010; 103: 535-544.
• 11 Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discovery 2010; 09: 154-169.
• 12 Zürn C. et al. ADP-receptor blockade: A case for personalised pharmacotherapy?. Thromb Haemost 2010; 103: 496-506.
• 13 Savi P. et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000; 84: 891-896.
• 14 Savi P. et al. The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. Proc Natl Acad Sci 2006; 103: 11069-11074.
• 15 Ding Z. et al. Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues Cyst 17 and Cyst 270. Blood 2003; 101: 3908-3914.
• 16 Algaier I. et al. Interaction of the active metabolite of prasugrel, R-138727, with cysteine 97 and cysteine 175 of the human P2Y12 receptor. J Thromb Haemost 2008; 06: 1908-1914.
• 17 van Giezen JJ. et al. Ticagrelor binds to P2Y12 receptors independently from ADP but antagonizes ADP-induced receptor signalling and platelet aggregation. J Thromb Haemost 2009; 07: 1556-1565.
• 18 Storey RF. et al. Open multicentre study of the P2T receptor antagonist ARC69931MX assessing safety, tolerability and activity in patients with acute coronary syndromes. Thromb Haemost 2001; 85: 401-407.
• 19 van Giezen JJ, Humphries RG. Preclinical and clinical studies with selective reversible direct P2Y12 antagonists. Semin Thromb Hemost 2005; 21: 195-204.
• 20 Dovlatova N. et al. The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function. J Thromb Haemost 2010; 06: 1153-1159.
• 21 Paul BZ. et al. Distribution of prostaglandins IP and EP receptor subtypes and iso-forms in platelets and human umbilical artery smooth muscle cells. Br J Haematol 1998; 102: 1204-1211.
• 22 Iyú D. et al. The role of prostanoid receptors in mediating the effects of PGE2 on human platelet function. Platelets 2010; 21: 329-342.
• 23 Gessi S. et al. A2A Adenosine receptors in peripheral blood cells. Br J Pharmacol 2000; 129: 2-11.
• 24 Dovlatova N. et al. Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor. Thromb Haemost 2008; 100: 261-270.
• 25 Heptinstall S. et al. DG-041 inhibits the EP3 prostanoid receptor-a new target for inhibition of platelet function in atherothrombotic disease. Platelets 2008; 19: 605-613.
• 26 Singh J. et al. Antagonists of the EP3 receptor for prostaglandin E2 are novel anti-platelet agents that do not prolong bleeding. ACS Chem Biol 2009; 04: 115-126.
• 27 Dohlman HG. et al. Model system for the study of seven-transmembrane-segment receptors. Ann Rev Biochem 1991; 60: 653-688.
• 28 Narumiya S. et al. Prostanoid receptors: structures, properties and functions. Physiol Rev 1999; 79: 1193-1226.
• 29 Srinivasan S. et al. The P2Y12 antagonists, 2-methylthioadenosine 5^’-monophosphate triethyammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels. J Biol Chem 2009; 284: 16108-16117.
• 30 Wallentin L. et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2009; 361: 1045-1057.
• 31 Sugimoto Y, Narumiya S. Prostaglandin E receptor. J Biol Chem 2007; 282: 11613-11617.
• 32 Foudi N. et al. Vasorelaxation induced by prostaglandin E2 in human pulmonary vein: role of the EP3 receptor subtype. Br J Pharmacol 2008; 154: 1631-1639.
• 33 Dey I. et al. Prostaglandin E2 couples through prostanoid EP4 receptors to induce IL-18 production in human colonic epithelial cells. Br J Pharmacol 2009; 156: 475-485.
• 34 Haslam RJ, McClenaghan MD. Measurement of circulating prostacyclin. Nature 1981; 292: 364-366.
• 35 Horn EH. et al. A cross-sectional study of platelet cyclic AMP in healthy and hypertensive pregnant women. Clin Sci 1991; 80: 549-558.
• 36 Takasaki J. et al. Molecular cloning of the platelet P2TAC ADP receptor: Pharmacological comparison with another ADP receptor, the P2Y1 receptor. Mol Pharmacol 2001; 60: 432-439.
• 37 Hasegawa M. et al. Stereoselective inhibition of human platelet aggregation by R-138727, the active metabolite of CS-747 (Prasugrel, LY640315), a novel P2Y12 receptor inhibitor. Thromb Haemost 2005; 94: 593-598.
• 38 Nolte C. et al. Comparison of vasodilatory prostaglandins with respect to cAMP-mediated phosphorylation of a target substrate in intact human platelets. Biochem Pharmacol 1991; 42: 253-262.
• 39 Xiange B. et al. A Gi-independent mechanism mediating Akt phosphorylation in platelets. J Thromb Haemost. 2010 Epub ahead of print.
• 40 Ding Z. et al. Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor. Mol Pharmacol 2006; 69: 338-345.
• 41 Chee MJS. et al. The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor. J Biol Chem 2008; 283: 33337-33346.
• 42 Björkman JA. et al. AZD6140 inhibits adenosine uptake into erythrocytes and enhances coronary blood flow after local ischemia or intracoronary adenosine infusion. Circulation. 2007; 116 (Suppl) II-28 [Abstract 245].