A key role of the fast ATP-gated P2X₁ cation channel in the thrombosis of small arteries in vivo

Abstract number: OC209

Hechler^* B., Lenain^* N., Marchese† P., Vial‡ C., Heim^* V., Freund^* M., Cazenave^* J.-P., Cattaneo§ M., Ruggeri† Z., Evans‡ R., Gachet^* C.

^*INSERM U311, France; †Scripps Research Institute, USA; ‡University of Leicester, UK; §University of Milan, Italy

Purine nucleotides mediate signaling in platelets through three different P2 receptors, the well characterized ADP sensitive G protein-coupled P2Y1 receptor, the P2Y12 receptor and the ATP-sensitive ligand-gated P2X1 cation channel. The role of the P2X1 receptor in platelet function has long been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. We have used P2X1-deficient mice (P2X1–/– mice) to assess the involvement of the platelet P2X1 receptor in hemostasis and thrombosis. The selective P2X1 receptor agonist alpha,betaMeATP induced a transient calcium entry in wild type (WT) platelets, treated with apyrase to prevent desensitization, whereas no calcium entry could be detected in P2X1–/– platelets. In vitro, the collagen-induced aggregation of P2X1–/– platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the substrate density was low and the wall shear rate elevated (>1500 s^-1). P2X1–/– mice exhibited no spontaneous bleeding tendency but their bleeding time, a measure of primary hemostasis in the microcirculation, was nevertheless prolonged (median 107 s) as compared to WT (median 85 s). In vivo, in a model of acute thromboembolism induced by infusion of a mixture of collagen (0.3 mg kg^-1) and adrenaline (60 µg kg^-1), that leads to massive occlusion of the pulmonary microvessels with platelet aggregates, mortality was 91% in WT mice whereas it was only 50% in P2X1–/– mice. Moreover in a model of localized arterial thrombosis of the mesenteric arterioles triggered by laser-induced vessel wall injury, the size of mural thrombi was decreased in P2X1–/– mice (mean thrombus height 5.1 ± 1.1 µM) as compared to the WT (13.7 ± 1.7 µM) (^***P = 0.0005, Wilcoxon signed rank test), while the time for complete thrombus removal was shortened in P2X1–/– mice (70 s) as compared to the WT (120 s). In summary, in vivo models of thrombosis where platelet activation is critical revealed a contribution of the P2X1 receptor to thrombosis in the microcirculation, in which blood flow is characterized by a high shear rate, and was mimicked by the in vitro studies of platelet–collagen interaction under blood flow at high shear. Overall, besides the G protein-coupled ADP receptors, P2Y1 and P2Y12, known to be critically involved in hemostasis and thrombosis, this study points to a key role of the P2X1 receptor in arterial thrombosis under high shear conditions.