A key role of the fast ATP-gated P2 X 1 cation channel in the thrombosis of small arteries in vivo

Abstract number: P0782

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 P2 X 1 cation channel. The role of the P2 X 1 receptor in platelet function has long been difficult to assess due to its rapid desensitization and the lack of pharmacologic tools. We have used P2 X 1-deficient mice (P2 X 1^-/- mice) to assess the involvement of the platelet P2 X 1 receptor in hemostasis and thrombosis. The selective P2 X 1 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 P2 X 1^-/- platelets. In vitro, the collagen-induced aggregation of P2 X 1^-/- 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 was elevated (>1500 s^-1). P2 X 1^-/- 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 mg 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 P2 X 1^-/- 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 P2 X 1^-/- mice (mean thrombus height 5.1 ± 1.1 mm) as compared to the WT (13.7 ± 1.7 mm) (^***P = 0.0005, Wilcoxon's signed rank test), while the time for complete thrombus removal was shortened in P2 X 1^-/- 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 P2 X 1 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 P2 X 1 receptor in arterial thrombosis under high shear conditions.