We have generated transgenic mice overexpressing the human P2X₁ ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X₁ionotropic activity as determined by more prominent P2X₁-mediated Ca²⁺ influx and platelet shape change. P2X₁ overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A₂ mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds⁻¹ resulted in increased P2X₁-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X₁ ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A₂-, and shear stress–triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.