Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles. In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec(-1) (shear stress approximately 375 dyne/cm2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation.