AbstractA fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.