Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis

Abstract

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.