Identification and Targeting of a PAR1-PAR4 Complex in Human Platelets and Thrombosis.

Thrombin is the most potent activator of platelets and plays a crucial role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, a high-affinity PAR1 and low-affinity PAR4. Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. PAR1-PAR4 co-factoring was shown by acceleration of thrombin cleavage and signaling of PAR4 upon co-expression with PAR1. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small molecule antagonists and pepducins was effective against carotid artery occlusion. Co-immunoprecipitation and fluorescence resonance energy transfer studies demonstrated that PAR1 and PAR4 associate as a hetero-oligomer in human platelets and recombinant fibroblasts. Together these studies show that PAR1 and PAR4 form a stable hetero-oligomer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 hetero-oligomer may present a novel therapeutic opportunity in the prevention of arterial thrombosis.