Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood

BACKGROUND: Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptors 1 (PAR1) and 4 (PAR4). PAR1 has higher affinity for thrombin and the first PAR1 antagonist, vorapaxar, was recently approved for use as an anti-platelet agent. However, vorapaxar is contraindicated in a significant number of patients due to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. OBJECTIVES: To determine the specific anti-platelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. METHODS & RESULTS: We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4 and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b β3 activation, alpha-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (FRET-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. CONCLUSIONS: These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.