Blockade of Lipid Receptor GPR31 Suppresses Platelet Reactivity and Thrombosis with Minimal Effect on Hemostasis

Objective:Platelet agonist-activated 12-lipoxygenase (12-LOX)/12-Hydroxyeicosatetraenoic acid (12-HETE)/G protein-coupled receptor 31 (GPR31) signaling has been proposed to regulate platelet reactivity. While inhibition or genetic ablation of 12-LOX supports an important role of 12-HETE in response to platelet agonists thrombin and collagen, the participation of GPR31 in platelet lipid signaling has not been examined. We developed a potent pepducin inhibitor, GPR-310, to test the downstream involvement of GPR31 in thrombin and collagen mediated platelet activation and thrombosis.

Approach and Results:Treatment of mice with GPR-310 reversibly inhibited ex vivo platelet aggregation in response to thrombin and the PAR4 agonist, AYPGKF. There was significant protection (P<0.002) against FeCl₃-induced carotid artery injury in mice by extending occlusion time from 100% occlusion at 27 min in the vehicle cohort to 20% occluded at 45 min in the GPR-310 cohort. GPR-310 treatment did not affect tail bleeding time. In human platelets, GPR-310 significantly (P<0.001) inhibited PAR4 agonist and collagen-mediated platelet aggregation and PAR4 calcium release. GPR-310 inhibited 12(S)-HETE- and PAR4-mediated RAP1 activation, with no effect on the PAR1-RAP1 signal. Accordingly, PAR1-mediated aggregation of human platelets was not affected by either GPR-310 or the 12-LOX inhibitor, ML355. GPR-310 caused a 5-fold shift in thrombin-mediated human platelet aggregation, comparable to a direct P2Y₁₂ inhibitor, AZD1283. Dual GPR31 and P2Y₁₂ inhibition showed synergy and protected against thrombin-mediated human platelet aggregation with a 19-fold shift. Blockade of GPR31 was more effective than the P2Y₁₂ inhibitor in a thrombin-mediated clot retraction assay. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems.

Conclusions:GPR31 may serve as a new therapeutic target in platelet-dependent arterial thrombosis and aggregation in humans.