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

Abstract

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. Disclosures No relevant conflicts of interest to declare.

Eicosanoids in inflammation in the blood and the vessel

“…In platelets, 12(S)-HETE-GPR31 signals through G αi to induce platelet activation and thrombosis. Activation of the GPR31 inhibits AC activity and results in Ras-related protein 1 (Rap1) and p38 activation ( Van Doren et al, 2019 ). 20-HETE affects vascular function by binding to G-protein coupled receptor 75 (GPR75) coupled to G αq/11 in endothelial cells which results in PLC-IP 3 -mediated increases in intracellular Ca 2+ ( Garcia et al, 2017 ), activation of the Rho kinase ( Randriamboavonjy et al, 2003 ) and the mitogen activated protein (MAP) kinase pathways ( Muthalif et al, 2000 ) ( Table 1 ).…”

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“…P2Y12 Crystal structures of the human P2Y12 receptor shown in figure 1 have as of late been settled. Structures of the receptor in a complex with the agonists 2-methylthio-adenosine diphosphate (2MeS-ADP) and 2-methylthio-adenosine triphosphate (2MeS-ATP) 10,11 and with the nonnucleotide antagonist ethyl 6-(4-[(benzylsulfonyl) carbamoyl] piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) were obtained 12,13 . The P2Y12 receptor represents a successful drug target, with several clinical drugs on the market, including clopidogrel, prasugrel, ticlopidine, cangrelor, and ticagrelor, and more drugs in clinical trials 14 .…”