Figure 6. GPR31 (G-protein–coupled receptor 31) regulates PAR (protease-activated receptor)-4–mediated platelet activation resulting in orchestrated control of Rap1-RASA3–mediated α_IIbβ₃ activation.

A, Fura-2–labeled transiently transfected GPR31/Chinese hamster ovary (CHO) cells were stimulated with 10 and 20 μM 12(S)-HETE, and Ca²⁺ flux was measured over time. B, Fura-2–labeled human platelets were stimulated with 10 μM 12(S)-HETE, and Ca²⁺ flux was measured over time. C, GPR310 (G-protein–coupled receptor 310) blocks PAR4-mediated calcium flux on human platelets. Platelets were preincubated with 3 μM GPR310 or vehicle before stimulation with 160 μM AYPGKF. D, Immunoblot showing 3 nmol/L thrombin activation of pAKT or pERK (phospho extracellular signal-regulated kinase) phosphorylation after 15 min in human platelets pretreated with 1 μM GPR310 as indicated. β-actin is used as loading control. E, Gel-purified platelets were treated with GPR310 (3 μM), AZD1283 (10 μM), or dual inhibition before thrombin stimulation (3 nmol/L) for 15 min, and Rap1 activation was measured. The bottom panels represent total Rap1 as loading control. F, Human platelets were treated with GPR310 (1 μM), AZD1283 (10 μM), dual inhibition, or LY294002 (10 μM), before thrombin stimulation with 3 nmol/L thrombin for 5 min. After membrane preparation, samples were immunoblotted with RASA3 ab. β-actin is used as loading control. G, Schematic of the PAR4, GPR31, and P2Y₁₂ signaling in human platelet. Molecular weight markers (kDa). 12-LOX indicates 12-lipoxygenase; GTP, guanosine triphosphate; pAkt, protein kinase B; pERK, phospho extracellular signal-regulated kinase; PLCβ, phospholipase C beta; Rap1, Ras-related protein 1; and RASA3, Ras P21 protein activator 3.