A, Top, Expression of mRNA encoding PAR (protease-activated receptor)-1, PAR4, and GPR31 in human platelets. Human platelets were gel purified, and total RNA was reverse transcribed using an oligo(dT), and cDNA was amplified with polymerase chain reaction (PCR) using specific sets of primers for PAR1, PAR4, GPR31, and actin described in the Methods section. RT-PCR products were run on a 1% agarose gel. A, Bottom, Gel-purified platelets were analyzed for GPR31 expression by flow cytometry using GPR31 specific ab. B, Chinese hamster ovary (CHO)-K1 cells expressing GPR31 and Cre (cAMP-responsive element) reporter stimulated with 10 μM forskolin in the presence or absence of 300 nmol/L 12(S)-HETE. C, Predicted GPCR (G-protein–coupled receptor) structural model of GPR31 and model of GPR310 (G-protein–coupled receptor 310) pepducin using GPCR-I-TASSER58 (CCR5 [C-C chemokine receptor type 5] PBID ID: 4MBS). D, Time course of Rap1 (Ras-related protein 1) activation in human platelets stimulated with 100 nmol/L 12(S)-HETE in the absence (−) or presence (+) of 1 μM GPR310. The lower panel shows Rap1 as loading control. E, Dose response of human platelets pretreated with GPR310 as indicated and activated with 100 nmol/L 12(S)-HETE. The lower panel shows Rap1 as loading control. F, Immunoblot showing 100 nmol/L 12(S)-HETE rapidly (15 min) activates p38 MAPK (mitogen-activated protein kinase) phosphorylation in human platelets. *P<0.05, unpaired 2-tailed t test.