Abstract 275: Gene Expression Profiling of G-protein Coupled Receptors in Human Platelets
G-protein coupled receptors (GPCRs) play an important role in platelet homeostasis. The aim of thi study was to establish the relative expression of known GPCRs as well as to identify novel GPCRs in circulating platelets. An improved platelet purification process was used to isolate circulating human platelets, and a platelet gene expression profile was generated using Affymetrix HG-U133 arrays. All detected GPCR transcripts were validated using quantitative real-time PCR (Q-RT-PCR) and CD45, a leukocyte specific transcript was also quantified to exclude leukocyte contamination. In total, 3,408 transcripts were detected, resulting in 24 validated GPCRs. Using Q-RT-PCR, the 12 most abundant platelet GPCRs are, in falling rank order (relative to P2Y1 expression): thrombin receptor (1865 ± 178%), ADP receptor P2RY12 (459 ± 88%), succinate receptor 1 (257 ± 48%), P2RY1 (100%), orphan receptor P2RY10 (68.2 ± 3.3%), LPA receptor GPR23 (48.2 ± 11%), orphan receptor GPR92 (26.1 ± 3.3%), alpha 2 adrenergic receptor (18.4 ± 4.4%), orphan receptor EBI2 (6.31 ± 0.42), adenosine receptors A2a (2.94 ± 0.24%) and A2b (2.88 ± 0.16%) and LPA receptor EDG2 (2.59 ± 0.39%). Interestingly, 11 of the 24 GPCRs detected are novel platelet GPCRs, including adenosine receptor A2b and succinate receptor 1. The effects on platelet activation of the adenosine A2b receptor and succinate receptor 1 were assessed using flow cytometry and the platelet activation markers PAC-1 (activated GPIIb/IIIa) and CD62P (P-selectin). Platelets activated by the stable ADP analogue 2-MeS-ADP (1 nM) showed a reduced platelet activation after addition of 1μM of the non-selective adenosine A2 agonist NECA (% change of activation marker expression): PAC-1: 21.4+-1.9 CD62P: 11.4+-1.8). This effect was partially abolished by 1μM of the selective adenosine A2b antagonist MRS1754 (PAC-1: 15.1+-2.7 and CD62P: 6.1+-2.4; P<0.05). Succinic acid had no effect on platelet activation. For the first time, a validated expression profile of all platelet GPCRs has been generated. Of the 24 GPCR genes detected, eleven GPCRs are reported in platelets for the first time, including five orphan receptors and a functional adenosine A2b receptor, revealing several new drug targets for the inhibition of platelet aggregation.