Diverse signaling of the platelet P2Y1 receptor leads to a dichotomy in platelet function

Abstract PlateletP2Y 1 receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no plateletaggregation is observed. We investigated signalling cascades involved in distinct P2Y 1 -dependent plateletactivities in vitro , using specific inhibitors for phospholipase C (PLC) (U73122, to inhibit the canonical pathway), and RhoGTPases: Rac1(NSC23766) and RhoA(ROCK inhibitor GSK429286). Human platelet rich plasma(for plateletaggregation) or isolated washed platelets(for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate(ADP) or the P2Y 1 specific agonist MRS2365. Aggregation, chemotaxis(towards f-MLP), or platelet-induced human neutrophil chemotaxis( PINC) towards macrophage derived chemokine (MDC) was assessed. Molecular docking of ADP and MRS2365 to P2Y 1 was analysed using AutoDockSmina followed by GOLD molecular docking in the Accelrys Discovery Studiosoftware. Inhibition of PLC, but not Rac1or RhoA, suppressed plateletaggregation induced by ADP and MRS2365. In contrast, platelet chemotaxisand PINC, were significantly attenuated by inhibition of platelet Rac1or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y 1 -induced aggregation, but a similar efficacy to stimulate platelet chemotaxisand PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y 1 receptor. PlateletP2Y 1 receptor activation during inflammation signals through alternate pathwaysinvolving Rho GTPasesin contrast to canonical P2Y 1 receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y 1 receptor.