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

Platelet P2Y₁ receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no platelet aggregation is observed. We investigated signalling cascades involved in distinct P2Y₁-dependent platelet activities 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 platelet aggregation) or isolated washed platelets (for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate (ADP) or the P2Y₁ 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 P2Y₁ was analysed using AutoDock Smina followed by GOLD molecular docking in the Accelrys Discovery Studio software. Inhibition of PLC, but not Rac1 or RhoA, suppressed platelet aggregation induced by ADP and MRS2365. In contrast, platelet chemotaxis and PINC, were significantly attenuated by inhibition of platelet Rac1 or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y₁-induced aggregation, but a similar efficacy to stimulate platelet chemotaxis and PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y₁ receptor. Platelet P2Y₁ receptor activation during inflammation signals through alternate pathways involving Rho GTPases in contrast to canonical P2Y₁ receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y₁ receptor.