P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation

P2X(1) receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X(1) receptors in platelet activation, the current study employed methods which maximally preserved P2X(1) functionality. In preliminary in vivo studies, P2X(1)-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X(1) inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X(1) with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X(1)-deficient murine platelets, and inhibition of P2X(1) in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X(1) antagonist MRS2159. In addition, P2X(1) stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X(1)-mediated priming, suggesting that the synergy between P2X(1) and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X(1) and thrombin receptors, with P2X(1) functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.