Platelet P2Y1 receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment

BackgroundPurinergic P2Y(1) and P2Y(12) receptors (P2Y(1)-R and P2Y(12)-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y(12)-R is the major target of antiplatelet drugs, no P2Y(1)-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y(1)-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y(1)-R antagonists constitutes an important preliminary step.ResultsHere, we investigated the pharmacology of P2Y(1)-R signaling through Gq and beta-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y(1)-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y(1)-R is constitutively active in physiological conditions. We showed that P2Y(1)-R also promotes constitutive recruitment of beta-arrestin 2 in HEK293T cells. Moreover, the P2Y(1)-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists.ConclusionsThis study sheds new light on P2Y(1)-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y(1)-R population in human platelets. Given the recent interest of P2Y(12)-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y(1)-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y(1)-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.

Automated summary

This study investigated the pharmacology of P2Y(1)-R signaling in various cells and platelets and found that P2Y(1)-R is constitutively active in physiological conditions. The study also identified P2Y(1)-R antagonists that can inhibit this constitutive activation. These findings suggest that targeting P2Y(1)-R constitutive activation could be a promising strategy for antiplatelet therapy.