Platelet P2Y Receptor Exhibits Constitutive G Protein Signaling and β-arrestin 2 Recruitment

Background: Purinergic P2Y and P2Y receptors (P2Y-R and P2Y-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-R is the major target of antiplatelet drugs, no P2Y-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y-R antagonists constitutes an important preliminary step.

Results: Here, we investigated the pharmacology of P2Y-R signaling through Gq and β-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-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y-R is constitutively active in physiological conditions. We showed that P2Y-R also promotes constitutive recruitment of β-arrestin 2 in HEK293T cells. Moreover, the P2Y-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists.

Conclusions: This study sheds new light on P2Y-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y-R population in human platelets. Given the recent interest of P2Y-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.