Loss of P2Y₁ receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity...

The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y₁ and Gi-coupled P2Y₁₂ receptors. After ADP exposure, the P2Y₁ receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth.

The objective of this study was to examine the role of rapid P2Y₁ receptor desensitization on platelet function and thrombus formation in vivo.

We analyzed a novel knock-in mouse strain expressing a P2Y₁ receptor variant that cannot be phosphorylated beyond residue 340 (P2Y₁^340-0P), thereby preventing the desensitization of the receptor.

P2Y₁^340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y₁^{340-0P/340-0P} and control mice. In vitro, P2Y₁^{340-0P/340-0P} platelets were hyperreactive to ADP, showed a robust activation response to the P2Y₁ receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions.

Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y₁ receptor desensitization in platelets and that impaired desensitization leads to increased P2Y₁ receptor signaling in vitro. Surprisingly, desensitization of the P2Y₁ receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.