Background: Hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, platelet aggregate formation has to be a self-limiting process in order to prevent thrombotic complications. The second wave mediator, ADP, activates platelets via the P2Y₁ and P2Y₁₂ receptors. Following ADP exposure, the P2Y₁ receptor undergoes a rapid desensitization, a negative feedback mechanism thought to be critical for limiting thrombus growth.

Aims: Here we analyzed a novel knock-in mouse strain expressing a P2Y₁ receptor variant that does not desensitize (P2Y₁^340-0P).

Methods: We analyzed platelet function of P2Y₁^340-0P platelets, using in vitro assays of platelet aggregation, integrin activation, microfluidics and calcium flux.  In vivo function was assessed by saphenous vein laser injury, ferric chloride carotid injury and collagen-induced pulmonary embolism.

Results: P2Y₁^340-0P mice did not follow a Mendelian inheritance pattern as homozygous mice exhibited early embryonic lethality. Both heterozygous (P2Y₁^+/340-0P) and hemizygous (P2Y₁^-/340-0P) mice were viable. Compared to controls, platelets from P2Y₁^-/340-0P mice were hyperreactive to ADP and the selective-P2Y₁ agonist, MRS2365, and they did not desensitize to repeated ADP challenge. P2Y₁^-/340-0P platelets also showed increased responsiveness to collagen and thrombin, consistent with the important role of released ADP at low concentrations of these agonists. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions, both in vitro and in vivo.

Conclusions: In summary, our studies demonstrate that receptor phosphorylation is critical for P2Y₁ desensitization in platelets, and that impaired P2Y₁ receptor desensitization leads to platelet hyperreactivity towards various agonists. Surprisingly, the mechanism of P2Y₁ receptor desensitization was not required for limiting platelet adhesion/aggregation at sites of vascular injury, while it was crucial for murine embryonic development.    

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/loss-of-p2y1-receptor-desensitization-does-not-impact-in-vivo-hemostasis-or-thrombosis-despite-increased-platelet-reactivity-in-vitro/