尽管体外血小板反应性增加，但 P2Y 受体脱敏丧失并不影响止血或血栓形成。
Loss of P2Y receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro.
机构信息
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: https://twitter.com/David_S_Paul.
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
出版信息
J Thromb Haemost. 2023 Jul;21(7):1891-1902. doi: 10.1016/j.jtha.2023.03.013. Epub 2023 Mar 21.
BACKGROUND
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 P2Y and Gi-coupled P2Y receptors. After ADP exposure, the P2Y receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth.
OBJECTIVE
The objective of this study was to examine the role of rapid P2Y receptor desensitization on platelet function and thrombus formation in vivo.
METHODS
We analyzed a novel knock-in mouse strain expressing a P2Y receptor variant that cannot be phosphorylated beyond residue 340 (P2Y), thereby preventing the desensitization of the receptor.
RESULTS
P2Y mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y and control mice. In vitro, P2Y platelets were hyperreactive to ADP, showed a robust activation response to the P2Y 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.
CONCLUSION
Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y receptor desensitization in platelets and that impaired desensitization leads to increased P2Y receptor signaling in vitro. Surprisingly, desensitization of the P2Y 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.
背景
血管损伤部位止血塞的形成强烈依赖于血小板的快速激活以及整合素介导的黏附和聚集。然而，为了防止血栓并发症，血小板聚集的形成必须是一个自我限制的过程。第二信使二磷酸腺苷（ADP）通过 Gq 偶联的 P2Y 和 Gi 偶联的 P2Y 受体激活血小板。ADP 暴露后，P2Y 受体发生快速磷酸化诱导的脱敏，这种负反馈机制被认为对限制血栓生长至关重要。
目的
本研究旨在研究快速 P2Y 受体脱敏在体内血小板功能和血栓形成中的作用。
方法
我们分析了一种新型表达不能磷酸化超过 340 位残基的 P2Y 受体变异体的敲入小鼠品系（P2Y），从而阻止受体脱敏。
结果
P2Y 小鼠遵循孟德尔遗传模式，P2Y 和对照小鼠的外周血小板计数相当。在体外，P2Y 血小板对 ADP 反应过度，对 P2Y 受体选择性激动剂 MRS2365 表现出强烈的激活反应，并且对重复 ADP 挑战不发生脱敏。我们观察到钙动员增加、蛋白激酶 C 底物磷酸化、α 颗粒释放、小 GTP 酶 Rap1 的激活以及整合素内-外向激活/聚集。然而，这种超反应性并没有导致在生理剪切条件下血小板黏附和过度塞形成增加。
结论
我们的研究表明，C 端受体磷酸化对于血小板中 P2Y 受体脱敏至关重要，并且脱敏受损会导致 P2Y 受体在体外信号转导增加。令人惊讶的是，P2Y 受体脱敏对于限制血管损伤部位的血小板黏附和聚集不是必需的，这可能是因为 ADP 很快在血液中降解或被冲走。
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