College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea.
College of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju 63243, Korea.
Int J Mol Sci. 2020 May 30;21(11):3932. doi: 10.3390/ijms21113932.
Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6 platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6 platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate G-coupled 5HT and G-coupled adrenergic receptors, respectively, was not affected in GRK6 platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6 platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y, P2Y, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6 platelets. Finally, GRK6 mice exhibited an enhanced and stable thrombus formation after FeCl injury to the carotid artery and shorter tail bleeding times, indicating that GRK6 mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.
血小板 G 蛋白偶联受体 (GPCR) 通过介导对各种激动剂的反应来调节血小板功能，包括二磷酸腺苷 (ADP)、血栓烷 A2 和凝血酶。虽然 G 蛋白偶联受体激酶 (GRK) 被认为在大多数 GPCR 功能中具有关键作用，但关于 GRK 在血小板中调节 GPCR 信号转导和 GPCR 脱敏的机制知之甚少。在这项研究中，我们研究了 GRK6 的功能作用以及 GRK6 调节血小板中特定 GPCR 脱敏的分子基础。我们使用 GRK6 敲除小鼠来评估 GRK6 在血小板激活中的功能作用。与野生型 (WT) 血小板相比，ADP 类似物 2-MeSADP、U46619、凝血酶和 AYPGKF 诱导的血小板聚集、致密颗粒和 -颗粒分泌以及纤维蛋白原受体激活在 GRK6 血小板中显著增强。然而，GRK6 血小板中胶原相关肽 (CRP) 诱导的血小板聚集和分泌不受影响。有趣的是，分别激活 G 偶联 5HT 和 G 偶联肾上腺素能受体的 5-羟色胺和肾上腺素的协同刺激诱导的血小板聚集在 GRK6 血小板中不受影响，表明 GRK6 参与了特定的 GPCR 调节。此外，GRK6 血小板中对 ADP 和 AYPGKF 的第二次刺激的血小板聚集得到恢复，而 WT 血小板中激动剂的再刺激不能诱导聚集，表明 GRK6 有助于 P2Y、P2Y 和 PAR4 受体脱敏。此外，GRK6 血小板中 2-MeSADP 诱导的 Akt 磷酸化和 AYPGKF 诱导的 Akt、细胞外信号调节激酶 (ERK) 和蛋白激酶 Cδ (PKC) 磷酸化显著增强。最后，GRK6 小鼠在 FeCl 损伤颈动脉后的血栓形成增强且稳定，尾巴出血时间缩短，表明 GRK6 小鼠更容易发生血栓形成和止血。我们得出结论，GRK6 通过选择性 GPCR 脱敏在调节血小板功能反应和血栓形成中发挥重要作用。
