Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University, Kingsville, TX 78363, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, The University of Texas El Paso, El Paso, TX 79902, USA.
Life Sci. 2020 Dec 1;262:118481. doi: 10.1016/j.lfs.2020.118481. Epub 2020 Sep 22.
G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αβγ heterotrimers, in which the βγ subunits are central players in downstream signaling. Herein, we investigated the role of Gβγ subunits in platelet function, hemostasis and thrombogenesis.
To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gβγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gβγ on α -granule secretion and αIIbβ3 activation. Clot retraction and the platelet spreading assay were used to examine Gβγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl-induced carotid-artery injury thrombosis model to determine Gβγ contribution to in vivo platelet function.
We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein's inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation.
Our findings demonstrate, for the first time, that Gβγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.
G 蛋白偶联受体 (GPCR) 通过与各种生理激动剂相互作用，紧密调节血小板功能。GPCR 信号的一个重要介质是 G 蛋白 αβγ 异三聚体，其中βγ 亚基是下游信号转导的核心。在此，我们研究了 Gβγ 亚基在血小板功能、止血和血栓形成中的作用。
为了实现这一目标，我们在小分子 Gβγ 抑制剂 gallein 的背景下使用了来自小鼠和人类的血小板。我们使用聚集仪检测聚集和致密颗粒分泌。我们还使用流式细胞术检测 P-选择素和 PAC1，以确定抑制 Gβγ 对 α-颗粒分泌和 αIIbβ3 激活的影响。使用血小板回缩和血小板铺展实验检测 Gβγ 在血小板信号转导中的作用，而使用 Western blot 检测其在 Akt 激活中的作用。最后，我们使用出血时间测定和 FeCl3 诱导的颈动脉损伤血栓形成模型来确定 Gβγ 对体内血小板功能的贡献。
我们观察到 gallein 抑制了激动剂刺激下的小鼠和人类血小板的聚集和分泌。此外，gallein 还抑制了整合素 αIIbβ3 的激活、血小板回缩、血小板铺展和 Akt 激活/磷酸化。最后，gallein 的抑制作用在体内表现出来，因为它能够调节生理止血并延迟血栓形成。
我们的研究结果首次表明，Gβγ 亚基直接调节 GPCR 依赖性血小板功能，无论是在体外还是体内。此外，这些数据突出了 Gβγ 作为治疗血栓性疾病的新的治疗靶点。
