a Department of Pathophysiology , Sechenov First Moscow State Medical University , Moscow , Russia.
b National Hemophilia Center , Sheba Medical Center , Tel-Hashomer , Israel.
Platelets. 2018 May;29(3):265-269. doi: 10.1080/09537104.2017.1295136. Epub 2017 Apr 13.
Mechanisms of platelet activation are triggered by thrombin, adenosine diphosphate (ADP), epinephrine, thromboxane A, and other soluble agonists which induce signaling via heterotrimeric Gαq, Gαi, and Gα12/13 proteins. We have undertaken a study addressing the contribution of these G proteins to platelet activation and clot formation in the presence of eptifibatide, thus excluding outside-in signaling provided by integrin αIIbβ3-fibrinogen engagement. Selective and combined activation of the G proteins was achieved by using combinations of platelet agonists and inhibitors. Platelet activation in platelet-rich plasma was evaluated by P-selectin expression using flow cytometry. Contribution of platelets to whole blood clotting was assessed by rotation thromboelastometry (ROTEM). Selective signaling of Gαq or Gαi but not Gα12/13 promoted P-selectin expression. Further enhancement of P-selectin expression was achieved by ADP-induced combined signaling of Gαq and Gαi, and to more extent by U46619 at high concentration (1.5 μM) induced combined signaling of Gαq and Gα12/13 while maximal P-selectin expression was achieved by thrombin receptor-activating peptide (TRAP)-induced combined signaling of Gαq, Gαi, and Gα12/13. In ROTEM, selective activation of Gαq, Gαi, or Gα12/13 failed to affect blood clotting. Combined signaling of Gαq and Gαi or Gαq and Gα12/13 or all three G proteins shortened the clotting time and stimulated clot strength. Pretreatment of platelets with acetylsalicylic acid did not change the effect of ADP but inhibited the effect of TRAP. Signaling of Gαq and Gα12/13 triggered by U46619 also stimulated clot formation. Combined signaling of either Gαq and Gαi or Gαq and Gα12/13 is sufficient to stimulate maximal platelet activation and enhanced clot formation in platelets treated with inhibitor of integrin αIIbβ3. It could be suggested that outside-in signaling is not necessarily required to fulfill these platelet functions.
血小板的激活机制是由凝血酶、二磷酸腺苷 (ADP)、肾上腺素、血栓烷 A 和其他可溶性激动剂触发的，这些激动剂通过异三聚体 Gαq、Gαi 和 Gα12/13 蛋白诱导信号转导。我们进行了一项研究，旨在探讨这些 G 蛋白在依替巴肽存在的情况下对血小板激活和血栓形成的贡献，从而排除整合素 αIIbβ3-纤维蛋白原结合引起的外向信号转导。通过使用血小板激动剂和抑制剂的组合，实现了对 G 蛋白的选择性和组合激活。使用流式细胞术通过 P-选择素表达评估富含血小板的血浆中的血小板激活。通过旋转血栓弹性测定法 (ROTEM) 评估血小板对全血凝血的贡献。选择性信号转导 Gαq 或 Gαi 但不是 Gα12/13 促进 P-选择素表达。ADP 诱导的 Gαq 和 Gαi 联合信号转导进一步增强了 P-选择素表达，而 U46619 在高浓度 (1.5 μM) 诱导的 Gαq 和 Gα12/13 联合信号转导则更显著，而最大的 P-选择素表达是由凝血酶受体激活肽 (TRAP) 诱导的 Gαq、Gαi 和 Gα12/13 的联合信号转导实现的。在 ROTEM 中，选择性激活 Gαq、Gαi 或 Gα12/13 均不能影响血液凝固。Gαq 和 Gαi 或 Gαq 和 Gα12/13 或所有三种 G 蛋白的联合信号转导缩短了凝血时间并刺激了血栓强度。用乙酰水杨酸预处理血小板不会改变 ADP 的作用，但会抑制 TRAP 的作用。U46619 触发的 Gαq 和 Gα12/13 信号转导也刺激了血栓形成。在抑制剂处理的血小板中，Gαq 和 Gαi 或 Gαq 和 Gα12/13 的联合信号转导足以刺激最大的血小板激活和增强血栓形成。可以认为，外向信号转导不一定是完成这些血小板功能所必需的。
