Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Thromb Haemost. 2023 Aug;21(8):2236-2247. doi: 10.1016/j.jtha.2023.04.008. Epub 2023 Apr 15.
Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported.
Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples.
Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer.
We confirmed the requirement of platelets, platelet contraction, and αIIbβ3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4 samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4 mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk.
Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.
血栓弹力描记术（TEG）用于实时确定有急性出血风险的患者的止血状态。凝血酶被认为通过生成聚合纤维蛋白和通过蛋白酶激活受体（PARs）激活血小板来驱动 TEG 中的凝血。然而，血小板激动剂受体和信号转导在 TEG 中的特定作用尚未报道。
在这里，我们使用小鼠和人血样本中血小板蛋白的遗传和药理学抑制，研究了 TEG 中血小板功能所需的特定受体和信号通路。
在重新钙化的、高岭土触发的柠檬酸化血液样本中，使用 TEG 5000 分析仪测定凝血参数（R 时间、α角[α]和最大振幅[MA]）。
我们证实了血小板、血小板收缩和αIIbβ3 整合素功能对正常α和 MA 的要求。巨核细胞/血小板中的整合素接头蛋白 Talin1（Talin1）缺失也降低了α和 MA，但在缺乏 Rap1 GTPase 信号的小鼠样本中仅观察到最小的缺陷。PAR4 样本显示α受损但 MA 正常。然而，在用缺乏血小板糖蛋白 VI 的 PAR4 小鼠样本或用 Syk 抑制剂处理的样本中，观察到与血小板耗竭样本相似的 TEG 轨迹受损。我们在人血中用 PAR1、PAR4 和 Syk 的联合抑制复制了这些结果。
我们的结果表明，标准 TEG 对整合素内-外激活和血小板止血功能至关重要的血小板信号通路不敏感。此外，我们首次提供证据表明 PARs 和糖蛋白 VI 在 TEG 中血小板介导的凝块收缩中起冗余作用。
