背景:血栓弹力图(TEG)用于实时确定急性出血风险患者的止血状态。凝血酶被认为通过产生聚合的纤维蛋白和通过PAR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在TEG中的具体作用尚未见报道.
目的:在这里,我们使用小鼠和人血液样品中血小板蛋白的遗传和药理抑制作用,研究了TEG中血小板功能所需的特异性受体和信号通路。
方法:凝血参数(R,α,MA)在重新钙化中确定,使用TEG5000分析仪的高岭土触发柠檬酸盐的血样。
结果:我们确认了血小板的需求,血小板收缩,和αIIbβ3整合素函数的正常α角(α)和最大振幅(MA)。巨核细胞/血小板(Talin1mKO)中整联蛋白衔接子Talin1的丢失也降低了α和MA,但在缺乏Rap1GTP酶信号的小鼠样品中仅观察到最小的缺陷。PAR4mKO样品显示α受损但MA正常。然而,在PAR4mKO小鼠的样本中观察到与血小板耗尽样品相似的TEG受损痕迹,这些样本在血小板上耗尽了GPVI或添加了Syk抑制剂.我们在人血液中复制了这些结果,同时抑制了PAR1,PAR4和Syk。
结论: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,我们提供了第一个证据,证明PAR和GPVI在TEG中血小板介导的凝块收缩中起冗余作用.
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 (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO 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.