National Medical Research Center of Cardiology, Russian Ministry of Health, Moscow, Russian Federation.
Platelets. 2023 Dec;34(1):2139365. doi: 10.1080/09537104.2022.2139365. Epub 2022 Nov 2.
Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.
活化的血小板具有在其表面表达的促凝活性磷脂酰丝氨酸 (PS)，这是组装凝血复合物的底物。我们研究了不同激动剂激活的血小板对纤维蛋白形成和凝血酶生成的影响，并将这些影响相互比较，与 PS 表达进行了比较。开发了改良的血浆再钙化测定法来评估血小板对纤维蛋白形成的影响。将未激活的人血小板或用 A23187 离子载体、胶原蛋白、花生四烯酸、ADP 或 TRAP（凝血酶受体激活肽）激活的血小板留在原处，并在 96 孔板中离心。然后加入血浆，再钙化，并通过吸光度监测纤维蛋白形成。以相同的方式制备的血小板用于测试其对凝血酶生成的影响。通过使用 annexin V 染色的流式细胞术评估 PS 表达。血小板明显加速了纤维蛋白形成和凝血酶生成。它们缩短了迟滞期并增加了血浆凝固的最大速率，并增加了凝血酶生成的峰值和最大速率。在这两种测试中，血小板可能通过在触发凝血反应后在血浆中形成的内源性凝血酶激活。然而，用外源性激动剂预处理会进一步增加血小板的促凝活性。在用 A23187 孵育后达到最大值，用胶原蛋白和花生四烯酸孵育时较低，用 ADP 和 TRAP 孵育时最低（后者可能由于与内源性凝血酶竞争而无效）。不同激动剂激活的血小板对纤维蛋白形成和凝血酶生成的影响相互关联，并与它们表面的 PS 表达相对应。
