A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation
A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation
复制标题
凝血酶/PAR-1在ADP诱导的血小板分泌和第二波聚集中的关键作用
DOI：
10.1111/jth.12168
复制
发表时间：
2013-05-01
影响因子：
10.4
通讯作者：
Hu, H.
中科院分区：
文献类型：
--
作者：
Jiang, L.;Xu, C.;Hu, H.
背景 在 ADP 刺激的富血小板血浆 (PRP) 中经常观察到稳定的或第二波血小板聚集，并人为降低细胞外钙水平，这归因于血栓素 A2 (TXA2) 生成的增强和核酸外切酶活性的抑制。然而，凝血酶在ADP诱导的血小板分泌和第二波聚集中的作用尚不清楚。目的和方法我们采用聚集测定法、流式细胞术、免疫印迹和 ELISA 来确定凝血酶是否以及如何参与 ADP 诱导的血小板分泌和第二波聚集。结果 ADP 诱导磷酸肌醇 3 激酶 (PI3K) 途径依赖性凝血酶生成，可能是由于 IIb3 相关凝血酶原的裂解所致。生成的凝血酶随后激活蛋白酶激活受体 1 (PAR-1)，并介导致密颗粒分泌和 ADP 刺激的柠檬酸 PRP 中的第二波血小板聚集。因此，ADP 诱导的致密颗粒分泌和 PRP 中的第二波血小板聚集同样被凝血酶抑制剂水蛭素、PAR-1 拮抗剂 SCH-79797 或 PI3K 抑制剂渥曼青霉素非累加性阻断。此外，ADP 刺激导致凝血酶原从 IIb3 解离，血浆凝血酶水平升高；两者均被渥曼青霉素预防。此外，ADP 引起的渥曼青霉素抑制的第二波血小板聚集可通过 PAR-1 激活肽 SFLLRN 的亚聚集浓度得以恢复。用吲哚美辛阻断 TXA2 的产生或将细胞外钙恢复至生理浓度不会影响这种凝血酶/PAR-1 依赖性。结论 PI3K 依赖性凝血酶生成和由此产生的 PAR-1 激活是传递 ADP 诱导的血小板激活过程中不可或缺的机制。
Background The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A2 (TXA2) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown. Objectives and Methods We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation. Results ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of IIb3-associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from IIb3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence. Conclusions A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.