Thrombosis and Atherosclerosis Research Institute, Hamilton, ON, Canada.
Departments of Medicine and Medical Sciences, McMaster University, Hamilton, ON, Canada.
J Thromb Haemost. 2020 Sep;18(9):2364-2376. doi: 10.1111/jth.14950.
Thrombin-activated platelets can promote fibrinolysis by binding plasminogen in a fibrinogen-dependent manner and enhancing its activation by tissue-type plasminogen activator (t-PA). Whether t-PA also binds to activated platelets and the mechanism for regulation of platelet-dependent fibrinolysis remain unknown.
Determine the mechanism of plasminogen and t-PA binding on thrombin-activated platelets and its regulation by activated thrombin-activatable fibrinolysis inhibitor (TAFIa).
Plasminogen and t-PA binding with or without TAFIa treatment was quantified using flow cytometry. Plasmin generation on platelets was quantified using a plasmin-specific substrate. Mass spectrometry analyses identified fibrinogen as a potential target of TAFIa. Thrombus formation was studied in mice lacking fibrinogen (Fg ) using intravital microscopy.
Plasminogen and t-PA bind to platelets activated by thrombin but not by other agonists, including protease-activated receptor agonists (PAR-AP). Plasminogen binds via its kringle domains because ε-aminocaproic acid eliminates binding, whereas t-PA binds via its finger and kringle domains. Plasminogen binding is fibrinogen-dependent because it is abolished on (a) Fg platelets, and (b) thrombi in Fg mice. Binding requires thrombin-mediated fibrinogen modification because addition of batroxobin to PAR-AP activated platelets has no effect on plasminogen binding but induces t-PA binding. TAFIa reduces plasminogen and t-PA binding to thrombin-activated platelets and attenuates plasmin generation in a concentration-dependent manner. Mass spectrometry identified K556 on the fibrinogen alpha-chain as a potential thrombin cleavage site that generates a TAFIa sensitive C-terminal lysine residue.
These findings provide novel mechanistic insights into how platelets activated by thrombin at sites of vascular injury can influence fibrinolysis.
凝血酶激活的血小板可以通过纤维蛋白原依赖性方式结合纤维蛋白溶酶原，并增强其组织型纤溶酶原激活物（t-PA）的激活，从而促进纤维蛋白溶解。t-PA 是否也与激活的血小板结合，以及调节血小板依赖性纤维蛋白溶解的机制尚不清楚。
确定纤维蛋白溶酶原和 t-PA 在凝血酶激活的血小板上结合的机制及其被激活的凝血酶可激活的纤溶抑制物（TAFIa）的调节作用。
使用流式细胞术定量测定有无 TAFIa 处理时纤维蛋白溶酶原和 t-PA 的结合。使用纤维蛋白溶酶特异性底物定量测定血小板上纤维蛋白溶酶的产生。质谱分析鉴定纤维蛋白原为 TAFIa 的潜在靶标。使用缺乏纤维蛋白原（Fg）的小鼠通过活体显微镜研究血栓形成。
纤维蛋白溶酶原和 t-PA 与凝血酶激活的血小板结合，但不与其他激动剂（包括蛋白酶激活受体激动剂（PAR-AP））结合。纤维蛋白溶酶原通过其 Kringle 结构域结合，因为 ε-氨基己酸消除了结合，而 t-PA 通过其指状和 Kringle 结构域结合。纤维蛋白溶酶原的结合依赖于纤维蛋白原，因为它在（a）Fg 血小板上被废除，并且（b）在 Fg 小鼠的血栓中被废除。结合需要凝血酶介导的纤维蛋白原修饰，因为向 PAR-AP 激活的血小板中添加巴曲酶对纤维蛋白溶酶原结合没有影响，但诱导 t-PA 结合。TAFIa 以浓度依赖的方式降低凝血酶激活的血小板上纤维蛋白溶酶原和 t-PA 的结合，并减弱纤维蛋白溶酶的产生。质谱分析鉴定纤维蛋白原α链上的 K556 为潜在的凝血酶裂解位点，产生 TAFIa 敏感的 C 末端赖氨酸残基。
这些发现为在血管损伤部位由凝血酶激活的血小板如何影响纤维蛋白溶解提供了新的机制见解。
