Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Department of Dentistry and Oral and Maxillofacial Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Thromb Haemost. 2023 Jan;123(1):16-26. doi: 10.1055/s-0042-1757407. Epub 2022 Oct 28.
Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen.
To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations.
Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma.
A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.
血栓调节蛋白（TM）通过凝血酶依赖性激活蛋白 C 和凝血酶激活的纤溶抑制物（TAFI）发挥双重调节剂-抗凝和抗纤溶潜能。激活的 TAFI 裂解部分降解纤维蛋白的 C 末端赖氨酸，并抑制纤维蛋白表面上纤溶酶原的结合及其激活。我们之前报道过，激活的血小板在组织型纤溶酶原激活物和纤溶酶原积累后，启动纤维蛋白网络的形成并触发纤维蛋白溶解。
分析 TM 结构域缺失变体在不同浓度下对凝血和纤溶的影响。
使用 TM 的结构域缺失变体，如 D123（所有细胞外区域）、E3456（蛋白 C 依赖性激活和 TAFI 的最小结构域）和 E456（仅蛋白 C 的最小结构域但不是 TAFI），浓度范围为 0.25 至 125 nM，用于浊度测定法确定凝血时间和纤维蛋白溶解时间，并可视化血小板富含血浆中纤维蛋白网络的形成和溶解。
低浓度的 D123 或 E3456，但不是 E456，会延长纤维蛋白溶解时间，并由于有效的 TAFI 激活而延迟荧光标记的纤溶酶原在激活的血小板/致密纤维蛋白区域的积累。相反，仅三种 TM 变体的最高浓度会延迟凝血时间，尽管激活的血小板附近的纤维蛋白网络形成几乎完整。TAFI 激活可能受到纤维蛋白形成阶段后凝血酶活性减弱的影响。这些发现表明，TM 的抗凝和抗纤溶潜能的时空平衡以结构域和浓度依赖性的方式受到控制。
