de Munk G A, Groeneveld E, Rijken D C
Gaubius Institute TNO, Leiden, The Netherlands.
J Clin Invest. 1991 Nov;88(5):1680-4. doi: 10.1172/JCI115483.
The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.
通过在有无可溶性兔血栓调节蛋白的情况下，将单链尿激酶型纤溶酶原激活剂（scu-PA）与不同浓度的人凝血酶孵育，研究了血栓调节蛋白对凝血酶使scu-PA失活的体外作用。在无血栓调节蛋白时，160 ng/ml凝血酶作用45分钟可使scu-PA发生50%失活；在有血栓调节蛋白时，4.6 ng/ml凝血酶作用45分钟可使scu-PA发生50%失活。在有无血栓调节蛋白的情况下，高分子量scu-PA与缺乏生长因子和kringle结构域的低分子量scu-PA的失活速率均无差异。用不同浓度的scu-PA进行酶动力学实验表明，血栓调节蛋白使凝血酶对scu-PA的Km从7.8 μM降至0.43 μM，kcat从0.30 s-1增至1.2 s-1，相应地二级速率常数kcat/Km增加了70倍。SDS-聚丙烯酰胺凝胶电泳显示，scu-PA被凝血酶失活后裂解为两条链，证实了血栓调节蛋白对scu-PA失活的加速作用。因此，血栓调节蛋白不仅具有抗凝特性，还具有抗纤溶特性。这种加速作用可能意味着细胞表面局部纤溶酶原激活剂活性调节的一种新机制。
