Wu Q, Tsiang M, Lentz S R, Sadler J E
Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri 63110.
J Biol Chem. 1992 Apr 5;267(10):7083-8.
Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
血栓调节蛋白是一种内皮糖蛋白，作为蛋白C激活的辅因子。为了研究人血栓调节蛋白的配体特异性，我们用人凝血酶、凝血酶S205A（其中活性位点丝氨酸被丙氨酸取代）、中凝血酶S205A和人因子Xa进行了平衡结合试验。在用表达细胞表面重组人血栓调节蛋白的CV-1(18A)细胞进行的竞争结合试验中，重组野生型凝血酶和凝血酶S205A以相似的亲和力抑制125I-二异丙基氟磷酸酯-凝血酶结合（Kd分别为6.4±0.5和5.3±0.3 nM）。然而，未检测到中凝血酶S205A或人因子Xa的结合抑制作用（Kd大于500 nM）。在直接结合试验中，125I标记的血浆凝血酶和凝血酶S205A与血栓调节蛋白结合，Kd值分别为4.0±1.9和6.9±1.2 nM。125I标记的中凝血酶S205A和人因子Xa未与血栓调节蛋白结合（Kd大于500 nM）。我们还比较了凝血酶和因子Xa激活人重组蛋白C的能力。在血栓调节蛋白存在的情况下，凝血酶对重组蛋白C的激活作用大大增强，而无论有无血栓调节蛋白，均未检测到因子Xa的显著激活作用。当用人脐静脉内皮细胞作为血栓调节蛋白的来源时，凝血酶和因子Xa也得到了类似的结果。这些结果表明，人源中凝血酶和因子Xa不太可能是重要的依赖血栓调节蛋白的蛋白C激活剂，凝血酶是人类内皮细胞血栓调节蛋白的生理配体。
