Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.
Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.
复制标题
凝血酶与重组人血栓调节蛋白的平衡结合：水蛭素、纤维蛋白原、Va 因子和肽类似物的作用。
DOI：
10.1021/bi00499a005
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Sadler,JE
中科院分区：
文献类型：
--
作者：
Tsiang,M;Lentz,SR;Dittman,WA;Wen,D;Scarpati,EM;Sadler,JE
霍华德休斯医学研究所，血液肿瘤学分部，医学系，生物化学和分子生物物理学系，华盛顿大学医学院，圣刘易斯，密苏里州63110接收日期：1990年1月8日;修订后的《指南》接收日期：1990年6月14日摘要：血栓调节蛋白是凝血酶的内皮细胞表面受体，可作为生理抗凝剂。在COS-7、CHO、CV-1和K562细胞系中研究了重组人血栓调节蛋白的性质。凝血酶调节蛋白在细胞表面表达，如通过获得凝血酶依赖性蛋白C活化所示。与天然血栓调节蛋白一样，重组血栓调节蛋白含有N-连接寡糖，MT约为100 000，并可被抗血栓调节蛋白抗体抑制或免疫沉淀。结合研究表明，由A549癌细胞表达的非重组血栓调节蛋白和由CV-1和K562细胞表达的重组血栓调节蛋白对凝血酶具有相似的Kf s，分别为1.3 nM、3.3 nM和4.7 nM。DIP-凝血酶与CV-1（18 A）细胞上重组血栓调节蛋白结合的Kd与凝血酶的Kd相同。水蛭素或纤维蛋白原浓度的增加逐渐抑制125 I-DIP-凝血酶的结合，而因子Va不抑制结合。测试三种合成肽抑制DIP-凝血酶结合的能力。水蛭素肽Hir 53 -64和血栓调节素第五-EGF结构域肽Tm 426 -444都从血栓调节素中取代了DIP-凝血酶，但与Hir 53 '* 4组成和电荷相似的因子V肽FacV 30 -43没有表现出结合抑制作用。这些数据排除了由凝血酶、血栓调节蛋白和水蛭素组成的三元复合物的显著形成。这些研究与血栓调节蛋白、水蛭素和纤维蛋白原在同一位点竞争结合DIP-凝血酶的模型一致。血栓调节蛋白是凝血丝氨酸蛋白酶凝血酶的内皮细胞表面受体（Esmon和Owen，1981）。凝血酶-血栓调节蛋白复合物激活蛋白C的速度至少比单独凝血酶快1000倍
Howard Hughes Medical Institute, Division of Hematology-Oncology, Department of Medicine, and Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110 Received January 8, 1990; Revised Manuscript Received June 14, 1990 abstract: Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 celllines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had MT~ 100 000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinomacells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kf s for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1 (18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 andthe thrombomodulinfifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53"* 4 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site. rombomodulin is an endothelial cell surface receptor for the blood clotting serine protease thrombin (Esmon & Owen, 1981). The thrombin-thrombomodulin complex activates protein C at least 1000-fold more rapidly than thrombin alone