Covalent binding of thrombin to specific sites on corneal endothelial cells.
Covalent binding of thrombin to specific sites on corneal endothelial cells.
复制标题
凝血酶与角膜内皮细胞上的特定位点共价结合。
DOI：
10.1021/bi00505a027
复制
发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Shuman,MA
中科院分区：
文献类型：
--
作者：
Isaacs,JD;Savion,N;Gospodarowicz,D;Fenton2nd,JW;Shuman,MA
用NaDodS04(十二烷基硫酸钠)-聚丙烯酰胺凝胶电泳法证实[125I]凝血酶与这些细胞上的特定部位结合，形成77000-Dalton复合体。[125I]凝血酶的结合被100倍的未标记的a-凝血酶和凝血酶抑制剂水飞蓟素所阻断。在细胞表面有大约100000个这种凝血酶结合位点。复合体的形成早在15岁的S就可以检测到，在接下来的20-30分钟内迅速增加，然后以较慢的速度持续到接下来的2.5h。酶的催化活性部位是形成稳定的复合体所必需的，这从二异丙基磷氟化物灭活的凝血酶不能与这些细胞形成稳定的复合体证明。该络合物在NaDodS04中与1.0M羟胺解离，表明该酶与细胞结合部位有酰基连接。凝血酶-内皮细胞复合体与凝血酶-抗凝血酶III复合体的凝胶电泳图不同，肝素不能促进凝血酶-内皮细胞复合体的形成。其他凝血酶-细胞复合体也被鉴定出来，然而，它们只占与细胞结合的凝血酶总量的一小部分。这些观察表明，α-凝血酶能够与角膜内皮细胞特异性反应，形成稳定的NaDod-S04复合体，这需要具有催化活性的酶。凝血酶是一种丝氨酸蛋白酶，当凝血酶原在血液凝固过程中被激活时，就会形成凝血酶。尽管凝血酶对精氨酸键表现出有限的选择性特异性(Elmore，1973)，但它具有多种生物学效应。在开始凝血后，凝血酶激活因子V、VIII和XIII，并将可溶性纤维蛋白原转化为纤维蛋白凝块(Ware等人，1947；Therriault等人，1957；Buluk等人，1961；Mo-rawitz，1905)。除第VIII因子外，这些糖蛋白的激活已被证明是有限蛋白分解的结果(Buluk等人，1961；Nesheim等人，1979；Esmon，1979；Bettelheim&Bailey，1952；Lorand，1952)。凝血酶对几种不同类型的细胞也有影响。包括刺激血小板分泌和聚集(Grette，1962)、成纤维细胞有丝分裂(Chen&Buchanan，1975)和B淋巴细胞(Chen等人，1976)，以及由内皮细胞刺激前列环素(PGI2)的形成和释放(Weksler等人，1978)。凝血酶导致内皮细胞激活纤溶酶原的能力显著降低，从而导致纤溶作用(Loskutoff，1979；Fehrenbacher等，1979)。凝血酶启动这些细胞效应的机制尚不清楚。凝血酶与血小板(Tollefsen等，1974；Gangully，1974)、血管内皮细胞(Awbrey等，1979)和成纤维细胞(Carney&Cun-Ingham，1978)的可逆和特异性结合已被证明。凝血酶与血小板的结合被认为是诱导血小板分泌的第一步。尽管天然凝血酶和凝血酶都在其丝氨酸活性部位结合失活
Binding of [125I] thrombinto a specific site on these cells with formation of a 77 000-dalton complex was demonstrated by NaDodS04 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I] thrombin was blocked by a 100-fold excess of unlabeled a-thrombin and by the thrombin inhibitor, hirudin. There are~ 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodS04-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated inNaDodS04 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr «= 90 000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes {M,< 77 000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that a-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-S04-stable complex which requires the catalytically active enzyme. rombin, a serine proteinase, is formed when the zymogen prothrombin is activated during blood clotting. Although it exhibits a limited preferential specificity for arginyl bonds (Elmore, 1973), thrombin has diverse biological effects. After initiation of coagulation, thrombin activates factors V, VIII, and XIII and converts soluble fibrinogen to a fibrin clot (Ware et al., 1947; Therriault et al., 1957; Buluk et al., 1961; Mo-rawitz, 1905). With the exception of factor VIII, activation of these glycoproteins has been shown to be theresult of limited proteolysis (Buluk et al., 1961; Nesheim et al., 1979; Esmon, 1979; Bettelheim & Bailey, 1952; Lorand, 1952). Thrombin also has effects on several different types of cells. These include stimulation of plateletsecretion and aggregation (Grette, 1962), mitogenesis of fibroblasts (Chen & Buchanan, 1975) and B lymphocytes (Chen et al., 1976), and stimulation of prostacyclin (PGI2) formation and release by endothelial cells (Weksler et al., 1978). Thrombin causes a marked re-duction in the capacity of endothelial cells to activate plasminogen and hence fibrinolysis (Loskutoff, 1979; Fehrenbacher et al., 1979). The mechanism by whichthrombin initiates these cellular effects is unknown. Reversible and specific binding of thrombin to platelets (Tollefsen et al., 1974; Ganguly, 1974), vascular endothelial cells (Awbrey et al., 1979), and fibroblasts (Carney & Cun-ingham, 1978) has previously been demonstrated. The binding of thrombin to platelets has been proposed as the first step in the induction of platelet secretion. Although both native thrombin and thrombin inactivatedat its serine active site bind