Bar-Shavit R, Eskohjido Y, Fenton J W, Esko J D, Vlodavsky I
Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel.
J Cell Biol. 1993 Dec;123(5):1279-87. doi: 10.1083/jcb.123.5.1279.
We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.
我们之前已经证明，化学修饰的凝血酶制剂通过精氨酸-甘氨酸-天冬氨酸（RGD）序列和αvβ3整合素诱导内皮细胞（EC）黏附、铺展和细胞骨架重组。然而，天然凝血酶不具有黏附特性，这与晶体结构分析一致，表明RGD表位的甘氨酸-天冬氨酸残基埋藏在分子内部。我们现在已经确定了一种将凝血酶转化为黏附蛋白的可能生理方式。纤溶酶是纤维蛋白溶解系统的主要终产物，它以时间和剂量依赖的方式将凝血酶转化为对内皮细胞有黏附作用的蛋白。凝血酶与纤溶酶孵育时产生的低分子量（约3000 D）裂解片段也能诱导内皮细胞黏附与铺展。该片段介导的细胞黏附被合成肽GRGDSP完全抑制。在肝素或硫酸乙酰肝素存在的情况下，凝血酶向黏附分子的转化显著增强，而其他糖胺聚糖（GAGs）（如硫酸皮肤素、硫酸角质素、硫酸软骨素）则无此作用。利用在GAG合成各方面存在缺陷的CHO细胞突变体研究了细胞表面硫酸乙酰肝素在凝血酶转化为内皮细胞黏附蛋白过程中的作用。在甲醛固定的野生型CHO-K1细胞表面孵育凝血酶和次优量的纤溶酶，随后内皮细胞黏附的诱导表明凝血酶有效地转化为了黏附分子。相比之下，在缺乏硫酸乙酰肝素和硫酸软骨素的固定CHO突变细胞，或不表达硫酸乙酰肝素但硫酸软骨素增加三倍的细胞中孵育凝血酶和纤溶酶则没有效果。在与培养的内皮细胞产生的天然而非肝素酶处理的细胞外基质（ECM）接触时孵育凝血酶和纤溶酶，也能获得类似的黏附特性增强。似乎细胞表面和与ECM相关的硫酸乙酰肝素通过其肝素结合位点调节凝血酶的黏附特性，使次优量的纤溶酶能够暴露RGD结构域。我们的结果首次证明了肝素对凝血酶分子有显著调节作用，使其转化为对内皮细胞有强大黏附作用的蛋白。这种转化在与细胞表面、基底膜和ECM接触时最为有效。
