THROMBIN ADHESIVE PROPERTIES - INDUCTION BY PLASMIN AND HEPARAN-SULFATE
THROMBIN ADHESIVE PROPERTIES - INDUCTION BY PLASMIN AND HEPARAN-SULFATE
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DOI：
10.1083/jcb.123.5.1279
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发表时间：
1993-12-01
影响因子：
7.8
通讯作者：
VLODAVSKY, I
中科院分区：
文献类型：
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作者：
BARSHAVIT, R;ESKOHJIDO, Y;VLODAVSKY, I
我们以前已经证明，化学修饰的凝血酶制剂诱导内皮细胞（EC）的粘附，扩散和细胞骨架重组通过精氨酸-甘氨酸-天冬氨酸（RGD）序列和α（V）β 3整合素。然而，天然凝血酶没有表现出粘附特性，与晶体结构分析一致，表明RGD表位的Gly-Asp残基被埋在分子内。我们现在已经确定了一种可能的生理手段，将凝血酶转化为粘附蛋白。纤溶酶是纤溶系统的主要终产物，它以时间和剂量依赖的方式将凝血酶转化为EC的粘附蛋白。EC粘附和扩散也诱导凝血酶与纤溶酶孵育后产生的低分子量（约3,000 D）的切割片段。该片段介导的细胞粘附被合成肽GRGDSP完全抑制。在肝素或硫酸乙酰肝素的存在下，凝血酶向粘附分子的转化显著增强，而其他糖胺聚糖（GAG）（例如，硫酸皮肤素、硫酸角质素、硫酸软骨素）没有效果。使用GAG合成各方面缺陷的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EC粘附蛋白中的作用。凝血酶和次优量的纤溶酶在甲醛固定的野生型CHO-KI细胞表面上的孵育导致凝血酶有效转化为粘附分子，如随后诱导EC附着所示。相比之下，凝血酶和纤溶酶与固定的缺乏硫酸乙酰肝素和硫酸软骨素的CHO突变体细胞或与不表达硫酸乙酰肝素且硫酸软骨素增加三倍的细胞孵育没有影响。凝血酶和纤溶酶与天然接触孵育后，获得了类似的粘附性能的增益，但不是肝素酶处理的细胞外基质（ECM）培养的EC产生。看来，细胞表面和ECM相关的硫酸乙酰肝素通过其肝素结合位点以使次优量的纤溶酶暴露RGD结构域的方式调节凝血酶粘附特性。我们的研究结果表明，第一次，肝素的凝血酶分子的显着调制，导致其转化为一个有效的粘附蛋白EC。这种转化在与细胞表面、基底膜和ECM接触时最有效。
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)beta3 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.