Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III.
Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III.
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DOI：
10.1006/abbi.1998.0963
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发表时间：
1999
影响因子：
3.9
通讯作者：
Y. Milev;D. Essex
中科院分区：
文献类型：
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作者：
Y. Milev;D. Essex
最近的研究表明，血小板表面存在一种蛋白质二硫键异构酶（PDI）并由其分泌，这提出了参与止血和伤口愈合的蛋白质也是这种酶的底物的可能性。在本研究中，使用血小板 PDI、血小板反应蛋白-1 (TSP)、α-凝血酶和抗凝血酶 III (AT) 的纯化制剂来证明 PDI 催化 TSP-凝血酶-AT 复合物的形成，与之前的上清液血小板活化结果一致。 1.25 微克/毫升浓度的 PDI 足以将近 50% 的凝血酶转化为 TSP-凝血酶-AT 复合物。复合物的形成需要低浓度的还原硫醇，N-乙基马来酰亚胺可以阻止该反应。该络合物被还原剂如巯基乙醇解离。不含 Ca2+ 且添加 EDTA 增加了复合物形成的速率，表明不含 Ca2+ 形式的 TSP 最有效。在不存在AT的情况下，形成少量的TSP-凝血酶复合物，其仅为在存在AT的情况下最大复合物形成的0-13%。该结果与显示凝血酶-AT复合物快速形成然后转化为三元复合物的动力学研究相结合，表明凝血酶-AT复合物是反应中必需的中间体。在最佳条件下，超过 70% 的凝血酶在 60 分钟内融入复合物中。肝素主要通过促进凝血酶-AT复合物的形成来加速反应，而对TSP影响不大。 PDI 与活化血小板上清液中的 TSP 共沉淀，表明 PDI 与其底物之间存在关联。总之，这些数据与 PDI 催化 TSP 与其他蛋白质形成二硫键连接的复合物的作用一致。
The recent demonstration of a protein disulfide isomerase (PDI) on the surface of and secreted from blood platelets raises the possibility that proteins involved in hemostasis and wound healing are also substrates of this enzyme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), alpha-thrombin, and antithrombin III (AT) were used to demonstrate that PDI catalyzes formation of a TSP-thrombin-AT complex consistent with previous results with supernatant platelet activation. Concentrations of 1.25 microg/ml of PDI were sufficient to convert almost 50% of thrombin to TSP-thrombin-AT complex. Complex formation requires low concentrations of a reduced thiol and the reaction can be prevented by N-ethymaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2+ and the addition of EDTA increased the rate of complex formation, indicating that TSP in the Ca2+-free form is most effective. In the absence of AT a small amount of TSP-thrombin complex formed which was only 0-13% of maximal complex formation in the presence of AT. This result, in combination with kinetic studies showing rapid formation of thrombin-AT complex followed by conversion to ternary complex, suggests that the thrombin-AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 70% of the thrombin is incorporated into the complex in 60 min. Heparin accelerated the reaction largely by enhancing formation of thrombin-AT complexes and had little effect on TSP. PDI coprecipitated with TSP from the supernatant solution of activated platelets, suggesting an association between PDI and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other proteins.