Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin
Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin
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DOI：
10.1021/bi990694s
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发表时间：
1999-08-10
期刊：
影响因子：
2.9
通讯作者：
Feinman, RD
中科院分区：
文献类型：
--
作者：
Essex, DW;Miller, A;Feinman, RD
在本研究中，使用血小板蛋白二硫键异构酶（PDI）、玻连蛋白、α-凝血酶和抗凝血酶（AT）的纯化制剂来证明PDI催化玻连蛋白-凝血酶-AT复合物的形成。复合物的形成需要还原型谷胱甘肽（GSH），N-乙基马来酰亚胺可以阻止复合物的形成，并且形成的复合物可以通过还原剂（例如巯基乙醇）解离。在没有 AT 的情况下，不会形成玻连蛋白-凝血酶复合物，表明凝血酶-AT 复合物是反应中的必然中间体。在最佳条件下，大部分凝血酶-AT 在 60 分钟内融入复合物中。 Thrombospondin-1，已知与凝血酶-AT 形成二硫键连接的复合物 [Milev, Y. 和 Essex, D. W. (1999) Arch.生物化学。生物物理学。 361, 120-126]，在低 Ca2+ 环境中与玻连蛋白竞争凝血酶-AT，有利于血小板反应蛋白的活性形式。这里提出的结果也可以解释之前的研究表明，玻连蛋白-凝血酶-AT 复合物在血浆（含有 PDI）中比在纯化蛋白（未使用 PDI）中形成得更好。我们能够从血浆中纯化出与血小板酶在免疫学上相同的 PDI。我们使用乱序 RNase 测定来证明添加的纯化 PDI 可以在血浆环境中发挥作用。 PDI 抑制剂可抑制血浆中复合物的形成。 PDI 在高 pH 值（生理范围附近）下以可溶形式从血小板表面释放，表明血浆 PDI 的来源。总之，这些研究表明 PDI 的功能是形成玻连蛋白与凝血酶-AT 的二硫键连接的复合物。
In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca2+ environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.