Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells.
Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells.
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DOI：
10.1016/s0021-9258(17)42691-3
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发表时间：
1984-12
期刊：
影响因子：
--
通讯作者：
J. V. van Mourik;D. Lawrence;D. Loskutoff
中科院分区：
文献类型：
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作者：
J. V. van Mourik;D. Lawrence;D. Loskutoff
培养的牛主动脉内皮细胞与异常稳定的纤维蛋白溶解抑制剂相关（Loskutoff，D. J.，货车穆里克，J.A.，洛杉矶的埃里克森和Lawrence，D.等人（1983）Proc. Acad. Sci. U.S.A.80，2956-2960）。这种抑制剂纯化到明显的同质性，从这些细胞的条件培养基的结合刀豆球蛋白A亲和层析和制备十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。它是一种单链糖蛋白，表观Mr为50，000 +/-2，500，等电点为4.5-5.0，抑制尿激酶和组织型纤溶酶原激活剂切割和激活纤溶酶原的能力。纤溶酶原激活物活性的这种抑制与酶-抑制剂复合物的形成有关，该复合物可在十二烷基硫酸钠存在下经聚丙烯酰胺凝胶电泳检测。在0.1%十二烷基硫酸钠存在下或在pH 2.7下孵育后，纯化的抑制剂保留全部活性，这两种处理迅速破坏蛋白酶连接蛋白（另一种纤维蛋白溶解的细胞抑制剂）的活性。在L-[3，4，5 - 3 H]亮氨酸存在下培养的克隆内皮细胞中纯化的抑制剂占24小时内细胞释放的总放射性标记蛋白的2.5-12%。这些结果表明，培养的牛主动脉内皮细胞合成和分泌的蛋白质，抑制纤溶酶原激活剂，是不同于蛋白酶连接蛋白。它是一种主要的内皮细胞产物，因此可能在调节这些细胞的纤溶系统中起重要作用。
Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.