McGowan E B, Yeo K T, Detwiler T C
Arch Biochem Biophys. 1983 Nov;227(1):287-301. doi: 10.1016/0003-9861(83)90373-9.
The action of exogenous calcium-dependent protease (CDP) on tritium-labeled surface glycoproteins was analyzed by incubation of labeled, washed human platelets with CDP partially purified from human platelets. Labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Incubation of the labeled platelets with the protease led to a loss (calcium-dependent) from the platelets of glycoproteins Ib and V and concomitant appearance in the supernatant solution of glycocalicin (a proteolytic fragment of glycoprotein Ib), glycoprotein V, and other, unidentified glycoproteins. These changes in surface label were accompanied by alterations in three parameters of platelet function. Compared to control platelets, the CDP-treated platelets were activated by thrombin more slowly and showed less saturable and nonsaturable binding of thrombin. The CDP-treated platelets, but not the controls, aggregated on addition of fibrinogen, indicating that treatment with CDP had exposed fibrinogen receptors. The alterations in surface glycoproteins and functional parameters were compared over a 1000-fold range of CDP treatment. The decreased binding of thrombin and the exposure of fibrinogen receptors were correlated with the release of surface glycoproteins to the supernatant solution, but the slow activation by thrombin was observed under conditions where no release of labeled glycoproteins was detected (i.e., brief incubations with low concentrations of CDP). Activation of the endogenous CDP with 2.5 mM calcium chloride plus the ionophore A23187 was accompanied by hydrolysis of actin-binding protein, a known substrate, and release to the supernatant solution of labeled glycocalicin and glycoprotein V plus a faster-migrating glycoprotein not released by exogenous protease. This effect was observed in the presence of leupeptin, which completely inhibited action of exogenous protease, suggesting that platelet calcium-dependent protease may modify the platelet surface in ways that can cause alterations of platelet function.
通过将标记并洗涤过的人血小板与从人血小板中部分纯化的钙依赖性蛋白酶（CDP）共同孵育，分析了外源性钙依赖性蛋白酶（CDP）对氚标记的表面糖蛋白的作用。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，随后进行荧光自显影来分析标记的糖蛋白。用蛋白酶孵育标记的血小板导致糖蛋白Ib和V从血小板中（钙依赖性地）丢失，并伴随糖链蛋白（糖蛋白Ib的蛋白水解片段）、糖蛋白V和其他未鉴定的糖蛋白出现在上清液中。表面标记的这些变化伴随着血小板功能三个参数的改变。与对照血小板相比，经CDP处理的血小板被凝血酶激活得更慢，并且显示出对凝血酶的可饱和和不可饱和结合较少。经CDP处理的血小板，而不是对照血小板，在添加纤维蛋白原时发生聚集，表明用CDP处理暴露了纤维蛋白原受体。在1000倍的CDP处理范围内比较了表面糖蛋白和功能参数的变化。凝血酶结合减少和纤维蛋白原受体的暴露与表面糖蛋白释放到上清液中相关，但在未检测到标记糖蛋白释放的条件下（即与低浓度CDP短暂孵育）观察到凝血酶激活缓慢。用2.5 mM氯化钙加离子载体A23187激活内源性CDP伴随着肌动蛋白结合蛋白（一种已知底物）的水解，以及标记的糖链蛋白和糖蛋白V加上一种未被外源性蛋白酶释放的迁移更快的糖蛋白释放到上清液中。在亮抑酶肽存在下观察到这种效应，亮抑酶肽完全抑制外源性蛋白酶的作用，表明血小板钙依赖性蛋白酶可能以能够导致血小板功能改变的方式修饰血小板表面。
