Analysis of the mechanism of endothelial cell -dependent activation of plasminogen activator inhibitor

内皮细胞（ECS）是止血、血栓形成和纤维蛋白溶解的调节剂。它们不仅产生凝血因子，而且产生组织型纤溶酶原激活物（t-PA），其是纤维蛋白和内皮细胞表面上纤溶酶原的生理学上重要的激活物。此外，最近已经清楚的是，t-PA活性由EC产生的特异性、快速作用的纤溶酶原激活物抑制剂1（派-1）控制。有趣的是，在培养的EC条件培养基中发现了两种不同形式的派-1：快速作用的活性形式（仅几%）和20倍过量的无活性但可被变性剂激活的潜伏形式。在试管中，即使在条件培养基中加入大量t-PA，也只能观察到少量t-PA/派-1复合物的产生。然而，我们发现，增加浓度的t-PA汇合EC产生了一个饱和的，剂量依赖性的增加激活剂/派-1复合物。我们通过使用放线菌素D阻止合成、[^<35>S] EC蛋白质的甲硫氨酸标记、多孔底部培养皿在培养的EC系统中分析了这些结果结合我们的实验结果和以前的观察，我们可以得出结论：在t-PA和EC存在的情况下，潜伏的派-1不能被激活，而派-1作为活性形式，与内皮细胞表面或细胞外基质上的结合蛋白结合，PA与派-1的反应主要发生在内皮细胞上，结合蛋白的一种可能的候选物可以是条件培养基中的玻连蛋白/总派-1。然而，我们不能直接显示内皮细胞相关的玻连蛋白的免疫组化方法和玻连蛋白是一个非常粘蛋白。因此，我们现在试图确定受体蛋白和玻连蛋白的身份，并阐明派-1和玻连蛋白之间的结合位点。

Endothelial cells (ECS) are modulators of hemostasis, thrombosis and fibrinolysis. Not only they produce coagulation factors but they produce tissue type plasminogen activator (t-PA) which is physiologically important activator of plasminogen on the surface of fibrin and ECs. In addition, recently, it has become clear that t-PA activity is controlled by a specific, fast- acting plasminogen activator inhibitor 1 (PAI-1) which is produced by ECs. Interestingly, two different forms of this PAI-1 have been found in medium conditioned by cultured ECs: a fast-acting active form (only a few %) and 20-fold excess of latent form that is inactive but can be activated by denaturants. In a test fube, even when we added a large amount of t-PA to conditioned medium, we could observe only a small amount of t-PA/PAI-1 complex generated in a test tube. However, we found that the addition of increasing concentrations of t-PA to confluent ECs produced a satorable, dose-dependent increase of the activator/PAI-1 complex. We analyzed these results in cultured ECs system by using actinomycin D to prevent synthesis, [^<35>S] Methionine labeling of ECs proteins, porous bottom culture dish (Transwell) to prevent t-PA directly interact with proteins on the surface of ECs.Taking our results and previous observations, we can conclude that latent PAI-1 could not be activated in the presence of t-PA and ECs but PAI-1, as an active form, bound to some binding protein on the surface of ECs or the extracellular matrix and the reaction between PA and PAI-1 mainly occurs on the ECs.Furthermore, one possible candidate of binding proteins may be vitronectin/total PAI-1 in conditioned medium. However, we were unable to demonstrate ECs-associated vitronectin directly bt immunohistochemical method and vitronectin is a very sticky protein. So, we are now trying to determine the identity of receptor protein and vitronectin and to clarify the binding site between PAI-1 and vitronectin.