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

内皮细胞是止血、血栓形成和纤溶的调节剂。它们不仅产生凝血因子，还产生组织型纤溶酶原激活物(t-PA)，它是纤维蛋白和内皮细胞表面重要的纤溶酶原激活剂。此外，最近发现，t-PA活性受内皮细胞产生的一种特定的、快速作用的纤溶酶原激活物抑制物1(PAI-1)控制。有趣的是，在培养的内皮细胞条件培养液中发现了两种不同的PAI-1形式：一种是快速作用的活性形式(只有几%)，另一种是20倍的潜伏形式，它是不活跃的，但可以被变性剂激活。在实验中，即使在条件培养液中加入大量的t-PA，我们也只能观察到在试管中产生少量的t-PA/PAI-1复合体。然而，我们发现，在融合的内皮细胞中加入浓度增加的t-PA会导致激活剂/PAI-1复合体饱和的、剂量依赖的增加。我们在培养的内皮细胞系统中使用放线菌素D来阻止合成，[^&lt；35&gt；[S]蛋氨酸标记内皮细胞蛋白，防止t-PA与内皮细胞表面的蛋白质直接相互作用。根据我们的研究结果和先前的观察，我们得出结论：t-PA和内皮细胞存在时，潜伏的PAI-1不能被激活，但PAI-1作为一种活性形式，与内皮细胞表面或细胞外基质上的某些结合蛋白结合，PA与PAI-1的反应主要发生在内皮细胞上。此外，条件培养液中可能的结合蛋白可能是Vitronectin/总PAI-1。然而，我们无法通过免疫组织化学方法直接证明内皮细胞相关的Vitronectin，而Vitronectin是一种非常粘性的蛋白质。因此，我们现在正试图确定受体蛋白和Vitronectin的同一性，并澄清PAI-1和Vitronectin之间的结合部位。

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.