| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1988: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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.
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