Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.
We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.
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Sznycer-Laszuk
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D002384:Catalysis; D002478:Cells, Cultured; D003470:Culture Media / Q000032:analysis; D004727:Endothelium / Q000201:enzymology / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015849:Plasminogen Inactivators; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis; D013917:Thrombin / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000096:biosynthesis
D003470:Culture Media; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D012313:RNA; D010960:Plasminogen Activators; D013917:Thrombin; D010959:Tissue Plasminogen Activator