THROMBIN DECREASES THE UROKINASE RECEPTOR AND SURFACE-LOCALIZED FIBRINOLYSIS IN CULTURED ENDOTHELIAL-CELLS

The endothelial cell (EC) urokinase receptor plays an important role i n the localization and receptor-mediated activation of EC-bound plasmi nogen and hence surface-localized fibrinolysis. Thrombin induced a rap id (<5 minute), time (0 to 30 minutes) and dose- (0.1 to 8 U/mL) depen dent decrease in the specific binding of I-125-labeled two-chain uroki nase-type plasminogen activator (tcu-PA) or diisopropylfluorophosphate -tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cu ltured ECs from various sources (range, 21% to 50%). The thrombin rece ptor activation peptide but not control peptide showed a similar but r educed decrease in the specific binding of I-125-labeled tcu-PA to u-P AR. Incubation of thrombin-treated cultures (10 to 12 hours) in comple te medium restored I-125-labeled tcu-PA ligand binding to normal level s. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 ho urs after thrombin treatment as analyzed by reverse transcriptase-poly merase chain reaction. Decreased thrombin-induced I-125-labeled tcu-PA binding correlated with the time-dependent decrease in surface-locali zed plasmin generation, as measured by the direct activation of I-125- labeled Glu-plasminogen and quantification of the 20-kD light chains o f I-125-labeled plasmin. After incubation with thrombin, plasmin gener ation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5X10(4) cells). Isolation of metabolically labeled S-35-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximate to 10- and approximate to 12-fold more 55-kD M(r), and app roximate to-6-fold more 35-kD M(r) S-35-labeled u-PAR forms than contr ol cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and t he glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) w ere also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway invol ving phospholipase C. These results establish a novel interrelation be tween thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-lo calized EC fibrinolytic activity.