Thrombin Decreases the Urokinase Receptor and Surface-Localized Fibrinolysis in Cultured Endothelial Cells

Abstract

Abstract The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (<5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125 I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluorophosphate–tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125 I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125 I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase–polymerase chain reaction. Decreased thrombin-induced 125 I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125 I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125 I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5×10 4 cells). Isolation of metabolically labeled 35 S-labeled u-PAR from the media of thrombin and phospholipase C–treated human aortic cultures yielded ≈10- and ≈12-fold more 55-kD M r and ≈6-fold more 35-kD M r 35 S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms ( M r , 54 kD) and the glycosyl-phosphatidylinositol–anchored protein CD59 ( M r , 20 kD) were 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 involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.