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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:410-419.)
© 1995 American Heart Association, Inc.

Articles

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

Xin-Nong Li; Vivek K. Varma; James M. Parks; Raymond L. Benza; Jay C. Koons; J. Robert Grammer; Hernan Grenett; Edlue M. Tabengwa; Francois M. Booyse

From the Division of Cardiovascular Disease, Department of Medicine, University of Alabama, Birmingham.

Correspondence to Francois M. Booyse, PhD, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, 809 BBRB, 845 19th St S, Birmingham, AL 35294-2170.

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 ¹²⁵I-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 ¹²⁵I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored ¹²⁵I-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 ¹²⁵I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of ¹²⁵I-labeled Glu-plasminogen and quantification of the 20-kD light chains of ¹²⁵I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5x10⁴ cells). Isolation of metabolically labeled ³⁵S-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 ³⁵S-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.

Key Words: thrombin • endothelial urokinase receptor • fibrinolysis • plasmin • ligand binding

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