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

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 diisopropylfluoro-phosphate-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 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 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.

Polyphenolics Increase t‐PA and u‐PA Gene Transcription in Cultured Human Endothelial Cells

“…HUVECs were obtained from fresh umbilical cords by mild collagenase treatment (Haddock et al, 1991;Jaffe et al, 1973;Li et al, 1995). HUVECs were seeded into plastic Petri dishes (9.6 cm 2 ) coated with human fibronectin and grown to confluency in complete 10% fetal bovine serum-containing culture medium, as we have described in detail previously (Aikens et al, 1997a,b;Booyse et al, 1981Booyse et al, , 1988.…”

Dependence of human vascular cell surface proteolysis on expression of the urokinase receptor

“…A growing body of information supports the concept that receptor-bound uPA plays a pivotal role in cell surface-localized generation of plasmin [1,18]. Thus, the invasive potential of uPAR-producing cells can be increased by uPA provided by neighboring cells [19].…”

Hypertriglyceridemic VLDL Downregulates Tissue Plasminogen Activator Gene Transcription Through cis -Repressive Region(s) in the Tissue Plasminogen Activator Promoter in Cultured Human Endothelial Cells

“…4,13 Because early-passage (passages 1 and 2) HUVECs do not synthesize urokinase, the results of this particular assay are largely attributed to the effects of tPA. 36 Postconfluent cultured HUVECs in 96-well plates (in triplicate) were incubated at 37°C for 8 hours in the absence/presence of HTG-VLDL or NTG-VLDL (20 g/mL) in serum-free culture medium, followed by removal of lipoproteins and further incubation in complete culture medium for an additional 16 hours.…”

“…ECs synthesize tPA and uPA as well as the major physiological regulator and inhibitor of PA-mediated fibrinolytic activity, Pmg activator inhibitor type (PAI)-1. 4,5 Therefore, it is conceivable that perturbation of the expression or activity of Ն1 of these fibrinolytic proteins may alter the hemostatic balance on the EC surface, thus promoting early initiation of fibrin deposition, atherogenesis, CAD, and eventual MI. 1 Several atherogenic lipoproteins may cause such a perturbation; eg, Lp(a), 6 oxidized LDL, 7,8 and acetylated LDL 9 increase PAI-1 levels and decrease tPA expression in cultured human ECs.…”