HYPERTRIGLYCERIDEMIC VLDL DECREASES PLASMINOGEN BINDING TO ENDOTHELIAL-CELLS AND SURFACE-LOCALIZED FIBRINOLYSIS

The effect of normo (NTG)- and hypertriglyceridemic (HTG)-VLDL on cult ured human umbilical vein endothelial cell (HUVEC) surface-localized f ibrinolysis was examined following preincubation with NTG-, HTG-VLDL, LDL (1-20 mu g/mL) or buffer (control). Ligand binding assays, using I -125-labeled tcu-PA, t-PA, or Glu-plasminogen (Glu-Pmg) were carried o ut in the absence/presence of lipoproteins. Scatchard analyses showed that HTG-VLDL decreased the B-max for I-125-labeled Glu-Pmg ligand bin ding similar to 35% [(2.11 +/- 0.39)-(1.40 +/- 0.32) x.10(6) sites/cel l, p < 0.05] and increased the K-d,K-app similar to 5-fold (0.32 +/- 0 .03 to 1.74 +/- 0.08 mu M, p < 0.01), while NTG-VLDL, LDL, and buffer had no effect. I-125-labeled PA ligand binding was unaffected by these lipoproteins. Receptor-bound PA activation of cell-bound I-125-labele d Glu-Pmg was measured by quantitation of either the M(r) 20 kDa light - or M(r) 60 kDa heavy-chain of I-125-labeled plasmin, following SDS-P AGE. Kinetic analysis of these data (HTG-VLDL vs controls) indicated t hat HTG-VLDL decreased the V-max of tcu-PA- and t-PA-mediated activati on of plasminogen similar to 2.7-fold (0.317 +/- 0.023 vs 0.869 +/- 0. 068 nM s(-1), p < 0.01) and similar to 2.9-fold (0.391 +/- 0.098 vs 1. 152 +/- 0.265 nM s(-1), p < 0.01), respectively. Increasing concentrat ions of the HTG-VLDL increased 1/V-max, yielding a series of parallel plots, typical for uncompetitive inhibition with a K-i for inhibition of similar to 10 mu g/mL. The combined ligand binding and kinetic data best fit an uncompetitive inhibition model in which the binding of th e large HTG-VLDL particle to the EC surface may directly affect Glu-Pm g binding and activation, thus contributing to early fibrin deposition and the increased thrombotic risk associated with HTG.