The effect of normo (NTG)- and hypertriglyceridemic (HTG)-VLDL on cultured human umbilical vein endothelial cell (HUVEC) surface-localized fibrinolysis was examined following pre-incubation with NTG-, HTG-VLDL, LDL (1−20 μg/mL) or buffer (control). Ligand binding assays, using 125I-labeled tcu-PA, t-PA, or Glu-plasminogen (Glu-Pmg) were carried out in the absence/presence of lipoproteins. Scatchard analyses showed that HTG-VLDL decreased the B max for 125I-labeled Glu-Pmg ligand binding ∼35% (2.11 ± 0.39)−(1.40 ± 0.32) × 106 sites/cell, p < 0.05 and increased the K d,app ∼5-fold (0.32 ± 0.03 to 1.74 ± 0.08 μM, p < 0.01), while NTG-VLDL, LDL, and buffer had no effect. 125I-labeled PA ligand binding was unaffected by these lipoproteins. Receptor-bound PA activation of cell-bound 125I-labeled Glu-Pmg was measured by quantitation of either the M r 20 kDa light- or M r 60 kDa heavy-chain of 125I-labeled plasmin, following SDS−PAGE. Kinetic analysis of these data (HTG-VLDL vs controls) indicated that HTG-VLDL decreased the V max of tcu-PA- and t-PA-mediated activation of plasminogen ∼2.7-fold (0.317 ± 0.023 vs 0.869 ± 0.068 nM s-1, p < 0.01) and ∼2.9-fold (0.391 ± 0.098 vs 1.152 ± 0.265 nM s-1, p < 0.01), respectively. Increasing concentrations 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 ∼10 μg/mL. The combined ligand binding and kinetic data best fit an uncompetitive inhibition model in which the binding of the large HTG-VLDL particle to the EC surface may directly affect Glu-Pmg binding and activation, thus contributing to early fibrin deposition and the increased thrombotic risk associated with HTG.