Hypertriglyceridemic VLDL decreases plasminogen binding to endothelial cells and surface-localized fibrinolysis.
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 micrograms/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 Bmax for 125I-labeled Glu-Pmg ligand binding approximately 35% [(2.11 +/- 0.39)-(1.40 +/- 0.32) x 10(6) sites/cell, p < 0.005] and increased the Kd, app approximately 5-fold (0.32 +/- 0.03 to 1.74 +/- 0.08 microM, 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 approximately 2.7-fold (0.317 +/- 0.023 vs 0.869 +/- 0.068 nM s-1, p < 0.01) and approximately 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 Ki for inhibition of approximately 10 micrograms/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.
Li
X N
XN
Department of Medicine, University of Alabama at Birmingham 35294-2170, USA.
Koons
J C
JC
Benza
R L
RL
Parks
J M
JM
Varma
V K
VK
Bradley
W A
WA
Gianturco
S H
SH
Taylor
K B
KB
Grammer
J R
JR
Tabengwa
E M
EM
Booyse
F M
FM
D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005342:Fibrinolysis; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000097:blood*; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding
D007457:Iodine Radioisotopes; D008079:Lipoproteins, VLDL; D010958:Plasminogen