Endothelial cell (EC) fibrinolysis is important in maintaining normal hemostasis and requires the regulated activation of plasminogen (pmg) on the EC surface. This process involves the synthesis, expression and complex multicomponent interactions of plasminogen activators (PAs), PAI-1 and PAs/Pmg receptor or binding proteins (Rs). Systemic factors that may impair EC surface-localized fibrinolysis would shift the hemostatic balance towards clot formation, promote the early initiation of coronary artery thrombosis and hence play a pivotal role in the pathogenesis of atherosclerosis, coronary artery disease, myocardial infarction (MI). The overall goal of these studies is to identify and define further the mechanistic link(s) between abnormal triglyceride-rich lipoproteins (TGRLP), in particular hypertriglyceridemiC(HTG-)-VLDL and postprandial[pp] TGRLP), as potential inducers of impaired EC fibrinolytic function, and the molecular mechanisms that underlie the early initiation of coronary artery thrombosis and atherogenesis. Specifically, the effects of abnormal TGRLP (including ppTGRLP and normal VLDL, LDL, HDL controls) will be examined on: the synthesis, activity, levels of PAs, PAI-1, PARs and PmgR in different cultured EC types, and whether TGRLP-induced effects on EC fibrinolysis correlate with changes in cellular lipid content (plasmin assays, ELISAs, SDS-PAGE/125I-fibrin plate assays, reverse transcriptase-PCR [RT-PCR] and Northern blot analysis, Scatchard analysis)(Aim 1); and surface-localized plasmin generation, including Pmg and scu-PA activation kinetics (K[m], k[cat], k[cat]/K[m]), PAPs/PmgR distribution and interrelationships (fluorogenic substrate/125I-Pmg activation analysis, immunofluorescence staining)(Aim 2). Individual human BC cultures will be PAI-1 genotyped, using a HindIII restriction fragment length polymorphism and (C-A)n dinucleotide repeat polymorphism as genetic markers, and the hypothesized relationship between PAI-1 genotype, PAI-1 levels and VLDL regulation examined in this culture model system (RT-PCR and Southern blot analysis, ELISAs, plasmin assays)(Aim 3). Finally, the altered expression of fibrinolytic proteins, Rs and activity in cultured BC morphologic phenotypes (giant vs small) will be defined and the presence of a giant EC precursor phenotype identified and isolated (flow cytometry, TGRLP ligand binding, plasmin assays)(Aim 4). These results will provide significant new insights into our understanding of the molecular mechanisms by which TGRLP may impair EC fibrinolysis and hence contribute to the early initiation of coronary artery thrombosis, atherogenesis, and myocardial infarction.
