: A potential major cause of vascular injury leading to the development of atherosclerosis is oxidative stress. Oxidative stress is the result of overproduction of reactive species that overwhelms the cellular antioxidant capacity leading to inactivation of key cellular functions and ultimately to cell death. Proteins are major targets of reactive species, and nitration of tyrosine residues is a selective protein modification induced by reactive nitrogen species in human disorders as well as animal and cellular models of disease. We have discovered that circulating fibrinogen is nitrated in patients with acute respiratory distress syndrome (ARDS). Nitration of fibrinogen significantly altered the function of fibrinogen by accelerating the rate of fibrin clot formation producing of fibrin clot with abnormal structure upon electron microscopic examination. Fibrin deposits are abundant in the lungs of patients with ARDS, a common complication of hemorrhagic injury and sepsis, and likely the result of abnormal clotting rather than failure in the fibrinolytic pathways, which are functioning normally in ARDS patients. A number of epidemiological studies have indicated that high levels of circulating fibrinogen is an independent predictor of coronary heart disease and in some cases of premature death from cardiovascular and heart disease although a causative correlation between high levels of fibrinogen and cardiovascular disease has not been established. Based on these data we developed the hypothesis that plasma protein nitration is a marker of oxidative stress and independent predictor of coronary heart disease and that nitration of fibrinogen is a critical post-translational modification responsible for abnormal functioning of the hemostatic system in atherosclerosis. To test the critical aspects of this hypothesis we propose to: 1) Quantify by the use of LC-MS the levels of 3-nitrotyrosine, dityrosine, the oxidation product of tyrosine and 3- chlorotyrosine, a marker of inflammation, in plasma proteins of smokers and nicotine users (Project 1), subjects in the prospective study of progression in coronary plaque burden (Project 5), the Apobec-l/LDLR double knock out mouse (Project 1) and in the plasma of mice with altered ApoA-I levels (Project 5). Evaluate the degree of nitration of specific proteins, fibrinogen, LDL/ApoB-100, and determine the site(s) of tyrosine nitration by the use of the Proteomics Core. 2) Evaluate the effects of nitration and/or oxidation on the biochemical, biophysical and viscoelastic properties of fibrinogen and fibrin clots by the use of scanning and transmission electron microscopy, and a Plazek torsion pendulum. 3) Determine the effect of nitration on critical functional aspects of fibrinogen and fibrin clot, ADP-induced platelet aggregation, endothelial cell gene expression (Genomics Core), endothelial-inflammatory cell interaction (in collaboration with Project 3), and kinetics of fibrinolysis. Overall this project is focused on investigating the possible biochemical and biophysical mechanisms that may underline abnormalities in the hemostatic factors that regulate critical pro- and anti-thrombotic functions in human cardiovascular disorders.
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