Pro-thrombotic State Induced by Post-translational Modification of Fibrinogen by Reactive Nitrogen Species

Abstract

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.Epidemiological studies have indicated that increased levels of circulating fibrinogen is an independent predictor of coronary heart disease and in some cases of premature death from cardiovascular disease, although a causative relationship between high levels of fibrinogen and cardiovascular disease has not been firmly established (1-4). Fibrinogen is a multifunctional protein essential for hemostasis. It is a 340-kDa glycoprotein, consisting of three non-identical peptide chains A␣, B␤, and ␥, which are linked together by 29 disulfide bonds (5). During coagulation, the soluble fibrinogen is converted to insoluble fibrin polymers. The process is initiated by thrombin, a serine protease, which catalyzes the cleavage first of two fibrinopeptides from the amino termini of the A␣ chains and then two fibrinopeptides from the amino termini B␤ chains. Upon release of the fibrinopeptides, the remaining fibrin monomers aggregate spontaneously to form ordered fibrin polymers (5). The clot is stabilized by the formation of covalent bonds introduced by the action of a transglutaminase, factor XIII (6). Under physiological conditions, fibrinolysis is dependent on the binding of circulating plasminogen and tissue-type plasminogen activator (tPA) 1 to fibrin clots. Urokinase and tPA convert plasminogen to the active protease plasmin, which then cleaves fibrin polymers to soluble fragments completing the coagulation and clot resolution cycle.A major cause...

Nitric oxide, oxidants, and protein tyrosine nitration

“…An alternative scenario is a gain-of-function modification, in which case a small fraction of nitrated protein can elicit a substantive biological signal. This attractive concept has been shown in a few proteins such as cytochrome c, which acquires a strong peroxidase activity after nitration (66), in nitrated fibrinogen, which accelerates clot formation (67) and in protein kinase C , which becomes activated and translocates on nitration (68).…”

Human Atherosclerotic Intima and Blood of Patients with Established Coronary Artery Disease Contain High Density Lipoprotein Damaged by Reactive Nitrogen Species

“…Moreover, elevated levels of nitrated plasma proteins associate with an increased risk of coronary artery disease, suggesting that oxidants derived from NO might modify circulating proteins or proteins that find their way into the bloodstream (30). Recent studies revealed that fibrinogen is one target for nitration in plasma (31). Also, exposing fibrinogen to nitrating oxidants in vitro accelerated clot formation, suggesting that fibrinogen nitration in vivo might be prothrombotic (31).…”

“…Recent studies revealed that fibrinogen is one target for nitration in plasma (31). Also, exposing fibrinogen to nitrating oxidants in vitro accelerated clot formation, suggesting that fibrinogen nitration in vivo might be prothrombotic (31). However, it is unclear whether NO and superoxide produced at physiologically plausible rates nitrate tyrosine efficiently in vitro (32,33), leaving open the possibility that 3-nitrotyrosine has a different origin in vivo.…”

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

“…This means that protein nitration can affect enzyme activity, protein-protein interaction and phospho-tyrosine signalling pathways [6][7][8][9][10][11][12].…”