Fibrinogen β-Chain Tyrosine Nitration Is a Prothrombotic Risk Factor

Abstract

Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the ␤-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob "B" mimetic peptides Gly-His-Arg-Pro am and Ala-His-Arg-Pro am suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.Activation of the coagulation cascade converts soluble fibrinogen to insoluble fibrin, which polymerizes to produce, along with platelets, the hemostatic clot (1, 2). Whereas the normal activation of the coagulation cascade is essential for life, inappropriate activation may result in thrombosis and complications that arise from the formation of fibrin clots. Indeed pathologically induced thrombogenesis is associated with adverse cardiovascular events (3, 4), thromboembolism in chronic obstructive pulmonary disease (5), and vascular complications in autoimmune diseases (6). Because fibrinogen is an acute phase reactant, epidemiological studies have documented elevated fibrinogen levels in subjects with these disorders (7-10). Moreover, increased levels of circulating fibrinogen (11, 12) and common polymorphisms (13, 14) have been associated with an increased risk for thromboembolic episodes in subjects with coronary artery disease and in some cases of premature death from cardiovascular disease. Despite these established associations, a causative correlation between high levels of fibrinogen or polymorphisms and cardiovascular disease has not been firmly documented.Recently it was shown that the levels of proteins modified on tyrosine residues to form 3-nitrotyrosine, a protein marker of nitric oxide-derived reactive nitrogen species, are enhanced in the plasma of coronary artery disease patients and independently predict risk for coronary artery disease (15). Using affinity capture of fibrinogen followed by stable isotope, liquid chromatography (LC) 3 /tandem mass spectrometry (LC/MS/MS) quantification we reported increased levels of nitrated fibrinogen in...

Fibrin Clot Structure and Function

“…68 Nitration of 2 ␤-chain tyrosines increases fibrin formation and stiffness, impairs clot lysis, and alters fibrin structure. 69 Data on the association between oxidative stress markers and fibrin clot properties in vivo are scarce. F 2 -isoprostanes, produced on nonenzymatic arachidonic acid peroxidation and a stable marker of oxidative stress, have been shown to associate with reduced clot permeability and fibrinolysis in cardiovascular patients.…”

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

“…The modification is of great interest as it may be used as a diagnostic biomarker for diseases caused by radical species [4 -6]. Examples include cardiovascular disease [7], Alzheimer's disease [8], and atherothrombotic diseases [9]. The development of methods for the identification of tyrosine-nitrated proteins and characterization of sites of 3-nitrotyrosine are key for the understanding of the associated biological processes.…”

“…The ECD and CID of the synthetic peptides GPLEnYGFAK, GPLEnYGFAKGPLAK, the synthetic fibrinogen ␤-chain peptide NYCGLPGEnYWLGNDK (known to be susceptible to tyrosine nitration at this site in vivo [9]), the myoglobin tryptic peptide nYLEFISDAIIHVLHSK (nY denotes 3-nitrotyrosine), and their unmodified counterparts were determined. The results show that for doubly-charged peptide ions the presence of 3-nitrotyrosine has a deleterious effect on ECD backbone cleavage.…”

The comparison of peroxynitrite action on bovine, porcine and human fibrinogens

“…Peroxynitrite induces changes in the function and the structure of human fibrinogen in vitro, generating nitrotyrosine, mostly in Aa-chain, acting on aC subunits [43], which in most vertebrates are natively unfolded regions of molecule with no stable structure [29]. Fibrinogen nitration in vivo results in increased velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness and reduced rate of fibrin clot lysis by plasmin [44]. Significantly increased nitration of fibrinogen has been reported in cardiovascular diseases [45].…”