Nitrated fibrinogen is a biomarker of oxidative stress in venous thromboembolism

Abstract

The pathogenesis of venous thromboembolism (VTE) is linked to inflammation and oxidant production, although specific markers for these pathways with pathological relevance to VTE have not been explored. The coagulant protein fibrinogen is posttranslationally modified by nitric oxide-derived oxidants to nitrated fibrinogen in both acute and chronic inflammatory states. Therefore, nitrated fibrinogen may serve as a marker of inflammation and oxidative stress in VTE. To test this hypothesis we enrolled subjects (n=251) presenting with suspected VTE at the University of Pennsylvania Hospital emergency department, 50 (19.9%) of whom were positive by imaging or 90-day follow-up. Mean nitrated fibrinogen was elevated in VTE-positive (62.7 nM, 95% CI 56.6–68.8) compared to VTE-negative patients (54.2 nM, 95% CI 51.4–57.1; P<0.01). Patients in the highest quartile of nitrated fibrinogen had an increased risk of VTE compared with patients in the lowest quartile (OR 3.30; 95% CI 1.25–8.68; P<0.05). This risk persisted after univariate adjustment for age, active cancer, and recent surgery, but not after multivariate adjustment. Mean fibrinogen levels measured either by the Clauss assay or by ELISA were not different between VTE-negative and VTE-positive patients. These data indicate that nitrated fibrinogen is an oxidative risk marker in VTE, providing a novel mechanistic link between oxidant production, inflammation, and VTE.

Introduction

Venous thromboembolism (VTE) is a common thrombotic disease that encompasses both deep vein thrombosis (DVT) and pulmonary embolism (PE). VTE affects around 1 per 1000 people per year within the United States, with increasing incidence for congenital and acquired thrombophilic risk factors [1], [2], [3]. VTE is a chronic disease with 30% of patients experiencing a recurrent event over 10 years [4], [5] and is estimated to account for between 100,000 and 180,000 deaths each year [6]. Thus, VTE represents a significant health problem that requires considerable attention.

The precise molecular and biochemical mechanisms of thrombus initiation in VTE remain unclear. Virchow proposed that changes in blood rheology, induction of a hypercoagulable state, or endothelial injury are conditions required for venous thrombosis [7]. More recent studies indicate that inflammation plays an important role in VTE. However, the interplay between inflammation and Virchow's triad is still unresolved. One possibility is that components of the triad activate or damage endothelial cells within the vein wall resulting in upregulation of receptors for inflammatory and procoagulant molecules. Electron micrographs of early thrombus formation show leukocyte adhesion to the vein wall [8], presumably through the binding of P- and E-selectins that are expressed on activated endothelial cells [9], [10]. Leukocytes shed tissue factor-bearing microparticles, which induce coagulation and the production of a fibrin clot [11]. Neutrophils and monocytes, two of the most prevalent leukocytes in venous thrombi, are also able to generate nitrating intermediates, capable of lipid peroxidation and nitration of proteins [12], [13].

One protein known to be modified by tyrosine nitration is the coagulant protein fibrinogen. Upon activation of the coagulation cascade, circulating plasma fibrinogen is cleaved by thrombin to fibrin monomers, which polymerize to form a fibrin clot. Recent studies demonstrate an emerging role for nitrated fibrinogen that links inflammation and oxidant production to coagulation. Nitrated fibrinogen is elevated in the plasma of patients with coronary artery disease (CAD) and in smokers [14], [15]. Additionally, in humans injected with small amounts of lipopolysaccharide, plasma levels of nitrated fibrinogen increase and remain elevated for at least 72 h postinjection [16]. This increase follows an elevation of circulating myeloperoxidase, an enzyme localized to the azurophilic granules of neutrophils, which is released during neutrophil activation and is capable of generating nitrating intermediates [12]. Although these data suggest that nitrated fibrinogen plays a key role linking inflammation, oxidant production, and arterial thrombotic diseases, its role in VTE remains undefined.

Moreover, nitrated fibrinogen is not simply a marker of inflammation and oxidative stress, but also has functional effects on fibrinogen and fibrin clotting. Fibrinogen nitration was shown to increase the rate of fibrin clot formation, diminish clot lysis rate, and alter fibrin clot structural and viscoelastic properties [14], [15], [16]. These functional effects suggest that nitrated fibrinogen may represent a risk factor for increased thrombotic tendency during inflammation and oxidant stress. However, the levels of nitrated fibrinogen in VTE have not been ascertained. Herein we quantified the levels of nitrated fibrinogen in patients presenting to the emergency department with suspected VTE and evaluated if nitrated fibrinogen is a biochemical risk factor that could explain associations of oxidative stress and inflammation with thrombotic complications in VTE.