Inflammation induces fibrinogen nitration in experimental human endotoxemia

Abstract

Elevated plasma fibrinogen is a prothrombotic risk factor for cardiovascular disease (CVD). Recent small studies report that fibrinogen oxidative modifications, specifically tyrosine residue nitration, can occur in inflammatory states and may modify fibrinogen function. HDL cholesterol is inversely related to CVD and suggested to reduce the oxidation of LDL cholesterol, but whether these antioxidant functions extend to fibrinogen modifications is unknown. We used a recently validated ELISA to quantify nitrated fibrinogen during experimental human endotoxemia (N=23) and in a cohort of healthy adults (N=361) who were characterized for inflammatory and HDL parameters as well as subclinical atherosclerosis measures, carotid artery intima-medial thickness (IMT) and coronary artery calcification (CAC). Fibrinogen nitration increased following endotoxemia and directly correlated with accelerated ex vivo plasma clotting velocity. In the observational cohort, nitrated fibrinogen was associated with levels of CRP and serum amyloid A. Nitrated fibrinogen levels were not lower with increasing HDL cholesterol and did not associate with IMT and CAC. In humans, fibrinogen nitration was induced during inflammation and was correlated with markers of inflammation and clotting function but not HDL cholesterol or subclinical atherosclerosis in our modest sample. Inflammation-induced fibrinogen nitration may be a risk factor for promoting CVD events.

Conflict of interest statement

There are no conflicts of interest.

Figures

Fig. 1

Fibrinogen nitration quantification by ELISA. (A) Plates were coated with polyclonal anti-nitrotyrosine antibodies and bound fibrinogen was detected with a polyclonal anti-human fibrinogen antibody that recognizes all chains of fibrinogen. The average ± SEM of 10 standard curves in the absence (○) and presence (□) of the 3-nitrotyrosine peptide used to raise the anti-nitrotyrosine antibodies is shown. Inset: The assay was validated by comparing the 3-nitrotyrosine/tyrosine ratio acquired with the ELISA with the ratio acquired by the established LC/ESI/MS/MS analysis in affinity-purified fibrinogen of the same individuals. The insert illustrates the results of Bland-Altman analysis described in the text. (B) To quantify plasma fibrinogen levels, plates were coated with a monoclonal fibrinogen antibody that recognizes the α-chain of native, oxidized, and nitrated human fibrinogen and bound fibrinogen was detected with the same antibody as in panel A. The average ± SEM of 10 standard curves is presented.

Fig. 2

Effects of human endotoxemia on plasma fibrinogen, nitrated fibrinogen, and ex vivo fibrin clotting kinetics. (A) Paired t test statistical comparison before and 72 h after LPS administration reveals a significant increase in nitrated fibrinogen levels after the inflammatory stimulus. (B) The same analysis reveals a significant increase in the levels of fibrinogen in plasma. (C) The fibrinogen concentration was adjusted to 0.5 mg/ml with TBS and fibrin clot formation was initiated by the simultaneous addition of calcium and thrombin. The curves represent the average ± SEM of 22 individual samples before (□) and 72 h after (Δ) LPS injection and demonstrate an increased velocity of fibrin clot formation after LPS (P=0.029). (D) Spearman correlation between levels of fibrinogen nitration and initial velocity of fibrin clot formation 72 h after endotoxin (r=0.42, P=0.04, N=23).