Study of structural and functional fibrinogen modifications in patients with Eosinophilic granulomatosis with polyangiitis (EGPA)

Eosinophilic granulomatosis with polyangiitis (EGPA), formerly called Churg–Strauss syndrome, is a rare systemic vasculitis characterised by peripheral eosinophilia, eosinophil tissue infiltration and disseminated necrotizing vasculitis with extravascular granulomas. In 30% of EGPA patients (especially during active phase of disease) cardiovascular complications represent a major cause of morbidity and mortality. Recent studies explored the role of oxidative stress in EGPA pathogenesis, suggesting that its clinical manifestations could be caused by an imbalance in redox homeostasis. Oxidative stress, a condition characterized by an imbalance between reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) production and antioxidant defences, has been widely implicated in several pathological disorders such as diabetes, cancer, degenerative diseases, chronic inflammatory diseases, cardiovascular diseases and thrombosis. It is now widely accepted that inflammation and oxidative stress display a prominent role in cardiovascular diseases pathogenesis. Proteins are the main targets for ROS, with consequent protein structure and function alterations. Compared with other plasma proteins, fibrinogen, which plays a key role in blood clotting, fibrinolysis, cellular and matrix interactions and in the inflammatory response, is particularly susceptible to oxidation, 20x than albumin. Fibrinogen oxidation can affect fibrinogen function, influencing fibrin formation, clot structure and fibrin susceptibility to lysis. Based on this background, the present project was undertaken to investigate, in EGPA patients, the effect of oxidative stress on fibrinogen structural/functional features. In 35 EGPA patients and 35 age-matched healthy controls we investigated plasma redox status, blood leukocyte ROS production and, in fibrinogen purified fractions, its structure and function. H2DCF-DA fluorescent probe was used to detect leukocyte intracellular ROS levels which were quantified by FACS analysis. Plasma malondialdehyde (MDA) concentration (colorimetric assay) was used as an index of lipid peroxidation and Oxygen Radical Absorbance Capacity (ORAC) for the assessment of plasma total antioxidant capacity. Fibrinogen was purified from patients and controls using the ethanol precipitation method. Furthermore, thrombin-catalyzed fibrin polymerization and plasmin-induced fibrinolysis were investigated in patients and controls. Fibrinogen conformational properties were explored by Intrinsic Fluorescence and Circular Dichroism Spectroscopy (CD). Fibrinogen oxidation was evaluated fluorimetrically by dityrosine content. Overall, the obtained results are indicative of an altered redox status in EGPA patients and suggest its association with impaired structural and functional fibrinogen properties. Our data demonstrate that oxidative-mediated fibrinogen structure modifications such as those observed in EGPA patients are associated with thrombosis tendency, suggesting new potential targets for innovative therapeutic approaches.