Post-translational oxidative modification of fibrinogen and its role in thrombotic risk in endometriosis

Fibrinogen is a 340 kDa glycoprotein synthetised in hepatocytes and secreted into the blood, where it plays a multifaceted role in many processes such as inflammatory responses, primary haemostasis and angiogenesis. However, its main role is to repair vascular damage through thrombin-mediated fibrin formation. In vivo, there is a careful balance between clot formation and fibrinolysis, which is important in preventing thrombotic and bleeding state, respectively. Moreover, inflammation-induced endothelial injury has emerged as a key factor linking oxidative stress and thrombosis1-7 . Oxidative stress (OS) is the result of many metabolic processes essential to the body. On the other hand, an imbalance between oxidant formation and endogenous antioxidant defences, in favour of the former, can cause many important cellular damages to all cellular components: DNA, membrane lipids, and proteins. Among plasma proteins, fibrinogen represents the major target for post-translational oxidative modifications. Consequences of oxidation consist in the addition of carbonyl groups in polypeptide side chains, dityrosine formation or nitrosylation that occurs in structural and functional changes1,2,6-8 . Recent studies of our research team have highlighted that fibrinogen post-translational oxidative modifications positively correlate with its structural and functional alterations, causing a thrombogenic fibrin clot in chronic inflammatory diseases, cardiovascular diseases and immune disease. Indeed, it is well known that immune system, inflammation and haemostasis are closely linked and play a key role in the thrombotic process. Moreover, these processes are associated with redox status imbalance9-12 . Endometriosis (EM) is a typical disorder of the female genital system characterized by an estrogen-dependent chronic inflammatory process and defined as the presence of endometrial tissue (glands or stroma) outside the uterine cavity. The etiopathogenesis of the disease is rather complex and still unclear, but recent studies highlighted that oxidative stress may be a potential factor involved in the pathophysiology of EM. Indeed, the literature has shown that the redox profile of women with EM is impaired and closely related to the pelvic inflammatory response. Furthermore, the association between endometriosis, systemic inflammation and oxidative stress is responsible for the onset of other comorbidities, particularly cardiovascular risk. In this regard, other studies have suggested that women with endometriosis have a hypercoagulable and inflammatory state associated with the disease, and consequently an increased risk of thrombotic events13-16 . However, the mechanism underlying this pathogenetic association remains to be elucidated; in fact, studies have not yet shown whether oxidative stress can affect the structure and function of fibrinogen. In this context, we investigated the in vitro post-translational fibrinogen structural and functional modification induced by OS. Subsequently, to confirm the results obtained in vitro, we assessed whether these changes also occur in an in vivo model. Here, we studied fibrinogen structure and function and plasma redox status in 15 women with EM and 15 age-matched healthy controls, in order to elucidate the possible mechanisms responsible for the increased cardiovascular risk in women with EM. In the first part of the study, human purified fibrinogen fraction purchased from SIGMA Aldrich was incubated either with increasing concentrations of AAPH (2,2′-Azobis(2-methylpropionamidine) dihydrochloride) (final concentrations of 0.05mM, 1mM, 2mM) or with PBS alone. In addition, fibrinogen was also treated with 1mM AAPH in the presence of 0.1mM Trolox, a water-soluble antioxidant analogue of vitamin E. Firstly, we have evaluated any functional alterations of in vitro fibrinogen in terms of both polymerization than plasmin- induced lysis. Afterwards, we assessed possible secondary and tertiary structural alterations of fibrinogen by Circular Dichroism (CD) and Intrinsic Fluorescence. In the second part of the study, plasma malondialdehyde (MDA) concentration was used as an index of lipid peroxidation and oxygen radical absorbance capacity (ORAC) to assess the total antioxidant capacity (TAC) of the plasma of EM and healthy controls. Then, in the purified fibrinogen fractions of both patients and controls, di-tyrosine content as a biomarker of oxidative stress was evaluated by fluorimetric method. To assess any functional and structural changes in fibrinogen in EM women, previously reported in vitro experiments on fibrinogen were used; specifically, thrombin-induced polymerization, susceptibility to plasmin-induced lysis, CD and Intrinsic Fluorescence. All the experiments were performed in triplicate and averaged. All statistical data were analyzed using the GraphPad Prism 10 software, considering a value of p<0.05 as statistically significant. In vitro functional experiments showed a significant impairment of fibrinogen; in particular, the ability to polymerize is significantly reduced proportional to increasing concentrations of AAPH, whereas simultaneous incubation of fibrinogen with AAPH 1mM + Trolox was able to prevent the observed changes. Similar results were obtained when the susceptibility of fibrin to plasmin lysis was analysed, showing resistance to the catalytic activity of the enzyme in parallel with the increase in AAPH levels; trolox-treated fibrinogen showed less resistance to lysis. Results about in vitro fibrinogen structure showed a decrease in terms of intrinsic fluorescence at the maximum emission point (347nm) of fibrinogen related to the increased fibrinogen + AAPH concentration. In parallel, CD spectra highlight a difference in protein secondary structure; in particular, non-oxidised fibrinogen shows a typical alpha-helix secondary structure with minima at 208 nm and at 222 nm, while fibrinogen + AAPH has a decreased negative peak in the 215 to 225 nm region, suggesting a reduction in α-helical content proportional to AAPH concentration. Fibrinogen treated with Trolox showed an improvement in fibrinogen structure with regard to both CD and intrinsic fluorescence. The obtained results on EM patients clearly indicate an overall impaired redox balance, explained by increased lipid peroxidation levels and reduced plasma TAC, if compared to healthy controls. Fibrinogen purified from EM women against to controls displayed: an increase in di-tyrosine content, a reduced susceptibility to plasmin-induced lysis and a slower rate of fibrin clot formation. Eventually, circular dichroism and intrinsic fluorescence experiments showed a secondary and tertiary altered fibrinogen structure in patients compared to controls. The results on plasma and fibrinogen of EM women are statistically significant at the p<0.0001 level. Our results showed that post-translational oxidative modifications of fibrinogen affect its physiological function and structure. Furthermore, we can confirm that these structural and functional changes also occur in EM women and this oxidative fibrinogen modifications are associated with thrombosis tendency. In addition, our results provide a biologically link between oxidative fibrinogen modifications and mechanism of thrombosis in women with EM. Further studies will allow us to elucidate the molecular mechanism behind thrombosis in EM women and to assess the potential benefits of therapy aimed at restoring redox status.