Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Plasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

Assessment Of Factors Of Vascular Wall Damage In Depressive Disorders In Patients With Myocardial Infarction

The study of pathogenetic predictors of the development of anxiety-depressive disorders in myocardial infarction will make it possible to develop ways of their correction, thereby reducing the frequency of complications of the postinfarction period. Clinical studies were based on the examination of 58 patients with MI (mean age 59.2 ± 4.7 years) who were admitted to the cardiological hospital for treatment, and the observation data for them at the stage of rehabilitation. On the basis of the presence of anxiety-depressive symptoms, the patients were divided into 2 groups. The first control group consisted of 14 patients with MI without depressive disorders. The second group included 44 age-matched patients who underwent MI with symptoms of anxiety and depression without comorbid cardiovascular pathology. The diagnosis of myocardial infarction was based on the results of clinical examination, ECG changes, laboratory parameters, and echocardiographic data. In order to assess the mental status of the subjects, subjective methods were used: the hospital anxiety and depression scale (HADS) for patients in a somatic hospital and recommended for use in patients with post-infarction period. Markers of endothelial dysfunction in blood plasma were determined by enzyme immunoassay using appropriate test systems. Since fibrinogen is one of the key factors in the blood coagulation process, disorders of which with a tendency to thrombotic changes are one of the central links in the pathogenesis of MI, the level of oxidized fibrinogen with parameters of the functional state of endothelial cells was studied. In the early stages after myocardial infarction, the level of oxidized fibrinogen was 1.7 times higher in the study group compared to the control, although, in general, the level of fibrinogen in the study group was within normal values. In the subsequent periods of the study, the level of oxidized fibrinogen was in high values and, on average, exceeded the control values by 1.64 times. Since one of the key roles in the development of dysfunction and endothelial destruction is assigned to the factors of oxidative stress, a correlation analysis of the relationship between the oxidative modification of fibrinogen and the parameters of endothelial function was carried out. A direct correlation was shown between the level of oxidized fibrinogen and the level of Endothelin-1 (r = 0.78, p <0.01), and a direct correlation with the level of von Willebrand factor (r = 0.365, p <0.01). Linear regression analysis confirmed the associations of oxidized fibrinogen with the indicated parameters of endothelial dysfunction. Based on the results obtained, it can be emphasized that with MI, in patients with developed DS, along with increased oxidative changes in lipids and plasma proteins, there is also a significant oxidative modification of fibrinogen, which does not depend on the concentration of fibrinogen. Oxidized fibrinogen potentiates potentially prothrombogenic changes in the vascular-platelet link of hemostasis, in particular, the acceleration of leukocyte-platelet aggregation. The revealed signs of thrombotic and hypercoagulant hemostasis disorders in patients with MI with depressive disorders, such as signs of endothelial dysfunction, elevated von Willebrand factor levels, are associated with oxidative changes in plasma fibrinogen in patients with MI with the development of DS, have a high diagnostic value.

The influence of oxidized fibrinogen on apoptosis of endothelial cells

Oxidative stress plays an important role in cardio-vascular diseases and atherosclerosis. Fibrinogen (FB), plasma coagulation protein, is a risk factor of atherosclerosis. Importantly, it can be readily oxidized during oxidative stress and in pathological conditions. FB can promote angiogenesis by supporting migration and proliferation of endothelial cells. On the other hand, recent reports demonstrated cytotoxicity of oxidized fibrinogen (oxFB). Endothelial dysfunction plays a critical role in the atherosclerosis development, therefore it is important to understand the effect of oxFB on human endothelial cells (hEC), and the mechanism of the cell death. Here, we studied influence of oxFB on hEC during 24 h incubation in two conditions: (1) at low serum level (0.1%) and in the absence of growth factors ("starvation"); (2) in full medium (5% FBS) with growth factor supplement. Apoptosis was evaluated using analysis of nuclear morphology, phosphatidylserine externalization on hEC surface and caspase-3 activation. In starvation, we observed significant cell death via apoptosis. FB prevented starvation-induced cell death and caspase activation. Caspase activity in the presence of oxFB was 1.5 times higher as compared to FB, yet oxFB demonstrated significant cell protection during stress. Similarly, in optimal cultivation conditions FB decreased the rate of apoptosis by three times, while oxFB supported cell viability to the lesser extent. Thus, FB can protect hEC in stress conditions (in starvation); oxidative modification of FB diminishes its antiapoptotic properties.

Oxidative Modification of Fibrinogen Affects Its Binding Activity to Glycoprotein (GP) IIb/IIIa

Aim: Proteins are sensitive biomarkers of human diease condition associated with oxidative stress. Alteration of protein structures by oxidants may result in partial or complete loss of protein functions. We have investigated the effect of structural modifications induced by metal ion catalyzed oxidation of fibrinogen on its binding capacity to glycoprotein IIb/IIIa (GpIIb/IIIa) and human platelets. Methods: We identified and quantified of binding capacity of native and oxidized fibrinogen to its receptor in vitro by flow cytometer. Dityrosine formation on oxidized fibrinogen were detected spectrophotometrically. Elevated degradation products of fibrinogen after oxidation were revealed in the HPLC analysis. The native and oxidized fibrinogen were analyzed on mass spectrum upon digestion with tyripsin. Results: Oxidatively modified fibrinogen showed less binding activity than native fibrinogen to GpIIb/IIIa coated micro beads and human platelets whereas slightly higher binding capaticity to ADP induced stimulated platelets. Formation of dityrosines in the amino acid side chains of fibrinogen were observed upon oxidation. Decreased binding capacity of oxidized fibrinogen correlated with intensities of dityrosine formation. Oxidized fibrinogen had more ion-mass intensities at higher than native fibrinogen. Clinical implications: Important point is decreased of binding capacity of the oxidized fibrinogen to own receptor. The decreased rate of binding, leading to effect in the diseases of clot formation may acount for the association between oxidation of fibrinogen and the incidence of effect in human diseases.