Characteristics of fibrin/fibrinogen degradation products in multiple sclerosis following SARS-CoV-2 infection

. Background. The purpose of this study was to investigate plasma levels of fibrinogen and products of its degradation in patients with multiple sclerosis (MS) with and without a history of coronavirus disease 2019 (COVID-19). Materials and methods. We examined 97 patients with MS. Based on the presence of COVID-19, all cases were divided into two groups. MS group included 56 patients who did not suffer from COVID-19 previously. MS + COVID group consisted of 41 cases who had a laboratory-verified diagnosis of COVID-19. The group of healthy controls included 30 healthy volunteers. Spectrophotometric techniques were used to measure the concentrations of fibrinogen, D-dimer, and soluble fibrin monomer complexes (SFMCs). Size-exclusion chromatography was applied to analyze the composition of SFMC fractions. Results. We found that concentrations of fibrinogen, D-dimer, and SFMCs were remarkably increased in plasma of all MS patients compared with healthy controls. The levels of D-dimer, and SFMCs did not differ between two MS groups, while plasma fibrinogen concentration was significantly increased in MS + COVID patients compared to MS group. Moreover, the development of MS was accompanied by the changes in both quantity and quality of SFMC composition compared to that of healthy controls. Our results demonstrated accumulation of high-molecular-weight SFMCs in plasma of MS patients. Conclusions. The findings indicated that MS patients had changed hemostasis characteristics; however, more research is required to determine the connection between particular hemostatic factors, namely fibrinogen, D-dimer, and SFMCs, and the pathophysiology of MS.


Introduction
Multiple sclerosis (MS) is an inflammatory-mediated demyelinating disease of the central nervous system (CNS) characterized by neuroinflammation and neurodegeneration [1].In the past decades, great efforts have been made in understanding the risk factors, and immune dysregulation mechanisms that are responsible for detecting MS and its progression.MS is thought to emerge as a result of a complex combination of genetic predispositions, environmental triggers, infectious events, and factors that lead to pro-inflammatory states, including smoking, obesity, etc. [2].MS lesions are widely acknowledged to originate from an autoimmune process that affects the myelination of the neurons present in the CNS.This leads to the formation of demyelinated plaques, which cause injury to neurons and their axonal extensions [3].Numerous studies have empha-sized the existence of relationship between the activation of the coagulation cascade and neuroinflammation, indicating that coagulation factors may play a more comprehensive role involving neurodegeneration and neuroinflammation in addition to being essential for the activation of the hemostatic cascade [4][5][6].In recent years, significant evidence has emerged implicating several of the major clotting factors, such as thrombin, or fibrinogen, in the pathogenesis of MS by triggering microglia activation and driving the progress of neuroinflammation [6].
Fibrinogen is a soluble 340 kDa dimeric glycoprotein that is synthesized in the liver by the hepatocytes.Each monomer made up of three polypeptide chains designated Aα, Bβ, and γ that are stabilized by disulfide bonds.Thrombin removes two small peptides, fibrinopeptides A and B, from the fibrinogen molecule, leading to exposure of multiple polymerization sites and initiating the formation of insoluble and stable fibrin clot [7].In addition to its major function in blood clotting, fibrin(ogen) plays an essential role in cellular and matrix interactions, inflammation, wound healing, angiogenesis, etc. Various studies have highlighted the contribution of fibrin(ogen) to the pathophysiology of MS [8][9][10][11].It was shown that the areas occupied by demyelinating lesions and characterized by axonal damage coincided with the areas of fibrin deposition in MS patients [12].Furthermore, fibrin deposition may precede the formation of demyelinating lesions [13].The role of fibrin in formation of demyelinating lesions may be associated with its proinflammatory function and ability to increase the release of several other immune mediators, which, in turn, can modulate immune process as well as both cell adhesion and migration [14].The other way by which fibrin(ogen) can be involved in the pathogenesis of MS is its direct triggering microglia activation.Ample evidence suggests that fibrinogen induces release of reactive oxygen species in microglia causing damage to nerve cells, which play a crucial role in the induction of axon degeneration in inflammatory demyelination [15].
Given the important role of fibrin(ogen) in the pathogenesis of MS, we hypothesized that factors affecting the concentration of circulating fibrinogen could be considered independent risk factors for deterioration in MS patients.Since coronavirus disease 2019 (COVID-19) is associated with both quantitative and qualitative changes in circulating fibrinogen, intra-and extravascular fibrin deposition, and fibrin degradation [16], COVID-19 infection can increase the risk of CNS demyelinating lesions leading to exacerbation of MS.Despite the expectation of a deleterious effect of COVID-19 on MS manifestations, the results regarding the relationship between these two disorders are quite controversial [17] and further studies are required.
The current research aimed to investigate plasma levels of fibrinogen and products of its degradation in MS patients with and without COVID-19 history.This new information should help clarify the underlying mechanisms of MS pathogenesis, and hopefully lead to new findings regarding contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to deterioration in MS patients.

Materials and methods
This research was conducted in accordance with the ethical standards and provisions of the Declaration of Helsinki (1964), and applicable regulatory requirements.The study protocol was approved by the ethics committees of both Taras Shevchenko National University of Kyiv (Kyiv, Ukraine) and Bogomolets National Medical University (Kyiv, Ukraine).A total of 97 MS patients (gender distribution: 34 male and 63 female) seen at the University Clinic of Bogomolets National Medical University between January 2021 and August 2022 were enrolled in this study.Average duration of disease was similar in all MS patients and ranged between 3 and 5 years.Participants were divided in two groups.MS group (n = 56) included MS patients who did not suffer from COVID-19 previously.MS + COVID group (n = 41) consisted of MS patients who had a laboratory-confirmed diagnosis of COVID-19 in the past 3-6-month period, but currently were negative, as determined by testing nasopharyngeal swabs.The healthy control (HC) group included 30 subjects, all of them were SARS-CoV-2-negative by nasopharyngeal swab at the time of blood sampling.We excluded all individuals who had cardiovascular and cerebrovascular diseases; were on hemostatic medications; had any acute or chronic disorders that can affect the hemostasis system.Written informed consent was obtained from all participants.
Whole blood samples were collected into vacutainer plasma tubes (tubes with K 2 EDTA).Immediately after drawing, tubes were gently inverted, and centrifuged at 2,500 g for 15 minutes.Plasma aliquots were separated immediately and stored at -20 °C until use.Prior to analysis, frozen plasma samples were placed into a +37 °C water bath, thawed for five to ten minutes, and mixed by gentle inversion.
The qualitative detection of immunoglobulins M (IgM) and G (IgG) antibodies against SARS-CoV-2 in blood plasma was done by chemiluminescence immunoassay using the reagent kit Maglumi 2019-nCoV IgM/IgG (Shenzhen New Industries Biomedical Engineering Co., Ltd., China).The results are expressed in absorbance unit (AU/mL).Acceding to the operating instructions, a result less than 1.00 AU/mL was considered to be non-reactive, while a result greater than or equal to 1.00 AU/mL was considered to be reactive.
Fibrinogen concentration was measured spectrophotometrically according to the method [18].Briefly, the fibrin clot formed after the addition of thrombin (2 NIH) was dissolved in 0.125% acetic acid.The optical density (OD) of samples was measured at wavelengths of 280 and 320 nm.The fibrinogen concentration (g/L) was calculated using the formula: (OD 280 -OD 320 ) × 255 ÷ 1.506, where 255 represents the conversion factor of the fibrinogen concentration in the sample volume to its plasma concentration, and 1.506 represents the fibrin extinction coefficient at 280 nm.
The concentration of D-dimer was determined by a sandwich-type immunochemiluminescent assay using D-dimer Test Kit for CLIA Maglumi (Snibe Co., Ltd., China).All manipulations were carried out according to the manufacturer's instructions.The analyzer automatically calculated the concentration of D-dimers in the samples based on the calibration curve.The results were expressed in μg FEO/mL.
The concentration of soluble fibrin monomer complexes (SFMCs) was determined using o-phenanthroline technique [18].This method is based on the estimation of time needed to form fibrin particles after addition of 0.78% o-phenanthroline solution (1 : 1).Further purification of the formed complexes was performed.
SFMCs were isolated from blood plasma according to the procedure described previously [19].The SFMCs were collected from 1 mL of blood plasma of each individual and kept at 4 ° till chromatographic analysis.Before chromatographic analysis, equal volume of SFMCs solutions obtained from five individuals of the same experimental group were randomly gathered to form pool of SFMCs, which was used in further analysis.Size-exclusion chromatography was performed on HiLoad 16/600 Superdex 200 pg column (GE HealthCare Life Sciences) using a BioLogic LP chromatographer (Bio-Rad, USA).The mobile phase was 0.05 M Tris-HCl (pH 7.4) containing 0.13 M NaCl, and the flow rate was 60 mL/hr.The pool of SFMCs gathered from five individuals was loaded onto the column in the volume of 1 mL, and the appearance of corresponding peaks (fractions) were monitored by means of UV detector.The peak areas were calculated using OriginLab v. 9.1.The chromatographic procedure was repeated at least 3 times for each experimental group.Every time we used freshly prepared SFMCs pool solution obtained by mixing of 5 different SFMC fractions isolated from individuals of the same experimental group.A standard ladder with known molecular weight was applied to the column for calibration.
Statistical processing of the obtained results was performed with methods of variational statistics using the computer program Statistica 12.0 (StatSoft Inc., USA).The hypothesis of the normal distribution was clarified with the Shapiro-Wilk and Kolmogorov-Smirnov tests.For studied variables, we reported means with SEM and medians with IQRs.The differences between the samples were determined using the Mann-Whitney U test or the Kruskal-Wallis test.A p < 0.05 was considered statistically significant.

Results
Basic characteristics of the multiple sclerosis and healthy control groups are summarized in Table 1.In this study, the MS patients (n = 97) and control subjects (n = 30) were well matched for age and gender distribution.The mean age of the MS and control groups was 40 ± 5 and 41 ± 4 years, respectively.The mean duration of disease in both MS and MS + COVID groups was similar and ranged from 3 to 6 years.As expected, MS + COVID subjects had significantly higher levels of circulating IgG antibodies against SARS-CoV-2, when compared to either HC or patients of MS group (p < 0.05).On the other hand, all participants had low levels of circulating anti-SARS-CoV-2 IgM that indicated the absence of an acute infection at the time of blood sampling.
Results from laboratory investigations showed that elevated fibrinogen (p < 0.01), D-dimer (p < 0.01), and SFMC concentrations (p < 0.01) were significantly associated with multiple sclerosis (Table 1).It should be noted that anti-SARS-CoV-2 IgG seropositive subjects had significantly higher fibrinogen concentration than seronegative individuals (p < 0.05) as well as healthy controls (p < 0.01).The other two parameters (D-dimer and SFMC) were comparable between the MS and MS + COVID groups, and no significant association with anti-SARS-CoV-2 IgG seropositivity was found (p > 0.05).
SFMC is a precursor of fibrin polymer formation at the early stage of blood coagulation.Thus, it could be used as a serological marker for the activation of the coagulation system and for the early detection of intravascular hypercoagulation conditions.Since our findings indicated that SFMC levels had been significantly elevated under multiple sclerosis, analysis of SFMC fraction composition was considered to be a logical continuation of our research.Our results showed that the examined SFMC pools contained a variety of protein molecules that had different molecular weight (up to 550 kDa).The difference between the results of fractionation of SFMC isolated from the plasma of MS patients, and HC was obvious (Table 2).As can be seen, the SFMC pool of healthy individuals consisted of 3 main fractions.The most abundant protein fraction in the SFMC pool of healthy controls was a fraction with a molecular weight of 330-340 kDa, which represented about 45 % of total SFMCs.Another majority fraction, which represented 35 % of the total SFMC content, corresponded to proteins whose molecular weight ranged from 100 to 110 kDa.Finally, 30 % of the SFMC pool was represented by 260-280 kDa protein complexes.

Characteristics
The development of MS was accompanied by changes in both the quantity and quality composition of the SFMC pool compared to the results obtained for HC (Table 2).Thus, in the group of anti-SARS-CoV-2 IgG seronegative MS subjects, we did not observe protein fractions ranging between 260-340 kDa.On the other hand, protein complexes with a molecular weight of 540-550 kDa were identified in the SFMC pool, and this fraction represented about 70 % of total SFMCs.The levels of the other two fractions, 100-110 and 140-160 kDa, were almost equal and represented 16 and 14 % of the total SFMC pool, respectively.

Discussion
COVID-19 poses a considerable threat to public health worldwide.The hallmark of COVID-19 pathogenesis is the cytokine storm, which may lead to multiorgan dysfunction and could influence inflammatory and degenerative processes in the CNS.Moreover, COVID-19 has been associated with hemostasis disbalance, which seems to be a crucial factor in neuroinflammation [16].Thus, the COVID-19 pandemic became an ongoing global challenge, especially for people with autoimmune diseases, such as multiple sclerosis.Indeed, there is a lot of evidence showing a relationship between COVID-19 and MS [17,[20][21][22].However, current findings are sometimes controversial, and more studies are needed to collect data on greater numbers of patients with SARS-CoV-2 infection since these cohorts are not yet large enough to make specific conclusions for patients with MS.
The results of our previous study demonstrated that SARS-CoV-2 IgG seropositive donors had increased levels of circulating fibrinogen, D-dimer, and SFMCs [19].Given the important role of fibrin(ogen) in the pathogenesis of MS, we hypothesized that SARS-CoV-2 infection could be a risk factor for deterioration in MS patients who suffered from COVID-19.
The major differences between the coagulation para-meters of healthy volunteers and MS patients were as follows: 1) circulating fibrinogen concentration was increased by 45 and 50 % in patients of MS and MS + COVID groups, respectively, compared to the controls; 2) the concentration of D-dimer was increased by 50 and 65 % in plasma of patients from MS and MS + COVID groups, respectively, compared to control subjects; and 3) the concentration of SFMCs was 2-fold higher in both MS groups than in the controls.Interestingly, according to the results obtained, a SARS-CoV-2 infection had a limited effect on the studied coagulation parameters in MS patients, causing statistically significant changes in only one of them, namely plasma fibrinogen concentration.
Our study confirms the findings of other researchers [8][9][10][11] that fibrinogen levels are elevated in MS patients.The severity of MS is assumed to correlate with fibrinogen level.This clotting factor is involved in the MS pathogenesis by triggering microglia activation and neuroinflammation [23].Since fibrinogen is an acute-phase protein, its accumulation in the bloodstream of MS patients may occur due to systemic inflammation, which takes place under this autoimmune disorder [11,23].Even more pronounced hyperfibrinogenemia in anti-SARS-CoV-2 IgG seropositive MS patients could develop due to thrombotic complications resulting from COVID-19 [24].
In this study, we demonstrated increased circulating Ddimer and SFMC values for both MS patient groups.The presented findings may be explained by the activation of the coagulation cascade under MS conditions.As a result of fibrinogen cleavage, accumulated fibrin mesh may be ul-  [25].
The increased circulating SFMC in MS patients appeared to be also related to the activation of the coagulation cascade [8].After thrombin cleaves the fibrinogen molecule, the fibrin monomers are formed, and during the early phase of thrombus formation, these monomers achieve stability by creating SFMCs with fibrinogen breakdown products.As the process of thrombosis continues, more fibrin monomers are created, and more complexes are formed.When the concentration of fibrin polymers reaches a threshold level, they combine with factor XIII to form stable clots.We hypothesized that SFMCs could also be used as a promising biomarker of intravascular hypercoagulation state, since these molecular complexes appear in plasma before actual "clot" formation occurs, in its early stages.
Subsequent examination of the composition of SFMC fractions isolated from the plasma of MS patients showed the formation of protein complexes with molecular weights different from those of healthy individuals.We think that abnormalities in the coagulation process under pathological conditions may be the reason for the variation in the SFMC composition.In MS patients, the formation of high-molecular-weight complexes (550-560 kDa) may be linked to increased plasma coagulation.The appearance in the bloodstream of high-molecular-weight complexes seems to be one of the promising diagnostic criteria for both coagulation and vascular thrombosis in MS patients.
It seems crucial to determine the exact links between MS pathogenesis and coagulation pathway dysregulation.Finding out how particular hemostatic factors, namely fibrinogen, D-dimer, and SFMCs, relate to the progression of neurodegeneration and neuroinflammation under MS will be the next task ahead.Further experiments on understanding the role of studied coagulation factors as promising biomarkers of MS severity may lead to improved diagnostic options, not only for demyelinating diseases, such as MS, but also for other neurodegenerative conditions.

Table 2 . SFMC fractions derived from the plasma of multiple sclerosis patients and healthy controls, %
disintegrated by plasmin into its degradation products, including D-dimer.The results of other studies have shown the elevated level of D-dimer in cerebrospinal fluid from MS patients.Moreover, it was proposed to use cerebrospinal fluid D-dimer as routine clinical marker of disease activity in MS patients Notes: values are expressed as mean ± SEM; * -p < 0.05 vs. HC; # -p < 0.05 vs. MS group.timately