Archives of Academic Emergency Medicine. 2021; 9(1): e26 https://doi.org/10.22037/aaem.v9i1.1128 OR I G I N A L RE S E A RC H Fibrinogen Dysregulation is a Prominent Process in Fatal Conditions of COVID-19 Infection; a Proteomic Analysis Mostafa Rezaei-Tavirani1, Mohammad Rostami Nejad2, Babak Arjmand3, Sina Rezaei Tavirani1, Mohammadreza Razzaghi4, Vahid Mansouri1∗ 1. Proteomics Research Center, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2. Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3. Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. 4. Laser Application in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Received: January 2021; Accepted: February 2021; Published online: 15 March 2021 Abstract: Introduction: Molecular pathophysiology of COVID-19 is not completely known. Expression changes in pa- tients’ plasma proteins have revealed new information about the disease. Introducing the key targeted plasma protein in fatal conditions of COVID-19 infection is the aim of this study. Methods: Significant differentially expressed proteins (DEPs) in the plasma of cases with a fatal condition of COVID-19 were extracted from an original article. These proteins were included in a network via STRING database along with 100 first neighbor proteins to determine central nodes of the network for analyzing. Results: Queried and added proteins were included in a scale free network. Three hub nodes were identified as critical target proteins. The top queried hub proteins were chains of fibrinogen; Fibrinogen Alpha chain (FGA), Fibrinogen gamma chain (FGG), and Fibrinogen beta chain (FGB), which are related to the coagulation process. Conclusion: It seems that fibrinogen dysregulation has a deep impact on the fatality of COVID-19 infection. Keywords: SARS-CoV-2; Proteomics; Proteins; Protein Interaction Maps; Fibrinogen Cite this article as: Rezaei-Tavirani M, Rostami Nejad M, Arjmand B, Rezaei Tavirani S, Razzaghi M, Mansouri V. Fibrinogen Dysregulation is a Prominent Process in Fatal Conditions of COVID-19 Infection; a Proteomic Analysis. Arch Acad Emerg Med. 2021; 9(1): e26. 1. Introduction Coronaviridiae family viruses possess a single RNA genome with a maximum of 32 kilobases (1). Coronaviruses have been found in many different animal cases (2, 3). Several coronaviruses are pathogenic to humans with mild clini- cal symptoms (1); however, in November 2002, severe acute respiratory syndrome (SARS), which was first reported in Guangdong (4), resulted in the death of 774 patients in 37 countries (5). Middle East respiratory syndrome (MERS) corona virus (MERS-CoV ), detected in Saudi Arabia for the first time in 2012, led to 858 fatalities (6). Recently, in 2019, a new type of corona virus called SARS CoV 2 was discovered, which leads to COVID-19 (7). WHO has reported millions ∗Corresponding Author: Vahid Mansouri; Proteomics Research Center, Fac- ulty of Paramedical Sciences, Darband Street, Tajrish Square, Tehran, Iran. Email: vm1343@yahoo.com, Tel: +982122718528, ORCID: 0000000230443342. of confirmed cases and hundreds of thousands of deaths due to COVID-19 pandemic around the world (8). In addi- tion to the lower respiratory tract, many other organs, such as nervous system, gastrointestinal tract, liver, kidney, and lymph node, have been infected by SARS-CoV 2 (9). There are many symptoms for COVID-19; including fever, pneumonia, and acute respiratory distress syndrome (10). Pathophysio- logical changes such as lymphopenia (11), microthrombo- sis (12), cytokine release syndrome (13), and vascular coag- ulation have been reported in severe COVID-19 cases (14). However the molecular pathogenesis of COVID-19 is poorly understood despite the extensive efforts of scientists (15). Pathophysiological changes during viral diseases and infec- tions lead to alteration of plasma protein expression (16). Identification of differentially expressed proteins (DEPs) in the plasma during COVID-19 could help us understand the molecular pathophysiology of disease. Understanding the molecular mechanism of the viral infection could contribute This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem M. Rezaei-Tavirani et al. 2 to finding different treatment methods. Proteomics, as a high throughput method, is applied to assess the effects of SARS Cov2 on patients’ plasma proteins. Proteomic findings could be assessed as an interactome unit, which is interesting for many investigators. In such studies, a limited number of critical proteins could be identified as critical DEPs (17, 18). There is a limited number of nodes, known as central nodes, which are discriminated from others by their connections to first neighbors or involvement in shortest pathways (19, 20). Identifying central proteins among the hubs, which are characterized by their connections with the first neighbors (21, 22) and central proteins, could assist us in gaining use- ful information for finding main disease biomarkers. In the present study, findings of a proteomic investigation by Ting Shu et al. (16), which was performed with the aim of iden- tifying plasma biomarkers of COVID-19 in fatal cases were assessed using network analysis to find the main targets of SARS-CoV 2. 2. Methods Considering fold change > 1.5 and p-value < 0.01, 42 differ- entially expressed proteins were extracted from data of the original article published by Ting Shu et al. (16). Since orig- inal data about differentially expressed proteins in serum of patients relative to the healthy controls have been previously published by Ting Shu et al. in Immunity (2020, 53 (5)), the details of data production and sampling are described in the authors report and here we only explain the methods of bioinformatic analysis. The data are related to the differen- tially expressed proteins of plasma in fatal cases of COVID- 19. The queried proteins were included in a network via “pro- tein query” of STRING database by Cytoscape software 3.7.2. Confidence score cutoff =0.4 was applied to construct the in- teractive network. Among the 42 queried proteins 32 were recognized by STRING. For better resolution the network was constructed by the 32 queried proteins and 100 first neigh- bors from STRING database. The main connected compo- nent of the constructed network was analyzed using “Net- work analyzer” application of Cytoscape. The analyzed net- work was visualized based on degree value and the identified hub nodes correspond to the degree value. 3. Results A total of 32 differentially expressed recognized proteins were assessed to construct a network using Cytoscape software via protein query of STRING database. For better resolution, 100 first neighbor proteins extracted from STRING were added to construct the network (Fig1). Network analyzer considered topological properties including Degree, betweenness cen- trality (BC) and Stress (Table 1). Hub-bottleneck nodes are identified based on highest value of degree and BC. Figure 1: The 32 queried proteins recognized by STRING database plus 100 first neighbors from STRING are included in a network. The nodes are laid out based on degree value; color from green to red and size increment are corresponding to increase of degree value. Figure 2: Network including top queried hub is extracted from STRING database. As the queried and added proteins were included in a scale free network, 32 hub nodes were determined as central pro- teins (Table 1). The three top hub proteins (FGA, FGG and FGF) were members of fibrinogen family. Other hubs, ar- ranged based on degree and BC, were ORM1, ORM2, PPBP, PF4, CRP, APOA2, SAA1, ACTB, CFB, and LCAT. Since central- ity values of the hub nodes were highly dispersed, the three This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem 3 Archives of Academic Emergency Medicine. 2021; 9(1): e26 Table 1: The list of 32 top hub-bottlenecks of COVID-19 fatality- based network with their corresponding degree (K) and betweenness centrality (BC) values Row Query proteins K BC Stress 1 FGA 108 0.032 7228 2 FGG 105 0.023 6398 3 FGB 102 0.021 5830 4 ORM1 92 0.014 3850 5 ORM2 91 0.016 3686 6 PPBP 79 0.007 1900 7 PF4 75 0.003 1212 8 CRP 52 0.004 1152 9 APOA2 49 0.003 898 10 SAA1 38 0.002 642 11 ACTB 37 0.013 3174 12 CFB 27 0.004 1418 13 LCAT 25 0.001 172 14 CETP 23 0 114 15 TLN1 22 0 96 16 SAA2 21 0 32 17 FGL1 20 0 38 18 CFI 17 0.01 892 19 YWHAZ 15 0 86 20 YWHAE 14 0.01 134 21 AZGP1 13 0 2 22 S100A8 10 0 56 23 CFHR1 9 0 158 24 CFHR3 7 0 8 25 PON3 7 0 0 26 PRDX6 7 0 44 27 ARHGDIB 4 0 6 28 TAGLN2 3 0 0 29 TRIM33 2 0 0 30 TUBB1 2 0 0 31 SH3BGRL3 0 0 0 32 UGP2 0 0 0 The prioritized proteins are arranged based on degree values. top queried fibrinogen hubs with highest degree values were determined as the central nodes of the analyzed network and discussed in the more detail (Table 1). Degree and stress of fibrinogen chain hubs were more than others. ORM1, ORM2, and PPDP proteins followed fibrino- gen chains, respectively. Neighbor proteins of queried hubs are shown in Figure 2. 4. Discussion COVID-19 patients with severe condition, present with in- tense inflammation, which is induced by acute respiratory syndrome (23) and leads to cytokine storm development. One of the main distinct features of COVID-19 is coagulopa- thy (24), which is commonly observed among patients and is accompanied with severe thromboembolic conditions (25). Coagulopathy increases D-dimer levels and leads to throm- boembolism (26). The guidelines of international society of thrombosis and haemostasis recommended anticoagulant therapy for COVID-19 patients (27). Several fold increase in fibrinogen level is reported in severe cases of COVID-19 (28). Our data analysis revealed that enough connections between the studied proteins, could form a scale free network. The first neighbors added to the queried proteins provide a scale free network (Figure 1). Assessments indicated that the scale free network could provide useful information to distinguish a limited set of proteins among a large number of proteins (Figure 2). Our results demonstrated that fibrinogen chains of FGA, FGG and FGB are top hub proteins related to COVID-19 fatalities (Table 1). On the other hand, neighbor proteins related to fibrinogen chains are APOA2, ORM2, ORM1 and CFP (Fig- ure 2). Considering molecular pathways of the coagulation process, in which fibrinogen chains are involved, may open a window to help in treatment of disease. The role of fibrino- gen in acute COVID-19 cases and clot formation has been considered in researches. Fibrinogen is a glycoprotein that is produced in liver as an anti-infective organ. Liver overreacts during acute inflammatory phase in hospitalized COVID-19 patients and secretes several reactants such as fibrinogen, C reactive protein (CRP), ferritin and plenty of cytokines (29, 30). Secretion of those reactants is the body’s defense mech- anism against invading pathogens. In this regard, the dual function of fibrinogen is important, as it regulates antimicro- bial activity of the immune cells and clot formation. Mac- 1(CD11b/CD18) is a leucocyte integrin receptor, regulating inflammatory responses, and fibrinogen is a ligand for Mac- 1, in addition to having coagulator functions (31). Mac-1 is a receptor for COVID-19 RNA strand and increase in fibrino- gen secretion could impregnate Mac-1 to reduce the negative effects of the virus (32, 33). Ko YP et al. summarized several host defensive mechanisms of fibrinogen, in summary two main mechanisms are fibrin matrix barrier formation and immune protective functions of host (34). Formation of thrombosis could limit pathogen spread as a defensive mechanism and the researchers believed that lo- calized formation of lungs thrombosis could restrict SARS Cov-2 virus from spreading (35). D-dimer is a product of fibrin degradation in blood after clot fibrinolysis. Increase in D-dimer is accompanied by reduction of fibrinogen re- lease from the platelets (25). A hypothesis states that in pa- tients with COVID-19 and other infectious diseases, increase in D-dimer and decrease in the secretion of fibrinogen, lead to activation of immune responses. On the other hand, de- crease in D-dimer and increase in the secretion of fibrino- gen, activates the coagulation mechanism and thrombi for- mation (36). Among the key genes that interact with D- dimer, fibrinogen level, and coagulation process, FGA, FGG, and FGB are prominent. Although additional gene clusters help govern D-dimer and fibrinogen count and thrombosis This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem M. Rezaei-Tavirani et al. 4 in COVID-19 patients (37). High circulating level of fibrino- gen has been linked to COVID-19 coagulopathy; however, Jecko Thachi et al. believed that in COVID-19 patients fib- rinogen is probably increased to protect the host (36). C reactive protein (CRP) released by liver is anti-infective and increases in acute phase of COVID-19, along with ferritin and fibrinogen, as a defense mechanism against pathogens (30). Our results revealed the indirect connection of CRP with FGB via ORM2 protein (Fig2). CPR forms a complex with histones to protect them from endothelial damage resulting from edema and thrombosis in hosts suffering from COVID- 19 (38). Researches believed that evaluating CRP or fibrino- gen levels in addition to other conventional markers could be useful for prediction of cardiovascular disease in patients with intermediate risk factors (39). Orosomucoid isoforms (ORM1 and ORM2) are inducers of M2 macrophages and increase in various infections (40)(41). Our results also revealed a connection between ORM1 and ORM2 and fibrinogen chains (Figure 2). This connection may be related to severe infection in fatal COVID-19 cases. Overall, the role of acute infections in COVID-19 patients, with regard to the secretion of fibrinogen and other promi- nent proteins, can be evaluated in future investigations. 5. Conclusion It can be concluded that activation of the clotting and em- bolism mechanisms along with fibrinogen secretion in pa- tients with COVID-19 are the prominent processes in fatal cases. 6. Declarations 6.1. Acknowledgment This project was supported by Shahid Beheshti University of Medical Sciences. 6.2. Authors’ contributions All authors have contributed equally in the project admin- istration. Final revision was done by Vahid Mansouri and Mostafa Rezaei Tavirani and verified by all authors. 6.3. Conflict of interest There is no conflict of interest. 6.4. Funding and supports This research was supported by shahid Beheshti univer- sity of medical sciences with the Ethics code number: IR.SBMU.RETECH.REC.1399.356 References 1. Su S, Wong G, Shi W, Liu J, Lai AC, Zhou J, et al. Epidemi- ology, genetic recombination, and pathogenesis of coro- naviruses. Trends in microbiology. 2016;24(6):490-502. 2. Cavanagh D. Coronavirus avian infectious bronchitis virus. Veterinary research. 2007;38(2):281-97. 3. Ismail M, Tang Y, Saif Y. Pathogenicity of turkey coronavirus in turkeys and chickens. Avian diseases. 2003;47(3):515-22. 4. Peiris J, Guan Y, Yuen K. Severe acute respiratory syn- drome. Nature medicine. 2004;10(12):S88-S97. 5. Chan-Yeung M, Xu R. SARS: Epidemiology. 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This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem Introduction Methods Results Discussion Conclusion Declarations References