Coagulopathy and thromboembolic events a pathogenic mechanism of COVID‐19 associated with mortality: An updated review

Abstract During 2019, the SARS‐CoV‐2 emerged from China, and during months, COVID‐19 spread in many countries around the world. The expanding data about pathogenesis of this virus could elucidate the exact mechanism by which COVID‐19 caused death in humans. One of the pathogenic mechanisms of this disease is coagulation. Coagulation disorders that affect both venous and arterial systems occur in patients with COVID‐19. The possible mechanism involved in the coagulation could be excessive inflammation induced by SARS‐CoV‐2. However, it is not yet clear well how SARS‐CoV‐2 promotes coagulopathy. However, some factors, such as pulmonary endothelial cell damage and some anticoagulant system disorders, are assumed to have an important role. In this study, we assessed conducted studies about COVID‐19‐induced coagulopathy to obtain clearer vision of the wide range of manifestations and possible pathogenesis mechanisms.


| INTRODUC TI ON
In December 2019, a new coronavirus which named SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) emerged in Wuhan, Hubei province, China. The conducted disease by this virus is coronavirus disease 2019 (COVID-19) rapidly spread in many countries around the world. [1][2][3][4] After a while, this novel coronavirus caused one of the most important health issues worldwide. [5][6][7][8] Clinical manifestation spectrum associated with COVID-19 is various and not completely clear. The disease presentations can range from an asymptomatic infection or mild illness to severe pneumonia, sepsis, loss of organs function and even death. [9][10][11][12] Previous important coronaviruses, SARS-CoV (SARS-CoV-1) and the Middle East respiratory syndrome (MERS-CoV), responsible for the outbreak in 2002-2003 and 2012, respectively, and associated with severe and fatal diseases. [13][14][15] Since the start of the pandemic by COVID-19, different therapeutic approaches are suggested. [16][17][18][19][20][21][22] Although most people with COVID-19 will face mild manifestations, some patients will demonstrate severe clinical signs that lead to hospitalization and intensive care unit (ICU) admission. These clinical complications include severe lung dysfunction, sepsis, shock or multiple organ failure. 23,24 There is a wide range of literature about the severity of COVID-19 and its potential life-threatening clinical conditions. 25 Coagulation disorders and thrombotic complications were reported in the COVID-19 patients. 26,27 The mechanism of coagulation disorders is not precise in the COVID-19 patients. In this regard, there are valuable researches available. 28 Furthermore, in this study, we conducted studies in COVID-19-associated coagulopathy to obtain a clearer vision of this wide range of manifestations and possible pathogenesis mechanisms. In addition, we tried to review the current literature in this particular field to provide an update for clinicians and researchers.

| P OSS IB LE MECHANIS MS FOR COAG UL ATI ON D ISORDER S IN COVID -19
Disorders in coagulation system such as myocardial infarction and thrombotic complications that affect both venous and arterial systems occur in COVID-19. 29,30 The coagulopathy conditions of COVID-19 are not only limited to the primary course of the disease and could be during post-infection or long COVID-19 conditions. 31 These coagulation disorders are due to excessive induction of inflammation, disturbing gas exchange, immobilization and dissemination of intravascular coagulation (DIC). 26,27,32 It is not yet completely clear how SARS-CoV-2 promotes coagulopathy. However, some factors, such as damage to pulmonary endothelial cells and some disorders in the fibrinolysis system, are assumed to have an important role. It is demonstrated that the level of factor VIII, fibrinogen, and also von Willebrand (VWF) in the COVID-19 disease are higher than in normal cases. Considering this, there is a growing body of research reports, which shows coagulation disorders despite the use of anticoagulant agents. [32][33][34][35] Some laboratory findings can support a poor prognosis in a patient infected by a novel coronavirus. Among those, some data obtained from coagulation parameters indicate that elevated levels of D-dimer (>3000 ng/mL) can be considered to predict unfavorable outcomes include death in severe COVID-19. 1,9,[36][37][38][39] Besides arterial and venous thrombosis, pro-inflammatory cytokines, which lead to cytokine storm (with unknown causes), are also associated with higher mortality risk. 40,41 The relationship between these two processes, thrombosis and inflammation, which strengthen each other, has been shown before and is compatible with the above findings. 42,43 It has been shown that anticoagulant factors like heparin and platelets, besides their original function, also act as anti-inflammatory and can modulate immune response. 44 would induce statistically significant decrease in mortality rate of severe disease and who present sepsis-induced coagulopathy (SIC) score ≥4 or D-dimer levels >6-fold upper from normal rang. 49 Disturb coagulation function, including elevated fibrinogen degradation products, factor VIII (FVIII), VWF levels and fibrinogen levels, reported in th COVID-19 patients. 50 Derangement of hemostasis associated with proteins involved in the coagulation process can be caused by cytokine storm which leads to a fundamental change in pro and anticoagulant proteins activity. 41,51 In one study, Christophe et al. report that reactivation of the immune system stimulates coagulation disorders. Moreover, increasing fibrinogen and FVIII levels and disturbing the fibrinolysis process have been shown in all patients infected by  Disorderliness in other thrombosis and fibrinolysis factors are also associated with severe thrombotic involvement. Levels of tPA (tissue-type plasminogen activator), TAFIa/I (thrombin activatable fibrinolysis inhibitor) and PAI-1 (plasminogen activator inhibitor 1) have a remarkable increase in the number of the critical patient than those patients who did not experience a severe form disease. High levels of TAFI antigen and PAI-1 seems to be associated with mortality in cardiovascular disease and arterial thrombosis. 32

| IMMUNE SYS TEM AND COAG UL ATI ON
Initiating immune response to invasive viral pathogens leads to disturbing the harmony between factors involved in the homeostatic mechanism associated with coagulation. Regardless of forming a clot, platelets mediate interaction between TLRs (toll-like receptors) and activate inflammatory cascade to eliminate viral infections. 32 There are some pieces of evidence about the importance of TLRs in the COVID-19 pathogenesis and induction of coagulopathy effects. [53][54][55] Too much-activated platelet and the cascade of events leads to coagulation blood, responsible for elevated D-dimer and thrombocytopenia. Apart from these, damages of the vascular endothelium, immune deregulation, increasing von Willebrand factor, and accumulation of macrophage, monocyte, platelet and lymphocyte have significant roles in coagulopathy after viral infection. 32,[56][57][58] There is a relationship between endothelial cell activation and increasing in VWF level. Some observations suggest the elevation of VWF in COVID-19. 50,59,60 ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) Enzyme, or VWFCP (von Willebrand factor-cleaving protease), regulates the VWF multimers size during synthesized by endothelial cells and megakaryocytes. A low level of ADAMTS13 can cause thrombotic thrombocytopenic purpura (TTP). [61][62][63][64] One study reported that ADAMTS13 and ADAMTS13/VFW: Ag ratio among survivors and non-survivors patients indicated a remarkable decrease in the deceased patients than survivors. 35 Also, poor clinical prognosis in the COVID-19 patients with coagulation and death is frequently occurs in patients representing myocardial infarction than in patients without cardiac involvement. 65 Additionally, level of Troponin-T in the COVID-19 patients shows statistically significant association with increase PT, PTT and D-dimer elevation.  Table 1.

| Venus thromboembolism (VTE)
VTE events, in the form of the DVT, cerebral venous thrombosis (CVT), or pulmonary embolism (PE), are estimated as the cause of death in 5%-10% of hospitalized individuals with SARS-CoV-2. 73,74 VTEs are includes DVT, PE, CVT, or even a combination of these conditions. The incidence of VTE in the COVID-19 patients was in range of 7%-26% based on the different clinical conditions of patients. 75 There are a significant number of studies about VTE and COVID-19. Conducted studies on VTE suggested a higher incidence of the VTE in ICU (Intensive Care Units) admitted patients. 76 Regardless of other conducted reports in the field of coagulopa- Alonso-Fernández and colleagues 80 concluded an increased age and D-dimer >1 μg/mL are frequent in COVID-19 with PE presentation. The importance of D-dimer monitoring was also suggested by other studies. [81][82][83] Regardless of the D-dimer level, the factor V activity is suggested as a biomarker for PE monitoring during COVID-19 by Stefely et al. 84 Meanwhile, investigation of cerebral venous sinus thrombosis represents good outcomes in the most COVID-19 patients with CVT. 85 In addition, the gender assessment indicates the female gender as the dominant portion of the patients. Furthermore, Cavalcanti and colleagues 86 study suggested that the median duration between the COVID-19 onset of symptom to development of thromboembolic events in patients with CVT is 2-7 days.

| Anticoagulant treatment
There are varieties of studies investigating the anticoagulant treatment guidelines in the COVID-19 patients. [87][88][89][90] Highlighted researches are listed in Table 2. These studies indicate that the Heparin/LMWH is used in the most common anticoagulation strategy, including for a wide range of the coagulopathy events such as DVT and VTE. In addition, heparin's anticoagulant activity is also familiar with antiviral activity against enveloped viruses, including coronaviruses. 91

TA B L E 2 (Continued)
stage of COVID-19 could be concidered as a candidate for thromboprophylaxis if there was low-bleeding risk, represents high D-dimer levels, or require supplemental oxygen. 107

| SARS-CoV-2 variants and coagulopathy
Effects of different SARS-CoV-2 variants in the severity of disease, epidemiological pattern, and clinical presentation of patients are previously described. 108 These difference in the severity of disease between variants also affects coagulopathy patterns. There are limited studies about this field of study. The in-vitro study indicates more potential for coagulation in the alpha variant in compare with the Omicron. 109 In addition, in clinically evaluated samples, Omicron variants are less competent for micro clot formation or coagulation abnormalities in comparison with alpha, beta and delta variants. 110,111 As a point of view, it seems that one of the aspects of less sever Omicron variant infection in compare with previous variants is associated with its coagulation alteration profile.

| NOVEL RE S E ARCH FOR THE PATHOPHYS IOLOGY OF COAG ULOPATHY IN COVID -19
There is some novel pathophysiology for coagulopathy in COVID-19.
In this study, we only provide a view of some of the most important novel mechanisms for coagulopathy in COVID-19. Some important original researches about Endothelial cell dysfunction, Complement activation, or other important mechanisms are provided in Table 3.
More discussion about these mechanisms are provided in the next sections. However, it should be considered that these mechanisms are tight links.

| Endothelial cells dysfunction
The vascular endothelium constitutes the inner cellular layer of vessels, which forms a barrier between blood and tissue. 112 Since it is   higher levels of VWF were seen in the COVID-19 patients, which is related to reduced activity of disintegrins metalloproteinase with ADAMTS13. 118 All these events can eventually lead to the development of thrombotic microangiopathy. 119  reported an increased number of CECs in the COVID-19 patients, especially those who were admitted to ICU. They also showed that CECs are correlated to elevated plasma levels of soluble vascular cell adhesion molecule 1 (sVCAM1). 123 Falcinelli and colleagues also confirmed these results. In addition to sVCAM1, they reported an increased amount of soluble intercellular adhesion molecule 1 (sICAM1) in COVID-19. 124 Taken together, we highly suggest implementing preventing measures such as blocking proinflammatory cytokines and NLRP3 inflammasome as effective measures to reduce the risk of endothelium damage by COVID-19.

| Platelets activation in COVID-19
Since there is a considerable number of platelets circulating in the blood vessels, they might be the first cells to face the virus in the blood. 125 The platelet response against the RNA viruses is initiated by the interaction between the viral genome and the toll-like receptor 7 (TLR7) located in the endolysosomes of platelets. 126 NETs traps help the immune system in fighting different pathogens including viruses. 129 However, they are extremely prothrombotic and induce intravascular coagulation. Furthermore, C3 can also be activated by this thrombin and eventually continue the complement cascade, which plays a major role in inflammation. 130 Zuo and colleagues have investigated the NET markers expression level such as myeloperoxidase-DNA complexes, and citrullinated histone H3 in the COVID-19 patients and compared it with healthy controls. Their results showed that there are higher levels of these markers in the COVID-19 patients. 131 The effects of COVID-19 on platelet activation are comprehensively investigated by Canzano and colleagues. They found that the COVID-19 patients represent higher levels of tissue factor (TF) positive platelets than healthy individuals and patients with coronary arthritis disease. Also, they found more up-regulation of P-selectin and formation of platelet-leukocyte aggregate in these patients.
Interestingly, they have observed activation of platelets in healthy individuals' blood after mixture with the plasma of the COVID-19 patients. 132,133

| Complement activation and coagulopathy in COVID-19
Innate immunity concidered as the first line of the body's defense against infectious agents. The complement system is a member of innate immune system and is comprised of a course of steps called complement cascade that trigger inflammation against infections and some other events. 134 The complement can be activated by classic, alternative and lectin pathways. Initiation of all of the mentioned pathways leads to the cleavage of a protein named C3. After cleavage, the protein will be divided into two proteins named C3a, which is a proinflammatory protein, and C3b, which opsonizes the pathogens and also continue the next steps. 135 C3b mediates the C5 cleavage and release C5a (anaphylatoxin) and, C5b, which is responsible for the formation of a membrane attack complex (C5b-9). 136 Albeit the complement plays a major role in the protection of the body, its overreaction can inflict heavy damage to different organs. 136,137 Besides acting as an anaphylatoxin, C5a increases the tissue factor activity, 138,139 and plays important role in coagulation. 140 Furthermore, two proteins that start the lectin pathway, namely MASP1, and MASP2, can activate thrombin by prothrombin cleavage 141 and also activate fibrinogen and factor XIII. 142,143 Different studies showed that inhibition of complement cascade resulted in consequences of anticoagulation effects such as prolonged bleeding. 140,[144][145][146] Most of our knowledge about the role of complements in coronavirus infection was obtained from studies on SARS-CoV-1 and MERS. 137 Gralinski and colleagues showed less severe symptoms in C3 deficient mice than intact mice, who were infected with SARS-CoV-1. 147 Jiang and colleagues observed elevated levels of C5a and higher expression of C5a receptor (C5aR) in leukocytes and pneumocytes of MERS-infected mice. Also, blockage of C5a-C5aR axis lightened the tissue damage in those mice. 148 In the SARS-CoV-2, Magro and colleagues reported deposition of complement fragments including MASP-2, C5b-9 and C4d microvasculature of lung tissue of patients with severe symptoms. 149 According to Gao and colleagues, the N protein on the surface of coronaviruses binds the MASP2 and triggers the complement cascade. 150 Lam and colleagues used flow cytometry to survey the formation of bonds between red blood cells and some products during complement activation such as C3b, iC3b, C3dg and C4d in hospitalized patients of COVID-19 compared with healthy individuals and they found a considerable increase in the COVID-19 patients. 151 Cugno and colleagues monitored the levels of C5b-9 and C5a and also VWF in hospitalized the COVID-19 patients.
They observed high levels of these factors in the patients. Moreover, they saw that their concentration decreased during the remission of these patients. 152 Altogether, this results illustrate the pivotal role of the complement in tissue damage as well as the development of coagulopathies and underlines the importance of preventive measures to control overreaction of this wing of the innate immunity.  169 In addition, some systematic reviews and meta-analyses have been done, and they have not shown an increased risk of mortality and hospitalization in using RAAS inhibitors. 170

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest, financial or otherwise.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study is available within the article.