key: cord-0937663-12huw6zj
authors: Elieh Ali Komi, Daniel; Rahimi, Yaghoub; Asghari, Rahim; Jafari, Reza; Rasouli, Javad; Mohebalizadeh, Mehdi; Abbasi, Ata; Nejadrahim, Rahim; Rezazadeh, Farzin; Shafiei-Irannejad, Vahid
title: Investigation of the Molecular Mechanism of Coagulopathy in Severe and Critical Patients With COVID-19
date: 2021-12-16
journal: Front Immunol
DOI: 10.3389/fimmu.2021.762782
sha: c636e9170021e976754f0400b22e996c7f1ced02
doc_id: 937663
cord_uid: 12huw6zj

Coagulopathy is a frequently reported finding in the pathology of coronavirus disease 2019 (COVID-19); however, the molecular mechanism, the involved coagulation factors, and the role of regulatory proteins in homeostasis are not fully investigated. We explored the dynamic changes of nine coagulation tests in patients and controls to propose a molecular mechanism for COVID-19-associated coagulopathy. Coagulation tests including prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen (FIB), lupus anticoagulant (LAC), proteins C and S, antithrombin III (ATIII), D-dimer, and fibrin degradation products (FDPs) were performed on plasma collected from 105 individuals (35 critical patients, 35 severe patients, and 35 healthy controls). There was a statically significant difference when the results of the critical (CRT) and/or severe (SVR) group for the following tests were compared to the control (CRL) group: PT(CRT) (15.014) and PT(SVR) (13.846) (PT(CRL) = 13.383, p < 0.001), PTT(CRT) (42.923) and PTT(SVR) (37.8) (PTT(CRL) = 36.494, p < 0.001), LAC(CRT) (49.414) and LAC(SVR) (47.046) (LAC(CRL) = 40.763, p < 0.001), FIB(CRT) (537.66) and FIB(SVR) (480.29) (FIB(CRL) = 283.57, p < 0.001), ProC(CRT) (85.57%) and ProC(SVR) (99.34%) (ProC(CRL) = 94.31%, p = 0.04), ProS(CRT) (62.91%) and ProS(SVR) (65.06%) (ProS(CRL) = 75.03%, p < 0.001), D-dimer (p < 0.0001, χ (2) = 34.812), and FDP (p < 0.002, χ (2) = 15.205). No significant association was found in the ATIII results in groups (ATIII(CRT) = 95.71% and ATIII(SVR) = 99.63%; ATIII(CRL) = 98.74%, p = 0.321). D-dimer, FIB, PT, PTT, LAC, protein S, FDP, and protein C (ordered according to p-values) have significance in the prognosis of patients. Disruptions in homeostasis in protein C (and S), VIII/VIIIa and V/Va axes, probably play a role in COVID-19-associated coagulopathy.

Coagulopathy is a frequently reported finding in the pathology of coronavirus disease 2019 ; however, the molecular mechanism, the involved coagulation factors, and the role of regulatory proteins in homeostasis are not fully investigated. We explored the dynamic changes of nine coagulation tests in patients and controls to propose a molecular mechanism for COVID-19-associated coagulopathy. Coagulation tests including prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen (FIB), lupus anticoagulant (LAC), proteins C and S, antithrombin III (ATIII), D-dimer, and fibrin degradation products (FDPs) were performed on plasma collected from 105 individuals (35 critical patients, 35 severe patients, and 35 healthy controls). There was a statically significant difference when the results of the critical (CRT) and/or severe (SVR) group for the following tests were compared to the control (CRL) group: PT CRT (15.014) and PT SVR (13. 

Coagulation is a dynamic process that is driven by the regulated proteolytic activation of zymogens (commonly known as coagulation factors) in injured vessels. Coagulation factors, except for FVIII, which is produced by liver sinusoidal endothelial cells and lymphatic tissue, are all produced by hepatocytes (1) . The main mechanisms (pathways) that trigger blood clotting include intrinsic and extrinsic pathways, each including a set of coagulation proteins in which factors I, II, IX, X, XI, and XII are the main factors in the intrinsic pathway and factors I, II, VII, and X are the factors described in the extrinsic pathway. Activated partial thromboplastin time (aPTT) and prothrombin time (PT) tests primarily measure the activity of the factors involved in the intrinsic and extrinsic pathways, respectively (2, 3) . Moreover, the common pathway is composed of factors I, II, V, VIII, and X (4). The proper proteolytic activation of coagulation factors controlled by a variety of regulatory proteins results in the conversion of soluble fibrinogen to insoluble fibrin strands (5) . Fibrinogen, a 340-kDa glycoprotein, is an acute-phase protein consisting of three polypeptide chains, Aa, Bb, and g, and becomes upregulated in response to injury and inflammation (5) . The term lupus anticoagulant (LAC) is used to determine heterogeneous immunoglobulins, their function resulting in the inhibition of phospholipid-dependent coagulation reactions (6) . Moreover, LACs can prolong the PTT test; therefore, a LAC test is used to evaluate prolonged PTT (7) . Coagulation regulatory proteins such as antithrombin III (ATIII), protein C, and Ddimer are involved in the normal function and homeostasis of the coagulation system. ATIII, a crucial anticoagulant molecule in mammalian blood, benefits from its cofactor, heparin, to inhibit the coagulation proteases, mainly thrombin and factor Xa (8) . Proteins C and S are vitamin K-dependent glycoproteins. Protein S, the cofactor for protein C, supports the activated protein C in the presence of phospholipids and calcium in the inactivation of membrane-bound factors V (FVa) and FVIIIa (9) . The mechanistic pathways through which protein C exerts its effects on the coagulation cascades include degrading factors V/ Va and VIII/VIIIa, releasing a tissue-type plasminogen activator, and stimulating fibrinolysis by interacting with the plasminogen activator inhibitor (10) . Fibrinolysis is an essential step in homeostasis that is finely controlled by a set of cofactors and inhibitors. Plasmin acts as the primary fibrinolysin and is activated from plasminogen in the presence of a tissue plasminogen activator (tPA) or urokinase (uPA) (11) . Plasmin, after being produced, lyses the cross-linked fibrin polymers and consequently forms fibrin degradation products (FDPs) such as D-dimer, which is widely used as a specific marker for thrombosis and physiological fibrinolysis (12) (Figure 1) . The coagulopathy and abnormal results in coagulation tests have become common features reported in patients with COVID-19 from the very early days of the emergence of the new coronavirus strain. We listed both the common coagulation tests, including PT, PTT, fibrinogen, and D-dimer, and those rarely investigated, such as regulatory proteins C and S as well as ATIII, in patients with COVID-19 along with the main results in Table 1 . COVID-19-dependent coagulopathy gained attention when PT, aPTT, fibrinogen, and D-dimer tests were recommended by researchers to evaluate the proper homeostasis of the system associated with the prognosis of patients. Moreover, the prophylactic use of anticoagulants was proven to be effective in lowering the mortality rate and highlighted the role of the coagulation system in COVID-19 (31) . The link between thrombosis and COVID-19 as an inflammatory disease has been investigated (32, 33) . In the present study, we used a coagulation panel of nine coagulation tests to assess the coagulation pathways in 105 included individuals to determine the molecular mechanism through which COVID-19 disrupts the homeostasis of the coagulation system.

We followed the guidelines for Corona Virus Disease 2019 edited by the Iranian National Health Commission (similar to the WHO guidelines and the New Coronavirus Pneumonia Prevention and Control Program, 7th edition, published by the National Health Commission of China) to classify the patients into critical and severe groups (34, 35) . The criteria used for the inclusion of individuals into each group are summarized in Table 2 . All 70 included patients had a positive result of the nucleic acid test of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by RT-PCR using primers targeting the RNA-dependent RNA polymerase (RdRP) and either nucleocapsid (N), envelope (E), or spike (S) genes. A negative result (using the same probes) was used as the main inclusion criterion for the control (CRL) group. All patients were tested for lung involvement by CT imaging. Moreover, individuals in the CRL group had no physical features of The average fibrinogen levels in the severe group was 384 ± 100 mg/ml and in the mild group was 311 ± 083 mg/ml (p = 0.14). 

Control a • Having a negative result for severe acute respiratory coronavirus 2 (SARS-CoV-2) by RT-PCR during the last 48 h (to exclude the chance of infection even at the earlier stage among enrolled healthy controls) •

No history of abnormal liver function tests (both direct and total bilirubin, SGOT, SGPT, ALKP) (considering that the majority of coagulation factors are produced in the liver, applying this criterion ensures no healthy control has a liver disease) •

No history of COVID-19 positivity reported from any immediate family member (to minimize the chance of getting infected from immediate family members during the time between PCR test and the time of blood collection. Additionally, it helps minimize the chance of being an asymptomatic carrier) •

Having no signs of fever, coughing, or other physical features of COVID-19 •

Having no history of hemorrhagic diseases in the past and present (any individuals with a history of recent hemorrhagic events such as a recent operation or menstruation in females were excluded to avoid impacts on the coagulation hemostasis) COVID-19 such as fever or coughing and never had a positive RT-PCR result before. We also checked immediate family history to exclude those who have and/or had a family member with a positive PCR test to exclude the possibility of including asymptomatic carriers as healthy controls. Considering that almost all coagulation factors are produced in the liver, any functional disorder in the organ may result in abnormal plasma levels of the factors; therefore, we performed liver functional tests (LFTs) for all 105 included individuals. were found with diabetes, one with kidney disease, and two with pulmonary disease. These patients were using metformin, glibenclamide, captopril, or losartan to control their chronic diseases, which have no effects on the coagulation factors. Due to the prophylactic guidelines for the administration of anticoagulation drugs to patients with poor health conditions, we collected the samples at admission before any medical intervention. Moreover, we checked for history of any drug use that could potentially interfere with our results by referring to medical insurance records and through collecting information using an enrollment form. Assuming an a value set at 0.05 (type I error), b at 0.10 (type II errors), a dropout rate of 5%, and considering the results of Gao et al. (15) , the sample size was set at a minimum of 35 patients in each group to compare the differences between the means. Patients in the severe and critical groups were selected from hospitalized patients in COVID and ICU wards, respectively, from either Urmia General Hospital or Taleghani Hospital in Urmia, Iran.

Approximately 1.8 ml of peripheral blood was collected into tubes containing 0.2 ml sodium citrate (3.2%). The tubes were immediately gently mixed and centrifuged (1,200 × g, 10 min), and the appearance of the plasma was checked to exclude icteric (abnormal function of the liver), lipemic (as a preclinical error especially in photometric assays) (36) , and hemolyzed specimens (37) or tubes with micro-clots (38) . Considering that prolonged storage of plasma specimens negatively affects the results of coagulation tests (39), we managed to perform this study at the peak of the fifth wave of the disease in Iran in order to include as many patients as possible. This strategy helped us collect all the required samples rapidly and to perform the coagulation tests quickly without freezing the plasma samples. All tests were run within 3 h after sample collection. According to the partially low stability of D-dimer in plasma (40), and using semi-quantitative kits to measure D-dimer and FDPs, we performed these tests before the other tests.

The 

After obtaining the samples, we checked them twice before and after putting them on the mixer to exclude any samples with visible signs of clotting or micro-clots. After 5 min of mixing the samples, they were centrifuged and the obtained plasma samples were analyzed for D-dimer and FDPs; then, the plasma samples were poured into conventional plastic tubes and loaded into the autoanalyzer. To perform semi-quantitative D-dimer and FDP tests, we used the glycine buffer to dilute each plasma sample in plastic test tubes. For the D-dimer test, we added 20 ml of reagent 1 (including ready-to-use latex particles coated with mouse antihuman D-dimer monoclonal antibody) to 20 ml of undiluted/ diluted plasma samples of each individual, mixed gently, and assessed the agglutination. The interpretation of the results was done using the protocol provided in Table 4 . A similar protocol was used for the FDP test, but with only two diluted concentrations/titers (1:2 and 1:8) assessed for agglutination according to the instruction of the manufacturer. We performed the rest of the tests using a fully automated STA 

The data obtained from the autoanalyzer for PT, PTT, fibrinogen, lupus anticoagulant, proteins C and S, and ATIII (quantitative tests) and for D-dimer and FDPs (semi-quantitative tests) were reported. Statistical analysis was performed using SPSS (ver. 21; IBM, Armonk, NY, USA). To compare the groups, we used one-way ANOVA, Fisher's exact test, and chi-square tests. A p-value <0.05 was considered to be statistically significant.

According to the results, there was no association between the age, weight, and BMI of individuals (p = 0.92, 0.03, 0.54, respectively). The elevated PT test results have been frequently reported in previous investigations worldwide. There was a significant difference among the three studied groups, in which the CTL group had the highest levels of D-dimer, whereas the CRL group had the lowest levels. The results for FDP also revealed that the majority of healthy controls (34 out of 35) had FDP levels below 5 mg/ml, while only 1 was found to have FDP levels between 5 and 20 mg/ml. In the SVR group, 31 (88.6%) patients had FDP levels below 5 mg/ml and 4 (11.4%) had FDP levels between 5 and 20 mg/ml. In contrast, only 23 patients in the CTL group had FDP levels below 5 mg/ml, and 9 (25.7%) were found to have levels between 5 and 20 mg/ml. There were 3 (8.6%) patients with FDP levels over 20 mg/ml. According to Figures 5D-F , the results for each group showed that patients in the CTL group had the highest levels of FDP, while healthy controls had the lowest levels.

We analyzed the data for the deceased individuals (11 out of 35 in the CTL group) and compared them with those of survivors in the same group in order to obtain a better understanding of the impact of abnormal coagulation test results on the fate of the patients. We marked these patients with black bullet points in 

In this section, we provide recommendations for further investigations ( Table 8 ) and address our limitations. We included 35 individuals in each group. Recruiting more patients will provide more accurate results in prospective studies. The enrollment of more patients provides the opportunity to determine cutoffs and design an alarm panel to be used in ICUs. We also applied a single-sampling strategy; however, monitoring the results by obtaining at least 2-3 samples in the CTL and SVR groups could more effectively monitor the test results and their association with the outcomes. One limitation of this study was the use of latex-based semiquantitative kits to assess D-dimer and FDPs. The results will be more reliable when both tests are performed using fully automated methods. Although the biofunctions of proteins S and C as biological regulators of factors V and VIII are well documented, we did not assess these two factors.

In the present study, we investigated the dynamic changes in 9 coagulation tests on 105 individuals classified into CTL, SVR, and CRL groups. Our study revealed significant aberrant coagulation changes among the studied groups in 4 aspects: extrinsic and intrinsic pathways, fibrinolysis, and the regulatory factors. Our results were consistent with those of the majority of 

Considering the low stability of D-dimer and fibrin degradation products (FDPs) over time, we strongly recommend performing these two tests immediately after plasma separation.

If the study is aimed to be performed on a high number of individuals or it is not possible to collect samples from all individuals in a short time, in which the plasma samples should be stored until running the tests, we recommend monitoring the effect of storage on samples. For this purpose, several samples with low, normal, and high results for the PT and partial thromboplastin time (PTT) tests can be frozen with other samples in separately labeled microtubes to evaluate the test results every 12 or 24 h by comparing the results with those from plasma samples before freezing.

Considering that pregnant women with physiological pregnancy have higher levels of D-dimer and fibrinogen, we recommend not including them as controls. Additionally, including them in the patient group may result in exaggerated results.

Fibrinogen levels may vary widely in several bio/pathologic situations, i.e., rise after menopause, rise in diabetes and hypertension, or decrease in alcoholics. We recommend considering such situations in the questionnaire to simply exclude unfit individuals.

Considering that PO 2 pressure is a critical factor in placing patients in the critical (CTL) and severe (SVR) groups and that it may vary during a single day in COVID-19 patients, we suggest placing patients with the lowest values into the CTL group and those with the highest into the SVR group and avoiding placing patients with PO 2 values near the cutoff.

Unmet questions C4b-BP has been reported to regulate proteins C and S. Since our results magnified the role of these two regulatory proteins in COVID-19-dependent coagulopathy, investigation of the association between the activity of proteins C and S and the concentration of C4b-BP can be helpful. (49, 50) The links between gene mutation and polymorphisms in coagulation regulatory proteins and coagulation disorders have been reported. Studying the association between the SNPs of proteins C and S and ATIII with the prognosis of COVID-19 in patients with coagulopathy could be beneficial.

Heparin therapy is widely recommended in patients with COVID-19. Considering that it acts as the cofactor for ATIII to inhibit thrombin and factor Xa, an investigation on the impact of heparin therapy on thrombin time (TT) and factor Xa activity may be an interesting theme for further research.

Proteins C and S regulate the conversion of V to Va and VIII to VIIIa. We suggest investigating these 6 factors for their possible association with the fate of critically ill patients. ) results when compared to the SVR and CRL groups, indicating a disruption in the extrinsic coagulation pathway. In addition, the prolonged PTT results in the CTL group and also similar results in the LAC test showed that not only the extrinsic pathway but even the intrinsic pathway was dysregulated. It should be considered that, in critically ill patients, lupus anticoagulant could be positive. An elevated fibrinogen level was one of the main findings in COVID-19-associated coagulopathy. We showed that there was a significant difference in the fibrinogen levels among the three groups and that the CTL group had the highest levels. It can be used as a common biomarker to predict the severity of the disease; however, the analysis of fibrinogen levels in deceased patients in the CTL group with the whole group showed that it had no significance in predicting death. Investigation of ATIII revealed that its activity was not significantly interrupted in COVID-19 patients (p = 0.321). However, proteins C and S, the other regulatory proteins, showed a significant decrease in their activity levels (p = 0.04 and p < 0.001, respectively). The difference between the reported p-values for these proteins was probably due to the low number of individuals recruited in each group; increasing the sample size will provide more accurate data. Considering that proteins C and S regulate the conversion of factors V and VIII to their active forms, we conclude that the disruption of homeostasis in protein C (and S) regulating the conversion of factors V and VIII to their active form could be a mechanism for COVID-19-associated coagulopathy. The fibrinolysis pathway was also affected in the presence of SARS-COV-2, in which the production of FDPs, mainly D-dimer, was accelerated, and according to our results, deceased patients were found to have significantly higher FDP and D-dimer levels when compared to survivors. The majority of coagulation factors are produced in the liver; to prevent the effects of hepatopathy on the levels of the coagulation factors and the corresponding tests, we enrolled normal controls and patients whose liver function tests were normal. Interestingly, factors including FVIII and vWF (which act as markers of endothelial activation) (53) were produced in the endothelial cells. Investigation of the levels of these factors in COVID-19 patients revealed that their levels increased and may correlate with poorer prognosis (54) (55) (56) . We showed that D-dimer, fibrinogen, PT, PTT, LAC, protein S, FDPs, and protein C (ordered according to their p-values) could effectively be used in the prognosis of the severity of the disease and that disruptions in proteins C and S regulating the conversion of factors V and VIII to their active form may interfere the homeostasis of the coagulation system.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the 

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We would like to thank the Cellular and Molecular Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences for the financial support.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.