Interplay between coagulopathy and inflammation in patients with COVID-19

Background: Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a systemic disease. Most patients have mild-to-moderate symptoms; however, severe respiratory symptoms develop in some. Although increased inflammatory markers and abnormal coagulation profile are related to the severity and mortality of COVID-19, no definitive relation has been established. Materials and Methods: The laboratory data of 199 patients with COVID-19 admitted to our dedicated COVID-19 care facility, categorized into mild (36), moderate (76), and severe (87) cases, were analyzed for a statistical association between the inflammatory markers and coagulation profile. IBM SPSS V23 software, Chicago, was used for data analysis. Results: A gradual increase in serum IL-6, ferritin, and D-dimer levels were observed from mild, moderate, and severe categories with statistically significant differences among all three groups. A positive correlation was observed between serum IL-6 and D-dimer (r² = 0.36, P < 0.01) only in the severe group. Among the three parameters, D-dimer had the best discriminating ability between severe and nonsevere disease with a serum cutoff level of 451 ng/mL. Conclusion: Thrombin has pro-inflammatory action. Similarly, IL-6 induces the release of acute-phase reactants like fibrinogen. Because of this interplay, patients with severe COVID-19 are at a dual risk of inflammatory and thromboembolic complications and must be monitored stringently.

Keywords: Coagulation-inflammation interplay; COVID-19; D; -dimer; ferritin; IL-6

INTRODUCTION

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first reported in December 2019 from Wuhan, China, has now become a global threat affecting more than half of the world population.[[1]] As of November 4, 2021, 247,968,227 cases and 5,020,204 deaths had been reported worldwide. The situation has been grave in India, too, with 34,321,025 infected cases and a mortality of 459,652. Delhi, the capital of India, was also severely hit by the COVID-19 waves.[[2]]

Although the SARS-CoV-2 primarily affects the lower respiratory tract, with the most common presenting complaints of COVID-19 disease being a runny nose, dry cough, sore throat, fever, fatigue, and myalgia, it has been seen that this virus can also affect other systems of the body, such as gastrointestinal system, cardiovascular system, hematopoietic system, immune system, and neurological systems. Most individuals recover quickly after suffering from a mild-to-moderate disease. Still, in some cases, the infection progresses rapidly to life-threatening conditions such as acute respiratory distress syndrome (ARDS), sepsis, or thromboembolic events. Many studies have been conducted to explore the risk and precipitating factors involved in the pathogenesis and progression of COVID-19 infection. Studies have shown a positive correlation between cytokine storms and clinical deterioration in patients with COVID-19.[[1]] Increased levels of serum IL-6 and ferritin levels, which are known inflammatory biomarkers, are found to be associated with the severe form of COVID-19 disease and poor prognosis. Also, it is evident from research that COVID-19 is a hypercoagulable state with an increased risk of thromboembolic events.[[3]] Raised plasma D-dimer levels have been linked with severe ARDS and increased mortality. Although increased levels of serum inflammatory biomarkers and an abnormal coagulation profile are individually related to severity and mortality in COVID-19, no definitive relationship has been established.[[1]]

Here, we aimed to characterize the inflammatory and coagulation profile of patients with mild, moderate, and severe COVID-19 disease, focusing on changes in serum levels of IL-6, Ferritin, and D-dimer.

MATERIALS AND METHODS

Study design and participants

One hundred and ninety-nine COVID-19-positive patients admitted to Lok Nayak Hospital, New Delhi, a dedicated COVID-19 care facility, were taken as study subjects. Based on the severity of illness, patients were classified as mild, moderate, and severe according to the clinical management protocol for COVID-19 given by All India Institute of Medical Sciences (AIIMS), New Delhi: "Clinical guidance for management of adult patients with COVID-19."[[4]]

Data collection

Their laboratory data from the Laboratory Information System was analyzed for a statistical association between the inflammatory biomarkers, IL-6 and ferritin, and D-dimer levels. The confidentiality of patients was maintained. Ethical approval was not needed per the Institutional Ethics Committee, as this was a retrospective study.

Laboratory methods

The serum samples were analyzed on Cobas e411 (Roche Diagnostics, Switzerland) for serum inflammatory biomarkers using an electrochemiluminescence assay. The coagulation profile was assessed in plasma samples on ACL Elite Pro (Instrumentation Laboratory, USA).

Statistical analysis

IBM SPSS V23 software, Chicago, Illinois was used for the statistical analysis of data. The serum IL-6, ferritin, and plasma D-dimer levels in different groups were analyzed using Kruskal–Wallis Test. Spearman's rho, a nonparametric test, was used to measure the strength of association among these parameters. A value of P < 0.05 (two-tailed) was considered statistically significant for all analyses.

RESULTS

One hundred and ninety-nine samples were used for statistical data analysis. They were categorized into three groups: mild, moderate, and severe, based on the severity of illness, according to the clinical management protocol for COVID-19 given by All India Institute of Medical Sciences (AIIMS), New Delhi: "Clinical guidance for management of adult patients with COVID-19."[[4]] The mild group included patients with COVID-19 with upper respiratory tract symptoms (and fever) without shortness of breath or hypoxia. In contrast, the moderate group had patients of COVID-19 with respiratory rate ≥24/min, SpO2 90%–93% on room air, and breathlessness. Those patients with COVID-19 with respiratory rate >30/min, SpO2<90% on room air, and breathlessness were categorized in the severe group. Therefore, our study included 36 mild cases, 76 moderate cases, and 87 severe COVID-19 cases.

Laboratory analysis

From descriptive statistics, the mean levels of IL-6, ferritin, and D-dimer in mild, moderate, and severe cases were calculated, which showed an increasing trend as the severity of the illness increased. The range of serum IL-6 levels was observed as 1 to 82 pg/mL, 2.2 to 197 pg/mL, and 7.9 to 3787 pg/mL in mild, moderate, and severe groups, respectively. The range of serum ferritin levels was between 7.41 to 1069 ng/mL, 37.2 to 2057 ng/mL, and 52.3 to 17990 ng/mL in mild, moderate, and severe groups, respectively. The mild, moderate, and severe groups had plasma D-dimer levels between 145 to 5000 ng/mL, 194 to 5000 ng/mL, and 192 to 5000 ng/mL, respectively.

The median and interquartile range (IQR) of serum IL-6, Ferritin, and D-dimer levels in the mild group was 8.15 pg/mL (4.19–23.32), 174 ng/mL (57.5–396) and 208 ng/mL (192–245), respectively. The moderate group included patients with median serum IL-6, Ferritin, and D-dimer levels of 29.4 pg/mL (9.7–58.95), 579 ng/mL (215–1204) and 497.5 ng/mL (310–972), respectively. The severe group included patients with the highest serum median levels of IL-6, Ferritin, and D-dimer, that is, 46.6 pg/mL (15.7–111), 598 ng/mL (362–1215) and 691 ng/mL (369–1137), respectively [Figure 1]. Kruskal–Wallis test was used to check for statistical differences across the three groups for all parameters measured. The examination revealed significant differences (χ2 = 24.9, P < 0.05, df = 2) in serum IL-6 levels among the three groups with H = 53 (mild), 97 (moderate), and 116 (severe). A significant difference (χ2 = 25, P < 0.05, df = 2) in serum Ferritin levels for the three groups with H = 48 (mild), 105 (moderate), and 110 (severe) was also noted with a significant difference (χ2 = 42.3, P < 0.05, df = 2) in D-dimer levels as well amidst the three groups with H = 33 (mild),102 (moderate), and 116 (severe).{Figure 1}

Correlation analysis

Spearman's rho analysis performed depicted a positive correlation between serum IL-6 and ferritin in the mild group (r2 = 0.60, P < 0.01) as well as the moderate group (r2 = 0.37, P < 0.01). In the severe group, a positive correlation was observed between serum IL-6 and D-dimer (r2 = 0.36, P < 0.01) [Figure 2] and [Table 1].{Figure 2} {Table 1}

Receiver operating characteristic (ROC) curve analysis

The receiver operating characteristic (ROC) curve was used to assess the diagnostic efficiency of inflammatory parameters and D-dimer in categorizing severe and non-severe COVID-19 disease. Nonsevere is inclusive of both the mild and moderate groups. The area under the curve (AUC), asymptomatic 95% confidence interval, and the P value is shown in the table. Among the three parameters, D-dimer had the best discriminating ability between severe and non-severe disease with a sensitivity of 65% and specificity of 57% with a serum cutoff level of 451 ng/mL [Figure 3] and [Table 2].{Figure 3} {Table 2}

DISCUSSION

The study found that serum IL-6 and D-dimer levels rise with the increasing severity of COVID-19 illness. However, a significant correlation between them was only seen in the severe group. As the progression of COVID-19 is affected by an immune response and the coagulative state of the body, it is critical to know the interdependence between them, thereby its consequences and possible management protocol for associated complications.

Compared with other RNA viruses such as Ebola, Lyssa, and Dengue, where hemorrhagic episodes are more common, coronaviruses predominantly cause thrombotic complications in 16%–49% of severe cases.[[3]] COVID-19 disease has a wide range of presenting symptoms, ranging from mild to severe. It affects several body organs, and the severity of the illness depends upon several factors. SARS-CoV-2 affects the lungs differently in different individuals—an increase in inflammatory cytokines known as cytokine storm has been reported in critically ill COVID-19 pneumonia patients. The activation of monocytes by granulocyte monocyte colony-stimulating factor (GM-CSF) leads to an increased or excessive production of IL-6 via a positive feedback mechanism, further accentuated by extracellular neutrophil traps.[[1]] Under normal physiological conditions, the cytokine release protects the body against viruses, but their overexpression has been seen to damage the body's systems. SARS-CoV-2 is a cytopathic virus that undergoes pyroptosis; it generates an inflammatory response, initially a local reaction but gradually progressing to a systemic inflammatory response.[[5]] The uncontrolled cytokine storm in COVID-19 pneumonia results in acute systemic inflammation characterized by high-grade fever and multi-organ dysfunction, called cytokine release syndrome (CRS). In patients with COVID-19, raised serum levels of pro-inflammatory cytokines (monocyte chemo-attractant protein (MCP-1), IFN-γ, IP-10, and IL-1β) and IL-10 and IL-4, which inhibits the inflammatory response, are found.[[1]] An imbalance between anti-inflammatory and pro-inflammatory markers in severe COVID-19 cases predisposes them to microthrombus formation, disseminated intravascular coagulopathy (DIC), and associated complications.[[3]]

Ferritin is well known to be an acute phase reactant apart from its main role in iron storage. It has two subunits, namely H and L, with H subunit expression regulated by inflammatory cytokines. Raised serum ferritin levels seen in patients with COVID-19 can have more than one underlying contributing factor, like a direct release from macrophages or increased synthesis under the influence of cytokines released by macrophages. The H subunit of ferritin has an immunomodulatory function; thus, ferritin induces pro-inflammatory and anti-inflammatory cytokines.[[6]] As reported by Chen et al., raised serum ferritin levels were found in 63% of patients with COVID-19.[[7]] In a study by Brandtner, a significant correlation was found between Ferritin and SOFA score (Sequential Organ Failure Assessment).[[8]] Also, plasma exchange therapy further discloses the role of Ferritin and IL-6, as symptoms have improved in most patients after receiving plasma exchange therapy.[[6]]

Another important complication in critically ill patients with COVID-19 is the increased risk of thromboembolic events evident from high D-dimer levels in admitted patients. D-dimer in plasma or whole blood is a marker for activated fibrinolysis and coagulation pathway. This helps early predict life-threatening complications like deep vein thrombosis (DVT) and pulmonary embolism (PE). Although consumptive coagulopathy is a prominent feature in sepsis-induced coagulopathy (SIC) and DIC, coagulopathy takes a different course in COVID-19 pneumonia.[[9]] The increased thrombin production seen in DIC results from increased expression of tissue factors from mononuclear cells and endothelial cells, which are induced by inflammatory cytokines such as IL-6. Although increased expression of IL-6 and other pro-inflammatory cytokines have also been observed in severe patients with COVID-19, overproduction of thrombin has not yet been established.[[7]]

Both coagulation and innate immunity play an important role in concert to counteract the invasion of the virus and the following cascade.[[7]] The activated coagulation is a possible explanation for raised D-dimer levels associated with increased mortality in patients with COVID-19. On the lookout to delineate molecules and mechanisms that connect coagulation and inflammation, the role of tissue factors in blood vessels was crucial. Thrombin acts via the coagulation–inflammation axis to bring out its hemostatic and thrombotic effects. The coagulation pathway occurs when it cleaves fibrinogen to produce an insoluble fibrin clot. Also, thrombin is a strong platelet activator, thus accelerating the path. However, it can also activate plasminogen activator inhibitor-1 (PAR-1) on endothelial cells and fibroblasts, which leads to the release of monocyte chemoattractant protein-1, TNFα, IL-1β, and IL-6.[[10]] Cross-talk between inflammation and coagulation is physiologically essential as it helps localize the infection and inflammation at the injury site through fibrins. However, inflammation-induced coagulation may sometimes contribute pathologically to critical conditions like coagulopathy associated with sepsis in cases of severe infection. The studies have shown evidence that the primary mediators of inflammation-induced coagulation are pro-inflammatory cytokines, with IL-6 playing a vital role in the initiation of coagulation activation and IL-1 having a significant role in the regulation of the physiological anticoagulation pathway. Antithrombin (AT), the protein C system, and tissue factor pathway inhibitor (TFPI) are necessary anticoagulants that keep a check on the coagulation system. Specific cell receptors on inflammatory cells and endothelial cells mediate the inflammatory activity driven by coagulation. AT binds to syndecan-4, a receptor on the cell surfaces of neutrophils, monocytes, and lymphocytes. As a result, it interferes with their interaction with endothelial cells and impairs inflammatory mediator release and activation.[[11]] As AT is a negative acute phase reactant, they are markedly decreased as a result of the inflammatory response, apart from being consumed in the coagulation process. Thus an impaired procoagulant-anticoagulant balance leading to a disbalance between the inflammatory and coagulation system can result in complications like microthrombus and multi-organ failure.

CONCLUSION

In patients with COVID-19, there is a synergistic interaction between the inflammatory response and the coagulation system. Thrombin (activated factor II), a participant in the coagulation pathway, also has pro-inflammatory action. Similarly, IL-6 induces the release of acute-phase reactants like C-reactive protein and fibrinogen. Although IL-6, ferritin, and D-dimer are raised in patients with COVID-19, it is significant in severe cases, as evident from this study. Because of this interplay, patients with severe COVID-19 are at a dual risk of inflammatory and thromboembolic complications and must be monitored stringently.

Ethical policy and institutional review board statement

Members of the Institutional Ethics Committee of Maulana Azad Medical College and Associated Hospital, New Delhi, India, have reviewed the larger research protocol entitled "Association Between Nutritional and Inflammatory Biomarkers in COVID-19 Patients with chronic kidney disease" in its meeting held on December 28, 2020 and found it suitable. Accordingly, they have approved the proposed research study communicated vide letter no. 1/IEC/MAMC/(182/10/2020/No. 234 dated January 14, 2021. The database of the above-stated research study was used to get data for the proposed study (IEC Registration no. ECR/329/Inst/DL/2023/RR-2019).

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.