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Interaction between blood coagulation and SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China in late December 2019. The virus causes multiple clinical complications in humans and can cause COVID-19 (Coronavirus Disease 2019). To date, SARS-CoV-2 has infected more than 132 million people and killed more than 2.88 million people worldwide.

COVID-19 is a heterogeneous disorder characterized primarily by fever, cough, acute respiratory distress syndrome (ARDS), and in-hospital death. Severe COVID-19 is associated with comorbidities such as cardiovascular disease, hypertension, diabetes and coagulopathy.

Coagulopathy-A condition in which blood’s ability to coagulate is impaired is recognized as an important aspect of morbidity in COVID-19 patients. In a recent study, Professor Lewis Cantley and a team of researchers investigated the role of coagulation factors in SARS-CoV-2 infection.Their recent research has been posted on bioRxiv*server.

“Coagulation-induced cleavage promotes spike activation and increases virus invasion into target cells.”

They found that proteases involved in coagulation could directly cleave SARS-CoV-2 glycoprotein spikes and promote viral entry. They also found that protease inhibitors indiscriminately inhibit spike cleavage by both transmembrane serine proteases and coagulation factors. Protease spike cleavage is essential for viral entry. Promotes membrane fusion and hijacking of host cells by the viral genome.

The· Spike protein It consists of S1 and S2 subunits. Host cell proteases act on the boundaries of the S1 / S2 subunits and in the S2’region proximal to the fusion peptide. This cut opens the spike trimer, exposing the S2’site. It must be cleaved to allow the release of the fusion peptide. One of the promising antiviral targets for SARS-CoV-2 is TMPRSS2, which is an important host cell factor in multiple proteolytic activations. Coronavirus..

Camostat and nafamostat are two broad-spectrum inhibitors that act on TMPRSS2. These two drugs are clinically approved in Japan (approved for other uses) and are not currently FDA approved in the United States. In this study, researchers found that nafamostat broadly inhibits cleavage of spike peptides by both transmembrane serine proteases and coagulation factors.

Even if the anti-SARS-CoV-2 vaccine is currently being administered worldwide, viral variants will emerge if large reservoirs of active cases continue to exist for extended periods of time.

The etiology of SARS-CoV-2 infection is associated with coagulopathy and thromboembolic events. Signs of dysregulated blood coagulation were evident in SARS-CoV-2 infected patients admitted to the hospital. These include 1) elevated D-dimer levels (indicators of fibrinolysis and coagulopathy), 2) decreased platelet counts (indicators of debilitating coagulopathy), and 3) systemic activity of coagulation factors V, VIII, and X. Was a rise.

The coagulation factor directly cleaves the SARS-CoV-2 spike. (A) Peptides from two known cleavage sites of the SARS-CoV-2 spike were designed using the C-terminal fluorophore 5-FAM and the N-terminal FRET quencher QXL-520. (B) Cleavage of a 10 μM S1 / S2 peptide substrate incubated with 125 nM TMPRSS2, factor Xa, or thrombin. The initial rate of cleavage of SARS-CoV-2 spikes S1 / S2 and S2'peptide substrates by (C) TMPRSS2, (D) Factor Xa, and (E) thrombin was measured in the 0-160 μM substrate range. From the initial rate values, the enzyme reaction rate constant (F) turnover rate Kcat (s-1), (G) affinity constant Km, and (H) specificity constant (Kcat / Km) include S1 / S2 and S2. Obtained for the indicated enzymes. 'peptide. The (IK) heatmap shows the initial rate of cleavage of the indicated peptide substrate V0 and the concentrations due to (I) TMPRSS2, (J) Factor Xa, and (K) thrombin.

The coagulation factor directly cleaves the SARS-CoV-2 spike. (A) Peptides from two known cleavage sites of the SARS-CoV-2 spike were designed using the C-terminal fluorophore 5-FAM and the N-terminal FRET quencher QXL-520. (B) Cleavage of a 10 μM S1 / S2 peptide substrate incubated with 125 nM TMPRSS2, factor Xa, or thrombin. The initial rate of cleavage of SARS-CoV-2 spikes S1 / S2 and S2’peptide substrates by (C) TMPRSS2, (D) Factor Xa, and (E) thrombin was measured in the 0-160 μM substrate range. From the initial rate values, the enzyme reaction rate constant (F) turnover rate Kcat (s-1), (G) affinity constant Km, and (H) specificity constant (Kcat / Km) include S1 / S2 and S2. Obtained for the indicated enzymes. ‘peptide. The (IK) heatmap shows the initial rate of cleavage of the indicated peptide substrate V0 and the concentrations due to (I) TMPRSS2, (J) Factor Xa, and (K) thrombin.

Because the coagulation cascade is regulated by a chain reaction of serine protease zymogens, each activated by proteolytic processing, researchers are wondering if the proteases involved in coagulation also play a role in SARS-CoV-2 spike cleavage. Was investigated.

They found that the coagulation factors serine protease factor Xa and thrombin directly cleave SARS-CoV-2 spikes and show more significant proteolytic activity on SARS-CoV-2 peptide substrates than TMPRSS2. did. They also found that the S1 / S2 boundary appears to be a factor Xa substrate that is even more optimal than a peptide substrate derived from a known physiological target of factor Xa in coagulation.

They reported that serine protease inhibitors suppress SARS-CoV-2 invasion through inhibition of TMPRSS2. They observed that TMPRSS2 is likely to be the primary cellular target for camostat and nafamostat.

In addition, they found that nafamostat is a versatile inhibitor of spike activation by various transmembrane serine proteases (TTSPs) and coagulation factors.

“We reduced the risk of artifacts by using multiple orthogonal platforms, including VSV-based pseudoviruses, HIV-based pseudoviruses, and syncytium formation.”

The researchers elaborated on the potential antiviral activity of anticoagulants, the risk of bleeding, and the clinical relevance of the limitations of this study.

COVID-19 related events (such as acute lung damage due to the cytopathic effect of the virus, etc. Cytokine storm, Complement activation, and antiphospholipid autoantibodies) are known to cause a coagulation cascade. Although the coordination between inflammation and hemostasis is well understood, researchers in this study point out that the exact molecular mechanism linking SARS-CoV-2 infection and hemostasis dysregulation remains unclear.

This study uses multiple platforms of FRET-based enzyme assays and pseudoviruses and cells.

In the fusion assay, researchers investigated and confirmed that circulating proteases involved in blood coagulation contribute to SARS-CoV-2 spike cleavage activation and thus enhance viral entry. Based on their study, researchers suggested that nafamostat is the most suitable multipurpose inhibitor for spike cleavage by TTSP and coagulation factors among the drugs currently available.

This study provides relevant data for investigating early intervention with carefully selected anticoagulant therapy for SARS-CoV-2 infection. Researchers emphasize that it is important to be prepared to mitigate future SARS-CoV-3 epidemics by understanding the interaction between the coronavirus and the host.

“Perhaps SARS-CoV-2 has been selected to induce and utilize a locally abundant environment for coagulation proteases, causing a positive feedback loop to promote entry into additional host cells. I am. “

Journal reference:

Interaction between blood coagulation and SARS-CoV-2 infection

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