Interaction between blood clotting 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 triggers multiple clinical complications in humans and can lead to COVID-19 (coronavirus disease 2019). To date, SARS-CoV-2 has infected over 132 million individuals and caused over 2.88 million deaths worldwide.

COVID-19 is a heterogeneous disease, characterized primarily by fever, cough, and acute respiratory distress syndrome (ARDS), and nosocomial mortality. Severe COVID-19 has been associated with comorbidities such as cardiovascular disease, hypertension, diabetes mellitus, and coagulopathy.

Coagulopathy – a condition in which the ability of blood to coagulate is reduced, is recognized as an important aspect of morbidity in patients with COVID-19. In a recent study, prof. Lewis Cantley and a team of researchers investigated the role of coagulation factors in SARS-CoV-2 infection. Their recent study was published on bioRxiv* server

“Coagulation-induced cleavage enhances spike activation and increases virus entry into target cells.”

They found that proteases involved in clotting can also directly separate the SARS-CoV-2 glycoprotein class, increasing virus entry. They also found that protease inhibitors promiscuously inhibit spike cleavage by both transmembrane serine proteases and coagulation factors. Cleavage of the spike with proteases is crucial for virus entry; facilitates membrane fusion and host cell abduction by the viral genome.

The spike protein consists of S1 and S2 subunits. Host cell proteases act at the S1 / S2 subunit and in the S2 ‘region proximal to the fusion peptide. This cleavage opens the spike trimmer and exposes the S2 ‘site, which must be cleaved to allow fusion peptide release. One of the promising targets of antiviral drugs for SARS-CoV-2 is TMPRSS2 which is an important host cell factor in the proteolytic activation of multiple coronaviruses.

Camostat and Nafamostat are two broad-spectrum inhibitors and act against TMPRSS2. Although these two drugs are clinically (approved for other applications) in Japan, they are not currently approved by the FDA for any of the indications in the United States. In this study, the researchers found that Nafamostat largely inhibited spike peptide cleavage by both transmembrane serine proteases and coagulation factors.

Although SARS-CoV-2 vaccines are currently being administered worldwide, the continued existence of a large reservoir of active cases over a long period of time will cause the emergence of viral variants.

The pathogenesis of SARS-CoV-2 infection has been associated with coagulopathy and thromboembolic events. Signs of blood clotting disorders were seen in patients infected with SARS-CoV-2 who were admitted to hospitals. These were 1) elevated levels of D-dimer (indicator of fibrinolysis and coagulopathy), 2) low platelet count (indicator of consuming coagulopathy), 3) increased systemic activity of coagulation factors V, VIII and X.

Coagulation factors directly cleave the SARS-CoV-2 spiral.  (A) Peptides derived from two known cleavage sites of the SARS-CoV-2 spike were designed with a C-terminal fluorophore 5-FAM and an N-terminal FRET quencher QXL-520.  (B) Cleavage of 10 μM peptide substrate S1 / S2 incubated with 125 nM TMPRSS2, factor Xa or thrombin.  The initial cleavage rates of S1 / S2 and S2 'peptides of the SARS-CoV-2 spike using (C) TMPRSS2, (D) factor Xa and (E) thrombin were measured in the range of 0-160 μM of substrate.  From the initial velocity values, the kinetic constants of the enzyme (F) of the rotational speed Kcat (s-1), (G) the affinity constant Km and (H) the specificity constant (Kcat / Km) for the indicated enzymes with S1 / S2 and S2 'peptides were obtained.  (IK) Heat charts show the initial rate of V0 cleavage of the indicated peptide substrates and the concentration of (I) TMPRSS2, (J) factor Xa and (K) thrombin.

Coagulation factors directly cleave the SARS-CoV-2 spiral. (A) Peptides derived from two known cleavage sites of the SARS-CoV-2 spike were designed with a C-terminal fluorophore 5-FAM and an N-terminal FRET quencher QXL-520. (B) Cleavage of 10 μM peptide substrate S1 / S2 incubated with 125 nM TMPRSS2, factor Xa or thrombin. The initial cleavage rates of S1 / S2 and S2 ‘peptides of the SARS-CoV-2 spike using (C) TMPRSS2, (D) factor Xa and (E) thrombin were measured in the range of 0-160 μM of substrate. From the initial velocity values, the kinetic constants of the enzyme (F) of the rotational speed Kcat (s-1), (G) the affinity constant Km and (H) the specificity constant (Kcat / Km) for the indicated enzymes with S1 / S2 and S2 ‘peptides were obtained. (IK) Heat charts show the initial rate of V0 cleavage of the indicated peptide substrates and the concentration of (I) TMPRSS2, (J) factor Xa and (K) thrombin.

Because the coagulation cascade is organized by a zymogen chain reaction of serine protease, each activated by proteolytic processing, the researchers investigated whether proteases involved in coagulation and a role in cleavage of SARS-CoV-2 scores.

They found that coagulation factors, serine protease factor Xa and thrombin, directly cleave the SARS-CoV-2 class, with more significant proteolytic activity against SARS-CoV-2 peptide substrates than TMPRSS2. They also found that the S1 / S2 boundary appears to be an even more optimal factor Xa substrate than peptide substrates derived from known physiological targets of factor Xa in coagulation.

Serine protease inhibitors have been reported to suppress the entry of SARS-CoV-2 by inhibiting TMPRSS2. They observed that TMPRSS2 was probably the primary cellular target of camostat and nafamostat.

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

“We mitigated the risk of artifacts using multiple orthogonal platforms, including VSV-based pseudovirus, HIV-based pseudovirus, and syncytial formation.”

The researchers explained in detail the clinical importance of the potential antiviral activity of anticoagulants, the risks of bleeding, and the limitations in this study.

Events associated with COVID-19 (such as acute lung injury from viral cytopathic effects, cytokine storm, complement activation, and antiphospholipid autoantibodies) are known to induce a coagulation cascade. Although the coordination between inflammation and hemostasis is well understood, the researchers in this study point out that the precise molecular mechanism linking SARS-CoV-2 infection and the disorder of hemostasis regulation is not yet clear.

In this study, Using FRET-based enzyme assay and multiple pseudovirus and cell platforms

by fusion tests, the researchers investigated and confirmed that the circulating proteases involved in blood clotting contribute to the activation of SARS-CoV-2 cleavage and thus promote virus entry. Based on their study, the researchers suggested that nafamostat, among currently available drugs, is the most suitable as a multi-purpose inhibitor against spina bifida by TTSP and coagulation factors.

This study provides relevant data for the study of early intervention with reasonably selected anticoagulant treatment for SARS-CoV-2 infection. The researchers emphasize that preparedness to mitigate a future SARS-CoV-3 epidemic is crucial to achieving an understanding of host-coronavirus interactions.

“Perhaps SARS-CoV-2 was subjected to selection to induce and utilize an environment locally enriched with coagulation proteases, encouraging positive feedback to promote entry into additional host cells.”

Journal reference:

  • Coagulation factors directly cleave the SARS-CoV-2 jump and enhance virus entry, Edward R. Kastenhuber, Javier A. Jaimes, Jared L. Johnson, Marisa Mercadante, Frauke Muecksch, Yiska Weisblum, Yaron Bram, Robert E. Schwartz, Gary R. Whittaker, Lewis C. Cantley, bioRxiv 2021.03.31.437960; doi: https://doi.org/10.1101/2021.03.31.437960, https://www.biorxiv.org/content/10.1101/2021.03.31.437960v1

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