Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (ScreenIT)
Abstract
Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.
Article activity feed
-
-
SciScore for 10.1101/2021.03.31.437960: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cell Culture: Calu3, A549, Caco2, and Vero cells were tested for mycoplasma (Lonza MycoAlert detection kit) and human cell line identity was authenticated by ATCC. Table 2: Resources
Antibodies Sentences Resources Prior to infection of target cells, the viral stock was incubated with anti-VSV-G antibody (3ug/ml) for 1 h at 37°C to neutralize contaminating rVSVΔG/NG/NanoLuc/VSV-G particles. anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell Culture: Calu3, A549, Caco2, and Vero … SciScore for 10.1101/2021.03.31.437960: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cell Culture: Calu3, A549, Caco2, and Vero cells were tested for mycoplasma (Lonza MycoAlert detection kit) and human cell line identity was authenticated by ATCC. Table 2: Resources
Antibodies Sentences Resources Prior to infection of target cells, the viral stock was incubated with anti-VSV-G antibody (3ug/ml) for 1 h at 37°C to neutralize contaminating rVSVΔG/NG/NanoLuc/VSV-G particles. anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell Culture: Calu3, A549, Caco2, and Vero cells were tested for mycoplasma (Lonza MycoAlert detection kit) and human cell line identity was authenticated by ATCC. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)A549 and Vero cells were grown in DMEM, supplemented with 10% FBS, 100U/ml Penicillin, and 100ug/ml Streptomycin. A549suggested: NoneCalu3 and Caco2 cells were grown in MEM, supplemented with 10% FBS, 100U/ml Penicillin, 100ug/ml Streptomycin, 1% MEM NEAA and 1mM sodium pyruvate. Caco2suggested: NoneLentiviral vectors were co-transduced with MD2G and PAX2 in 293T cells (5 million cells/10cm plate) with 25ul of XtremeGene9 (Millipore Sigma, #6365787001) and supernatant was harvested at 48hr and 72hr post transfection. 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Calculation of enzyme constants was performed with Graphpad Prism software (version 9.0). Graphpad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GFP+RFP+ cells were quantified using a BD Attune NxT Flow cytometer (Thermo Fisher Scientific) and data was analyzed in FlowJo version 10.7.1. FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Limitations: The experiments of this study, like prior studies using similar techniques, have some limitations. Protease enzymatic assays on peptide substrates allow for detailed biochemical characterization of a specific site, but peptide substrates may not have the equivalent three-dimensional conformation or post-translational modifications of the full-length protein produced in appropriate cells. For instance, SARS-CoV-2 S is extensively glycosylated (Watanabe et al., 2020). Pseudovirus and syncytia formation provide complementary functional assays, dependent on homotrimeric spike protein produced in human cells. The possibility of additional spike cleavage sites and potential pro- and anti-viral consequence of proteases acting on cell surface proteins including ACE2 cannot be excluded. The amount, density, and accessibility of spike protein could be different between these cell-based surrogate assays and wild type SARS-CoV-2 infection. However, antibody neutralization is highly correlated between authentic virus and corresponding pseudotyped viruses, suggesting similar conformation (Schmidt et al., 2020). We have mitigated the risk of artifact by using multiple orthogonal platforms, including VSV-based pseudovirus, HIV-based pseudovirus, and syncytia formation.
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT02735707 Recruiting Randomized, Embedded, Multifactorial Adaptive Platform Trial… NCT04505774 Recruiting Anti-thrombotics for Adults Hospitalized With COVID-19 (ACTI… NCT04372589 Active, not recruiting Antithrombotic Therapy to Ameliorate Complications of COVID-… Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-
