Unique transcriptional changes in coagulation cascade genes in SARS-CoV-2-infected lung epithelial cells: A potential factor in COVID-19 coagulopathies

  1. Ethan S. FitzGerald
  2. Amanda M. Jamieson

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of COVID-19 (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19 associated coagulopathies are unknown. While there are many theories for the cause of this pathology, including hyper inflammation and excess tissue damage, the cellular and molecular underpinnings are not yet clear. By analyzing transcriptomic data sets from experimental and clinical research teams, we determined that changes in the gene expression of genes important in the extrinsic coagulation cascade in the lung epithelium may be important triggers for COVID-19 coagulopathy. This regulation of the extrinsic blood coagulation cascade is not seen with influenza A virus (IAV)-infected NHBEs suggesting that the lung epithelial derived coagulopathies are specific to SARS-Cov-2 infection. This study is the first to identify potential lung epithelial cell derived factors contributing to COVID-19 associated coagulopathy.

GRAPHICAL ABSTRACT

AUTHOR SUMMARY

Why was this study done?

  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic.

  • In addition to the acute pulmonary symptoms of COVID-19 (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients.

  • Currently, the molecular pathogenesis underlying COVID-19 associated coagulopathies are unknown. Understanding the molecular basis of dysregulated blood coagulation during SARS-CoV-2 infection may help promote new therapeutic strategies to mitigate these complications in COVID-19 patients.

What did the researchers do and find?

  • We analyzed three publicly available RNA sequencing datasets to identify possible molecular etiologies of COVID-19 associated coagulopathies. These data sets include sequencing libraries from clinically isolated samples of bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 positive patients and healthy controls. We also analyzed a publicly available RNA sequencing dataset derived from in vitro SARS-CoV-2 infected primary normal human bronchial epithelial (NHBE) cells and mock infected samples.

  • Pathway analysis of both NHBE and BALF differential gene expression gene sets. We found that SARS-CoV-2 infection induces the activation of the extrinsic blood coagulation cascade and suppression of the plasminogen activation system in both NHBEs and cells isolated from the BALF. PBMCs did not differentially express genes regulating blood coagulation.

  • Comparison with influenza A virus (IAV)-infected NHBEs revealed that the regulation of the extrinsic blood coagulation cascade is unique to SARS-CoV-2, and not seen with IAV infection.

What do these findings mean?

  • The hyper-activation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system in SARS-CoV-2 infected epithelial cells may drive diverse coagulopathies in the lung and distal organ systems.

  • The gene transcription pattern in SARS-CoV-2 infected epithelial cells is distinct from IAV infected epithelial cells with regards to the regulation of blood coagulation.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.06.182972: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 isolate USA-WA1/2020 (NR-52281) was propagated in Vero E6 cells, and viral titers were determined via plaque assay on Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Raw sequencing data were submitted to the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO Accession - GSE63473).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Read adapter and quality trimming was performed using the Trim Galore!
    Trim Galore
    suggested: (Trim Galore, RRID:SCR_011847)
    package41 and Sequencing quality was assessed using the FASTQC and MULTIQC packages.
    FASTQC
    suggested: (FastQC, RRID:SCR_014583)
    MULTIQC
    suggested: (MultiQC, RRID:SCR_014982)
    Sequence alignment to the GRCh38 reference transcriptome was performed using the Salmon read alignment software and differential gene expression analysis was performed using DESeq2 and the tximport packages.
    Salmon
    suggested: (Salmon, RRID:SCR_017036)
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Individual gene plots and heat maps were generated in R using the pheatmap or ggplot2 R packages to directly evaluate each gene’s differential expression.
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04333407RecruitingPreventing Cardiac Complication of COVID-19 Disease With Ear…
    NCT04365309Enrolling by invitationProtective Effect of Aspirin on COVID-19 Patients

    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.

    About SciScore

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  2. Version published to 10.1101/2020.07.06.182972v1 on bioRxiv