Platelet proteome analysis reveals an early hyperactive phenotype in SARS-CoV-2-infected humanized ACE2 mice

  1. Saravanan Subramaniam
  2. Ryan Matthew Hekman
  3. Archana Jayaraman
  4. Aoife Kateri O’Connell
  5. Paige Montanaro
  6. Benjamin Blum
  7. Devin Kenney
  8. Maria Ericsson
  9. Katya Ravid
  10. Nicholas A Crossland
  11. Andrew Emili
  12. Florian Douam
  13. Markus Bosmann

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Abstract

Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19.

Key Points

  • SARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation.

  • Complement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi.

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    SciScore for 10.1101/2021.08.19.457020: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Methods: Ethics statement: All experimental procedures with animals were approved by the Boston University Biomedical Research, Institutional Biosafety Committee and Institutional Animal Care and Use Committee.
    Sex as a biological variableSARS-CoV-2 infection: 14 week old C57BL6J and K18-hACE2 transgenic female mice were intranasally inoculated with 1×106 PFU of SARS-CoV-2 in 50 μl of sterile 1X PBS and sham mice were inoculated with 50 μl of sterile 1X PBS.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following antibodies were used: Mouse-SARS-CoV-2 spike protein (clone: E7U6O; Cell Signaling Technology, USA); rabbit-CD61 (clone: ARC0460; Invitrogen, USA)
    Mouse-SARS-CoV-2 spike protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    African green monkey kidney Vero E6 cells (ATCC® CRL-1586™, American Type Culture Collection, Manassas, VA) were used to propagate the viral isolate in vitro.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL/6J mice and heterozygous humanized ACE2 (K18-hACE2) mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from the Jackson Laboratory (Bar Harbor, ME).
    C57BL/6J
    suggested: RRID:MGI:3589388)
    SARS-CoV-2 infection: 14 week old C57BL6J and K18-hACE2 transgenic female mice were intranasally inoculated with 1×106 PFU of SARS-CoV-2 in 50 μl of sterile 1X PBS and sham mice were inoculated with 50 μl of sterile 1X PBS.
    C57BL6J
    suggested: None
    Recombinant DNA
    SentencesResources
    For absolute quantitation of viral RNA, a 389 bp fragment from the SARS-CoV-2 E gene was cloned onto pIDTBlue plasmid under an SP6 promoter using NEB PCR cloning kit (New England Biosciences, Ipswich, MA).
    pIDTBlue
    suggested: RRID:Addgene_117418)
    Software and Algorithms
    SentencesResources
    Desalted peptides (20 μg) were labeled with Tandem Mass Tags (TMT) using TMTPro-16plex isobaric tags (ThermoFisher Scientific; #A44520) as per manufacturer’s instructions.
    ThermoFisher Scientific
    suggested: None
    Analysis of raw mass spectrometry data: All acquired MS/MS spectra were simultaneously searched against the complete SwissProt mouse proteome (downloaded on 2020-10-20) and the Uniprot SARS-CoV-2 proteome (downloaded on 2020-05-03) using MaxQuant (Version 1.6.7.0), which integrates the Andromeda search engine.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    Whole slide image analysis: Slides were imaged on the HALO™ image analysis platform using a Mantra 2.0™ quantitative pathology workstation and unmixed with InForm software version 2.4.8 (Akoya Biosciences, Marlborough, MA, USA).
    InForm
    suggested: (inForm, RRID:SCR_019155)
    Statistical analysis: Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software, LLC) by one-way ANOVA (multiple groups) and Bonferroni multiple-comparisons test, or student-t test (2 groups).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Entrez and Ensembl IDs were mapped using biomaRt (v2.44.4) and only proteins with mapped IDs were used for visualization and downstream analysis.
    biomaRt
    suggested: (biomaRt, RRID:SCR_019214)
    Volcano plots were constructed using ggplot2 (v3.3.3).
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Venn diagrams were constructed using VennDiagram (v1.6.20) and VennDetail (v1.4.0).
    VennDiagram
    suggested: (VennDiagram, RRID:SCR_002414)
    Heatmaps were generated using ComplexHeatmap (v2.4.3) package with unsupervised clustering, and the dendrograms were reordered for a more meaningful visualization using dendsort (v0.3.3) and dendextend (v1.14.0) packages.
    ComplexHeatmap
    suggested: (ComplexHeatmap, RRID:SCR_017270)
    Gene Ontology (GO), including biological process (BP), molecular function (MF) and cellular component (CC), was analyzed using enrichR (v3.0) and gProfiler2 (v0.2.0).
    enrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Pathway analyses based on Kyoto Encyclopedia for Genes and Genomes (KEGG) and Reactome were performed using clusterProfiler (v3.16.1) and ReactomePA (v1.32.0), respectively.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    clusterProfiler
    suggested: (clusterProfiler, RRID:SCR_016884)
    To understand interactions between proteins in nine key processes and pathways that were enriched in either 2dpi or 4dpi data sets, an interactome was constructed and visualized using the stringApp (v1.6.0) in Cytoscape (v3.8.2).
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Data sharing statement: The proteomics data presented in this manuscript and raw data files will be deposited in the ProteomeXchange Consortium (http://www.proteomexchange.org) upon the acceptance of the manuscript.
    http://www.proteomexchange.org
    suggested: (ProteomeXchange, RRID:SCR_004055)

    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: 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: No clinical trial numbers were referenced.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.08.19.457020v1 on bioRxiv