Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19

  1. Sokratis A. Apostolidis
  2. Amrita Sarkar
  3. Heather M. Giannini
  4. Rishi R. Goel
  5. Divij Mathew
  6. Aae Suzuki
  7. Amy E. Baxter
  8. Allison R. Greenplate
  9. Cécile Alanio
  10. Mohamed Abdel-Hakeem
  11. Derek A. Oldridge
  12. Josephine R. Giles
  13. Jennifer E. Wu
  14. Zeyu Chen
  15. Yinghui Jane Huang
  16. Jonathan Belman
  17. Ajinkya Pattekar
  18. Sasikanth Manne
  19. Oliva Kuthuru
  20. Jeanette Dougherty
  21. Brittany Weiderhold
  22. Ariel R. Weisman
  23. Caroline A. G. Ittner
  24. Sigrid Gouma
  25. Debora Dunbar
  26. Ian Frank
  27. Alexander C. Huang
  28. Laura A. Vella
  29. The UPenn COVID Processing Unit
  30. John P. Reilly
  31. Scott E. Hensley
  32. Lubica Rauova
  33. Liang Zhao
  34. Nuala J. Meyer
  35. Mortimer Poncz
  36. Charles S. Abrams
  37. E. John Wherry

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Abstract

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

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  1. Version published to 10.3389/fimmu.2022.834988
  2. ScreenIT

    SciScore for 10.1101/2021.05.01.442279: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Patients, subjects, and clinical data collection: Patients admitted to the Hospital of the University of Pennsylvania with a SARS-CoV-2 positive result were screened and approached for informed consent within 3 days of hospitalization (COVID-19 inpatient group).
    IRB: All participants or their surrogates provided informed consent in accordance with protocols approved by the regional ethical research boards and the Declaration of Helsinki.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Neutralization and inhibition assays: Neutralizing monoclonal antibodies against C3a (Biolegend, cat # 518105), C5a (R&D, cat # MAB2037-SP), IL-6 (Biolegend, cat # 501101) and CD16/32 (Biolegend, cat # 101302) were used for the neutralization assays.
    C3a
    suggested: None
    IL-6
    suggested: (GenWay Biotech Inc. Cat# GWB-78CDF3, RRID:AB_10514648)
    CD16/32
    suggested: (BioLegend Cat# 101302, RRID:AB_312801)
    A) Violin plots of gMFI expression of CD62P, CD63, CD32 and C3aR on the surface of control platelets incubated with COVID-19 plasma (n=10 patients) in the presence or absence of neutralizing antibodies to FcγRIIa, IL6, C3a and C5a, as indicated.
    CD62P
    suggested: None
    CD63
    suggested: None
    CD32
    suggested: None
    IL6
    suggested: None
    C5a
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Platelet aggregation on photochemically-injured endothelium in a microfluidic system: 6 × 106 cells/channel of human umbilical vein endothelial cells (HUVECs, ATCC-PCS-100-013) were seeded into the fibronectin (50 μg/mL, Sigma-Aldrich cat. #
    HUVECs
    suggested: None
    Software and Algorithms
    SentencesResources
    Platelet accumulation in the injured endothelium field was captured by Zeiss Axio Observer Z1 inverted microscope using Montage Fluxion software and analyzed using ImageJ as described (51).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Other statistical analysis was performed using Prism software (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04579393CompletedFostamatinib for Hospitalized Adults With COVID-19
    NCT04374539RecruitingPlasma Exchange in Patients With COVID-19 Disease and Invasi…
    NCT04355494No longer availableSOLIRIS® (Eculizumab) Treatment of Participants With COVID-1…

    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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  3. Version published to 10.1101/2021.05.01.442279v1 on bioRxiv