Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19

  1. Yu Zuo
  2. Shanea K. Estes
  3. Ramadan A. Ali
  4. Alex A. Gandhi
  5. Srilakshmi Yalavarthi
  6. Hui Shi
  7. Gautam Sule
  8. Kelsey Gockman
  9. Jacqueline A. Madison
  10. Melanie Zuo
  11. Vinita Yadav
  12. Jintao Wang
  13. Wrenn Woodard
  14. Sean P. Lezak
  15. Njira L. Lugogo
  16. Stephanie A. Smith
  17. James H. Morrissey
  18. Yogendra Kanthi
  19. Jason S. Knight

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Abstract

Some patients with severe COVID-19 develop prothrombotic autoantibodies that are similar to antiphospholipid antibodies found in autoimmune diseases.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.15.20131607: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study complied with all relevant ethical regulations, and was approved by the University of Michigan Institutional Review Board (HUM00179409), which waived the requirement for informed consent given the discarded nature of the samples.
    IACUC: Experimental protocols were approved by the University of Michigan Institutional Animal Care and Use Committee (PRO00008113), and all relevant ethical regulations were followed.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMale C57BL/6 mice were purchased from The Jackson Laboratory and used for experiments at 10-12 weeks of age.
    Cell Line AuthenticationAuthentication: Neutrophil preparations were at least 95% pure as confirmed by both flow cytometry and nuclear morphology.

    Table 2: Resources

    Antibodies
    SentencesResources
    These various units are in accordance with the international consensus guidelines on aPL testing from the 13th International Congress on Antiphospholipid Antibodies (88).
    Antiphospholipid
    suggested: None
    First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human myeloperoxidase antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 μg/ml in coating buffer (Cell Death kit).
    anti-human myeloperoxidase
    suggested: (Bio-Rad Cat# 0400-0002, RRID:AB_617350)
    The plate was washed five times, and then incubated for 90 minutes at room temperature with 10x anti-DNA antibody (HRP-conjugated; from the Cell Death kit) diluted 1:100 in blocking buffer.
    anti-DNA
    suggested: None
    The primary antibody was against neutrophil elastase (Abcam 21595, diluted 1:100), and the FITC-conjugated secondary antibody was from SouthernBiotech (4052-02, diluted 1:250).
    neutrophil elastase (Abcam 21595
    suggested: (Abcam Cat# ab21595, RRID:AB_446409)
    Non-specific binding was blocked with 4% non-fat milk, followed by incubation with primary antibody directed against citrullinated histone H3 (Abcam 5103).
    citrullinated histone H3
    suggested: None
    Detection was with an HRP-labeled anti-rabbit secondary antibody and an HRP-labeled β-actin antibody, followed by detection using chemiluminescence.
    anti-rabbit
    suggested: None
    β-actin
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Male C57BL/6 mice were purchased from The Jackson Laboratory and used for experiments at 10-12 weeks of age.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Data analysis was with GraphPad Prism software version 8.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    This study has some limitations. We did not have access to the fresh plasma that would be required for lupus anticoagulant testing (which would have provided additional context and risk stratification to the aPL profiling). We speculate that many of the patients with positive aPS/PT would have displayed a lupus anticoagulant phenotype—as reported recently (26)—but proving that will require further study and prospective access to samples. We were also not able to define a clear link between circulating aPL and large artery/vein thrombosis. Eleven patients in our cohort had thrombotic events and 55% of those were positive for aPL. It should be noted that aggressive anticoagulation has been regularly employed at our institution including many patients treated prophylactically with therapeutic doses of anticoagulation. It should also be noted that aPL were not tested on a defined hospital day, but rather when a sample became available to the research laboratory; future studies should endeavor to systematically track aPL over the full course of a hospitalization, and perhaps especially at and after the time of discharge. In the meantime, and as we await definitive antiviral and immunologic solutions to the current pandemic, we posit that testing aPL, including aPS/PT, may lead to improved risk stratification and personalization of treatment for COVID-19, and that aPL are at least a part of the complex COVID-19 thromboinflammatory milieu.

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

    IdentifierStatusTitle
    NCT04391179Active, not recruitingDipyridamole to Prevent Coronavirus Exacerbation of Respirat…

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 28. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1126/scitranslmed.abd3876
  3. Version published to 10.1101/2020.06.15.20131607v2 on medRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.06.15.20131607: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThis study complied with all relevant ethical regulations , and was approved by the University of Michigan Institutional Review Board ( HUM00179409) , which waived the requirement for informed consent given the discarded nature of the samples .RandomizationInterestingly , a small study from China showed that dipyridamole significantly suppressed D-dimer elevation and improved platelet counts in patients with COVID-19; however , prospective , randomized clinical trials are needed to evaluate clinical outcomes42 . “Criteria” aPL are defined based on their inclusion in the updated Sapporo classification criteria: namely , anticardiolipin IgG and IgM , anti-Blindingnot detected.Power Analysisnot detected.Sex as a biological variableMale C57BL/6 mice were purchased from The Jackson Laboratory and used for experiments at 10-12 weeks of age.Cell Line AuthenticationNeutrophil preparations were at least 95% pure as confirmed by both flow cytometry and nuclear morphology.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antiphospholipid syndrome (APS) is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies (aPL) targeting phospholipids and phospholipid-binding proteins.
    aPL) targeting phospholipids and phospholipid-binding proteins.
    suggested: None
    We detected anticardiolipin IgM antibodies in 23%, anti-PS/PT IgG in 24%, and antiPS/PT IgM in 18%.
    anti-PS/PT IgG
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>antiPS/PT IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The majority of positives were associated with three antibodies: anti-PS/PT IgG , anticardiolipin IgM , and anti-PS/PT IgM.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>three antibodies: anti-PS/PT IgG</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anticardiolipin IgM</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-PS/PT IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DISCUSSION aPL are a heterogeneous group of autoantibodies that play an important role in the pathogenesis of APS via their interactions with plasma protein such as β2GPI , prothrombin , thrombomodulin , plasminogen , antithrombin III , protein C , protein S , annexin II , annexin V , and likely others10-15 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>plasma protein such as β2GPI , prothrombin , thrombomodulin , plasminogen , antithrombin III , protein C , protein S , annexin II , annexin V</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Retrospective studies have suggested that anti-β2GPI IgA antibodies are significantly associated with thrombosis in lupus patients ( OR 2.8 , 95 % CI 1.3-6.2)44 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-β2GPI IgA</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">While the clinical significance of non-criteria aPL such as anti-PS/PT during viral infection remains to be fully defined , we demonstrate here that IgG fractions containing high levels of these antibodies trigger NET release in vitro and accelerate thrombosis in vivo .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-PS/PT</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Defining the extent to which these samples may contain aPL or other autoantibodies , in addition to protective anti-COVID-19 antibodies , is another potential area for future investigation .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-COVID-19</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">First , a high-binding EIA/RIA 96-well plate ( Costar ) was coated overnight at 4ºC with anti-human myeloperoxidase antibody ( Bio-Rad 0400-0002) , diluted to a concentration of 1 µg/ml in coating buffer ( Cell Death kit) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human myeloperoxidase</b></div>
        <div>suggested: (Bio-Rad Cat# 0400-0002, <a href="https://scicrunch.org/resources/Any/search?q=AB_617350">AB_617350</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plate was washed five times , and then incubated for 90 minutes at room temperature with 10x anti-DNA antibody ( HRP-conjugated; from the Cell Death kit ) diluted 1:100 in blocking buffer .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-DNA</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primary antibody was against neutrophil elastase (Abcam 21595, diluted 1:100), and the FITCconjugated secondary antibody was from SouthernBiotech (4052-02, diluted 1:250).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>neutrophil elastase (Abcam 21595</b></div>
        <div>suggested: (Abcam Cat# ab21595, <a href="https://scicrunch.org/resources/Any/search?q=AB_446409">AB_446409</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analysis was with GraphPad Prism software version 8.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    • This study has several limitations.
    • We did not have access to the fresh plasma that would be required for lupus anticoagulant testing (which would have provided additional context and risk stratification to the aPL profiling).
    • We speculate that many of the patients with positive anti-PS/PT would have displayed a lupus anticoagulant phenotype-as reported recently

    Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  5. Version published to 10.1101/2020.06.15.20131607v1 on medRxiv