Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination

  1. Melissa Klug
  2. Olga Lazareva
  3. Kilian Kirmes
  4. Marc Rosenbaum
  5. Marina Lukas
  6. Simon Weidlich
  7. Christoph D. Spinner
  8. Moritz von Scheidt
  9. Rosanna Gosetti
  10. Jan Baumbach
  11. Jürgen Ruland
  12. Gianluigi Condorelli
  13. Karl-Ludwig Laugwitz
  14. Markus List
  15. Isabell Bernlochner
  16. Dario Bongiovanni

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet–leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

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  1. Version published to 10.1055/s-0041-1733934
  2. ScreenIT

    SciScore for 10.1101/2021.05.18.21257324: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All participants gave written informed consent.
    IRB: The study was approved by the local ethics committee (approval number 4/21 S-KH) and complies to the Declaration of Helsinki.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Platelets were stained with a custom-made CyTOF panel of 18 antibodies for 30 minutes (containing anti-CD3-170Er, anti-CD9-171Yb, anti-CD29-156Yb, anti-CD31-145Nd, anti-CD36-152Nd, anti-CD40-142Nd, anti-CD41-89Y, anti-CD42a-141Pr, anti-CD42b-144Nd, anti-CD47-209Bi, anti-CD61-146Nd, anti-CD62P-161Dy, anti-CD63-150Nd, anti-CD69-162Dy, anti-CD107a-151Eu, anti-CD154-168Er, anti-PAC1-155Gd and anti-Par1-147Sm antibodies; see major resources table for antibody information).
    anti-CD3-170Er
    suggested: None
    anti-CD9-171Yb
    suggested: None
    anti-CD29-156Yb
    suggested: None
    anti-CD31-145Nd
    suggested: None
    anti-CD36-152Nd
    suggested: None
    anti-CD40-142Nd
    suggested: None
    anti-CD41-89Y
    suggested: None
    anti-CD42a-141Pr
    suggested: None
    anti-CD42b-144Nd
    suggested: None
    anti-CD47-209Bi
    suggested: None
    anti-CD61-146Nd
    suggested: None
    anti-CD62P-161Dy
    suggested: None
    anti-CD63-150Nd
    suggested: None
    anti-CD69-162Dy
    suggested: None
    anti-CD107a-151Eu
    suggested: None
    anti-CD154-168Er
    suggested: None
    anti-PAC1-155Gd
    suggested: None
    anti-Par1-147Sm
    suggested: None
    Software and Algorithms
    SentencesResources
    After acquisition at the Helios CyTOF system (Fluidigm), samples were normalized, processed and pre-gated using the Cytobank™ software (www.cytobank.org, Beckman-Coulter, Brea, CA, USA)17
    Cytobank™
    suggested: (Cytobank, RRID:SCR_014043)
    Computational Analysis and clustering analysis with FlowSOM algorithm: We performed computational analysis with Cytobank and statistical tests were performed on the transformed data using the scipy python package (version 1.6.3)18.
    scipy
    suggested: (SciPy, RRID:SCR_008058)
    FlowSOM analysis was performed using the implementation in Cytobank.
    Cytobank
    suggested: (Cytobank, RRID:SCR_014043)
    FlowSOM analysis was run separately for each timepoint and condition using the same minimum spanning tree and seed with 10 000 events per sample.
    FlowSOM
    suggested: (FlowSOM, RRID:SCR_016899)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Avoiding the spectral limitation of flow-cytometry, this method allows a precise quantification of transmembrane proteins expression and it can be used to investigate the platelet expression phenotype with high-definition20. We here provide the first mass cytometric analysis of protein expression after SARS-CoV-2 vaccination. A recent study suggested a direct effect on thrombocytes of the spike protein S, sought to be mediated through the ACE2 receptor9. Zang et al. reported that the spike protein alone (without presence of the virus) can dose-dependently enhance platelet aggregation, ATP release and dense granule secretion. Moreover, they reported that the spike protein induces GPIIb/IIIa activation and P-Selectin expression even in the absence of agonists9. In contrast to these findings, we observed that the expression level of the activated form of GPIIb/IIIa (PAC-1) as well as of other activation markers - including P-Selectin - does not change after the spike protein upregulation mediated by vaccination with BNT162b2 (Figure 1A-B and supplemental figure S1). Of note, the expression profiles after vaccination differed from COVID-19 patients characterized by the same experimental pipeline and remained similar to an independent control cohort (Figure 2B). Platelet reactivity to physiological stimulus with TRAP was not altered after vaccination in contrast to the dysregulation observed during SARS-CoV-2 infection8,21. Clustering analysis of non-stimulated platelets did not d...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 19. 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.

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

    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.

  3. Version published to 10.1101/2021.05.18.21257324v1 on medRxiv