SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19

  1. Lize M. Grobbelaar
  2. Chantelle Venter
  3. Mare Vlok
  4. Malebogo Ngoepe
  5. Gert Jacobus Laubscher
  6. Petrus Johannes Lourens
  7. Janami Steenkamp
  8. Douglas B. Kell
  9. Etheresia Pretorius

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.

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  1. Version published to 10.1042/bsr20210611
  2. ScreenIT

    SciScore for 10.1101/2021.03.05.21252960: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethical clearance: Ethical clearance for the study was obtained from the Health Research Ethics Committee (HREC) of Stellenbosch University (South Africa) (reference: N19/03/043, project ID: 9521).
    Consent: The experimental objectives, risks, and details were explained to volunteers both verbally and in text and informed consent were obtained prior to blood collection.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableSample demographics and considerations: Blood was collected from healthy volunteers (N=11; 3 males, 8 females; mean age 43.4 ± 11.7) to serve as controls.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    A total of 1 ⍰g trypsin (New England Biosystems) was added to the plasma for 1:50 enzyme to substrate ratio.
    New England Biosystems
    suggested: (Penn Cell Center Stockroom, RRID:SCR_010003)

    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:
    A further limitation of this exploration is the use of PPP in investigating clot formation at a scale appropriate to the microvasculature. While the protocol enables the study of fibrin microclots, which are of interest in COVID-19, it excludes the influence of RBCs, which are known to heavily influence the non-Newtonian flow behaviour of blood at that scale (McHedlishvili, 1998). The inaccuracy of the flow regime arising from this exclusion and from the variability of viscosity introduces error into the results. Nonetheless, the inclusion of flow an appropriate spatial scale has enabled us to observe COVID-19 PPP clot formation over space and time, under dynamic conditions, and has given insights which would otherwise prove difficult to glean. Mass spectrometry confirmed that spike protein causes structural changes to β and γ fibrin(ogen), complement 3 and prothrombin. These proteins become less resistant to trypsinization and changes the conformation, in such a way that there is a significant difference in peptide structure before and after spike protein addition.

    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.
    • No funding statement was detected.
    • 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.

  3. Version published to 10.1101/2021.03.05.21252960 on medRxiv