Dysregulation of circulating protease activity in Covid-19-associated superinfection

  1. Fernando Dos Santos
  2. Joyce B. Li
  3. Nathalia Juocys
  4. Rafi Mazor
  5. Laura Beretta
  6. Nicole G. Coufal
  7. Michael T.Y. Lam
  8. Mazen F. Odish
  9. Maria C. Irigoyen
  10. Anthony J. O’Donoghue
  11. Federico Aletti
  12. Erik B. Kistler

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Abstract

Infection by SARS-CoV-2 and subsequent COVID-19 can cause viral sepsis and septic shock. Several complications have been observed in patients admitted to the intensive care unit (ICU) with COVID-19, one of those being bacterial superinfection. Based on prior evidence that dysregulated systemwide proteolysis is associated with death in bacterial septic shock, we investigated whether protease activity and proteolysis could be elevated in COVID-19-induced sepsis with bacterial superinfection. In particular, we sought to assess the possible implications on the regulation of protein systems, such as for instance the proteins and enzymes involved in the clotting cascade.

Blood samples collected at multiple time points during the ICU stay of four COVID-19 patients were analyzed to quantify: a) the circulating proteome and peptidome by mass spectrometry; b) plasma enzymatic activity of trypsin-like substrates and five clotting factors (plasmin, thrombin, factor VII, factor IX, factor X) by a fluorogenic assay.

Of the four patients, one was diagnosed with bacterial superinfection on day 7 after beginning of the study and later died. The other three patients all survived (ICU length-of-stay 11.25±6.55 days, hospital stay of 15.25±7.18 days). Spikes in protease activity (factor VII, trypsin-like activity) were detected on day 7 for the patient who died. Corresponding increases in the total intensity of peptides derived by hydrolysis of plasma proteins, especially of fibrinogen degradation products, and a general reduction of coagulation proteins, were measured as well. A downregulation of endogenous enzymatic inhibitors, in particular trypsin inhibitors, characterized the non-surviving patient throughout her ICU stay. Enzymatic activity was stable in the patients who survived.

Our study highlights the potential of multiomics approaches, combined with quantitative analysis of enzymatic activity, to i) shed light on proteolysis as a possible pathological mechanism in sepsis and septic shock, including COVID-19-induced sepsis; ii) provide additional insight into malfunctioning protease-mediated systems, such as the coagulation cascade; and iii) describe the progression of COVID-19 with bacterial superinfection.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.20.21265115: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The University of California, San Diego Institutional Review Board (IRB) provided approval of the study conduct (IRB# 190699, Protocol #20-0006); informed consent was obtained for the collection of blood samples and de-identified use of the data.
    Consent: The University of California, San Diego Institutional Review Board (IRB) provided approval of the study conduct (IRB# 190699, Protocol #20-0006); informed consent was obtained for the collection of blood samples and de-identified use of the data.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Polymorphprep™ was added to collected samples and centrifuged at 500g x 30 mins to separate plasma from white blood cells per manufacturer’s instructions (Progen) and transferred to new Eppendorf tubes and centrifuged at 3731 x g for 5 minutes to remove any cellular debris.
    Polymorphprep™
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    There are some limitations of this study that need to be acknowledged. First, regarding the clinically (obtained from the patient record) derived data, the low sample size of the studied patient population and non-uniformity in the timing of some laboratory results hinders the ability to make stronger inferences than are made here; the robustness of the presented results would doubtlessly have been strengthened with a larger sample size and fewer missing data. However, even with this very limited sample size we were able to make some cautious conclusions as to new hypotheses of molecular mechanisms that might otherwise not be recognized with a larger patient population. Second, regarding data drop-out, this was a real-world observational study, and the clinical laboratory measurements and values reflect patient management of severely ill COVID+ patients in the ICU. Third, in our analytical protocol, the mass spectrometry approach was necessarily untargeted; if specific protein systems and products of their degradation were targeted, it is possible that the potential dysfunction in these systems (e.g., coagulation, complement, etc.) could be highlighted in greater detail. Finally, when comparing and integrating proteomics and peptidomics data, the availability of transcriptomics data, which were not part of the experimental design of this observational study, could add information on protein regulation, and help understand to a greater depth the impact of abnormal proteolysis ...

    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 10.1101/2021.10.20.21265115v1 on medRxiv