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

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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement -) California Breast Cancer Research Program of the University of California, RGPO Grant R01RG3766 (R00RG2541) -) US ARMY MEDICAL RESEARCH ACQUISITION ACTIVITY (USAMRAA) Award #W81XWH-17-2-0047. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of The University of California, San Diego gave approval for this study IRB# 190699, Protocol #20-0006). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data are available via ProteomeXchange with identifier PXD029181