The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII

The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Main protease (M pro ) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII Vincenzo De Filippis, Anna Pagotto, Federico Uliana, Giulia Nordio, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4959212/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract The connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic. Although available data indicate that systemic inflammation sustains a hypercoagulant state and increases thrombotic risk, the underlying molecular mechanisms of COVID-19 associated coagulopathy remain unclear. Here we show that SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factor zymogens VII and XII at their physiological activation sites, involving Arg-Ile/Val bonds, where FVII and FXII are strategically positioned at the beginning of the extrinsic or intrinsic pathways of blood coagulation. Even though the observed cleavage sites are not compatible with the known substrate specificity of the protease, involving Gln-X bonds, the results of High Throughput Protease Screen assay unveil an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by enzymatic assays with synthetic peptide substrates containing Arg at P1 position. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel, additional mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19. Biological sciences/Biochemistry/Proteases Biological sciences/Microbiology/Virology/SARS-CoV-2 COVID-19 SARS-CoV-2 Main Protease thrombosis proteolysis proteomics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx S1 SupplementaryTable2.xlsx S2 SupplementaryTable3.xlsx S3 SupplementaryTable4.xlsx S4 Cite Share Download PDF Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4959212","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":372948093,"identity":"97d6cee1-c22b-4337-926a-af4166e0713e","order_by":0,"name":"Vincenzo De 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