The main protease (M(pro)) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII.

Although the connection between COVID-19 and coagulopathy has been clear since the beginning of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain elusive. Available data support that the hyper-coagulant state is sustained by systemic inflammation. Here we show that the SARS-CoV-2 main protease (M(pro)) can play a direct role in the activation of coagulation. Adding M(pro) to human plasma increased clotting probability by 3-fold. Enzymatic assays and degradomics analysis indicate that M(pro) cleaves and activates coagulation factors VII and XII. This activity is compatible with an extended secondary specificity of M(pro) for R↓X that diverge from its well-established preference for LQ↓X. This finding is supported by HDX-MS characterization of the M(pro) complex with an Arg-containing inhibitor, as well as the proteolytic cleavage of the peptide FTRLR↓SLEN by M(pro). Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.