Platelets are hyper-activated in COVID-19. However, the mechanisms promoting platelet activation by SARS-CoV-2 are not well understood. This may be due to inherent challenges at discriminating the contribution of viral versus host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles, as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself, or components derived from SARS-CoV-2-infected human lung epithelial cells, could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions and the release of extracellular vesicles. We find that neither SARS-CoV-2 nor purified Spike activate platelets. In contrast, TF produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and extracellular vesicles were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with virus or extracellular vesicles, which were indistinguishable. Increased TF mRNA expression and activity were also found in lungs in a murine model of COVID-19 and in plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere in platelet activation and coagulation that is characteristic of COVID-19.
