SARS-CoV-2–SP-PV infection increases cell surface TF activity and releases TF⁺ vesicles in human MDMs. (A) MDMs were treated with control vehicle (CV) or infected with SARS-CoV-2–SP-PV (SP-PV) or vesicular stomatitis virus G protein envelope pseudotyped lentivirus, which was generated and isolated in the same fashion as of SARS-CoV-2–SP-PV, as a control pseudovirus (Con-PV). At the indicated time, the cell supernatants were removed, and the EVs released into the conditioned medium were isolated by centrifugation at 21 000g for 1 hour. TF activity associated with the cell surface or EVs was determined by adding FVIIa (10 nM) and the substrate factor X (175 nM) and measuring the rate of factor Xa generation in a chromogenic assay. (B-D) Analysis of total and cell surface TF protein expression in MDMs treated with CV or infected with SARS-CoV-2–SP-PV by immunoblot analysis (B), flow cytometry (C), or immunofluorescence confocal microscopy (D). (E-F) Intact MDMs infected with SARS-CoV-2–SP-PV (for 6 hours) or EVs released from the infected MDMs were incubated with polyclonal rabbit anti-human TF antibody (10 µg/mL) for 1 hour (E) or annexin V (400 nM) for 30 minutes (F) before measuring cell surface– or EV-associated TF activity as described in panel A. (G) SARS-CoV-2–SP-PV infection does not induce the externalization of phosphatidylserine. MDMs were treated with a control vehicle or infected with the pseudovirus for 6 hours and then stained with AF488–annexin V and subjected to fluorescence microscopy. As a positive control, MDMs were treated with HgCl₂ (5 µg/mL) for 5 minutes to externalize phosphatidylserine. All experiments were repeated at least 3 times, and data were expressed as mean ± standard error of the mean (SEM). Statistical significance between the 2 groups was calculated using the Mann-Whitney test and among the groups by 1-way analysis of variance followed by Tukey’s post hoc analysis. *P < .05; **P < .01; ns, not statistically significant (compared with values obtained with control vehicle treatment).