Figure 2.
SARS-CoV-2–SP-PV–induced increased TF activity is dependent on ASMase and ACE2. (A) SARS-CoV-2–SP-PV infection induces the translocation of ASMase to the cell surface and reduces SM content in the plasma membrane. MDMs were infected with a control vehicle (CV) or infected with SARS-CoV-2–SP-PV (SP-PV) for 0.5 or 6 hours. Top of image: plasma membrane was stained with PKH67 fluorescent dye (green). For immunostaining ASMase, fixed and permeabilized cells were incubated with rabbit anti-human ASMase antibody (2 µg/mL) at 4°C overnight. After removing the primary antibodies and washing the cells, the cells were incubated with AF546-conjugated donkey anti-rabbit IgG (2 µg/mL) for 90 minutes (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The yellow color in the plasma membrane (white arrow marks) represents the colocalization of ASMase staining with PKH67 fluorescence in the plasma membrane, indicating the translocation of ASMase to the outer leaflet of the plasma membrane. The fluorescence signaling intensity of ASMase in the plasma membrane was quantified by choosing 30 random areas in the plasma membrane and using ImageJ software (National Institutes of Health). The quantified data were shown in a bar graph. Bottom of image: to label plasma membrane SM, fixed and nonpermeabilized cells were incubated with an SM-binding protein, lysenin (1 µg/mL), for 2 hours at room temperature, and then cells were incubated with rabbit anti-human lysenin antibody (1:200) overnight, followed by secondary antibody, AF546-conjugated donkey anti-rabbit IgG for 90 minutes (red). Nuclei were stained with DAPI (blue). The immunofluorescence staining of SM (red) was analyzed by confocal microscopy. White arrowheads point out SM in the outer plasma membrane in control (noninfected) cells. (B) ASMase silencing attenuates the SARS-CoV-2–SP-PV–induced increased TF activity. MDMs were transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ASMase (100 nM) using X-tremeGene transfection reagent. After 48 hours, transfected MDMs were infected with the pseudovirus for 6 hours, and EVs released into the supernatant medium were isolated. Cell surface TF activity and TF activity associated with EVs were measured as described in panel C. (C) ASMase functional inhibitors attenuate SARS-CoV-2–SP-PV–induced increased TF activity in MDMs. MDMs were pretreated with an ASMase inhibitor, desipramine (1 μM) or imipramine (1 μM), for 1 hour and then infected with the pseudovirus for 6 hours. EVs released into the supernatant medium were isolated. TF activity on the cell surface and associated with EVs was determined by adding FVIIa (10 nM) and the substrate factor X (175 nM) and measuring the rate of factor Xa generation. (D) ACE-2 silencing attenuates SARS-CoV-2–SP-PV–induced increased TF activity. MDMs were transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ACE-2 (100 nM). After 48 hours, the transfected MDMs were infected with the pseudovirus for 6 hours, and EVs released into the supernatant medium were isolated. TF activity on cell surfaces or EVs was measured as described for panel C. (E) ACE-2 silencing blocks SARS-CoV-2–SP-PV–induced ASMase translocation to the outer leaflet of the plasma membrane. MDMs transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ACE-2 (100 nM) were infected with the pseudovirus for 6 hours. After that, cells were fixed, permeabilized, and stained with rabbit anti-human ASMase antibody as described in panel A (red). Cells were also immunostained for ACE-2 (magenta). Nuclei were stained with DAPI (blue) and plasma membrane was stained with PKH67 fluorescent dye (green). White arrowheads point out ASMase translocated to the plasma membrane in the vehicle-treated or scRNA-transfected cells that were infected with the pseudovirus. The fluorescence signaling intensity of ASMase staining in the plasma membrane was quantified using Image J software. These data were shown in a bar graph. Statistical significance between the 2 groups was calculated using the Mann-Whitney test. *P < .05; ****P < .0001; ns, not statistically significant.

SARS-CoV-2–SP-PV–induced increased TF activity is dependent on ASMase and ACE2. (A) SARS-CoV-2–SP-PV infection induces the translocation of ASMase to the cell surface and reduces SM content in the plasma membrane. MDMs were infected with a control vehicle (CV) or infected with SARS-CoV-2–SP-PV (SP-PV) for 0.5 or 6 hours. Top of image: plasma membrane was stained with PKH67 fluorescent dye (green). For immunostaining ASMase, fixed and permeabilized cells were incubated with rabbit anti-human ASMase antibody (2 µg/mL) at 4°C overnight. After removing the primary antibodies and washing the cells, the cells were incubated with AF546-conjugated donkey anti-rabbit IgG (2 µg/mL) for 90 minutes (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The yellow color in the plasma membrane (white arrow marks) represents the colocalization of ASMase staining with PKH67 fluorescence in the plasma membrane, indicating the translocation of ASMase to the outer leaflet of the plasma membrane. The fluorescence signaling intensity of ASMase in the plasma membrane was quantified by choosing 30 random areas in the plasma membrane and using ImageJ software (National Institutes of Health). The quantified data were shown in a bar graph. Bottom of image: to label plasma membrane SM, fixed and nonpermeabilized cells were incubated with an SM-binding protein, lysenin (1 µg/mL), for 2 hours at room temperature, and then cells were incubated with rabbit anti-human lysenin antibody (1:200) overnight, followed by secondary antibody, AF546-conjugated donkey anti-rabbit IgG for 90 minutes (red). Nuclei were stained with DAPI (blue). The immunofluorescence staining of SM (red) was analyzed by confocal microscopy. White arrowheads point out SM in the outer plasma membrane in control (noninfected) cells. (B) ASMase silencing attenuates the SARS-CoV-2–SP-PV–induced increased TF activity. MDMs were transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ASMase (100 nM) using X-tremeGene transfection reagent. After 48 hours, transfected MDMs were infected with the pseudovirus for 6 hours, and EVs released into the supernatant medium were isolated. Cell surface TF activity and TF activity associated with EVs were measured as described in panel C. (C) ASMase functional inhibitors attenuate SARS-CoV-2–SP-PV–induced increased TF activity in MDMs. MDMs were pretreated with an ASMase inhibitor, desipramine (1 μM) or imipramine (1 μM), for 1 hour and then infected with the pseudovirus for 6 hours. EVs released into the supernatant medium were isolated. TF activity on the cell surface and associated with EVs was determined by adding FVIIa (10 nM) and the substrate factor X (175 nM) and measuring the rate of factor Xa generation. (D) ACE-2 silencing attenuates SARS-CoV-2–SP-PV–induced increased TF activity. MDMs were transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ACE-2 (100 nM). After 48 hours, the transfected MDMs were infected with the pseudovirus for 6 hours, and EVs released into the supernatant medium were isolated. TF activity on cell surfaces or EVs was measured as described for panel C. (E) ACE-2 silencing blocks SARS-CoV-2–SP-PV–induced ASMase translocation to the outer leaflet of the plasma membrane. MDMs transfected with transfection reagent (vehicle), scrambled oligonucleotide (scRNA), or siRNA specific for ACE-2 (100 nM) were infected with the pseudovirus for 6 hours. After that, cells were fixed, permeabilized, and stained with rabbit anti-human ASMase antibody as described in panel A (red). Cells were also immunostained for ACE-2 (magenta). Nuclei were stained with DAPI (blue) and plasma membrane was stained with PKH67 fluorescent dye (green). White arrowheads point out ASMase translocated to the plasma membrane in the vehicle-treated or scRNA-transfected cells that were infected with the pseudovirus. The fluorescence signaling intensity of ASMase staining in the plasma membrane was quantified using Image J software. These data were shown in a bar graph. Statistical significance between the 2 groups was calculated using the Mann-Whitney test. *P < .05; ****P < .0001; ns, not statistically significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal