Figure 2.
FVIIa-mediated EV generation from HUVECs is dependent on the EPCR-PAR1 signaling axis. (A) HUVECs were transfected with scrambled RNA (Scr RNA; 200 nM) or EPCR-specific siRNA (EPCR siRNA; 200 nM) using Lipofectamine RNAiMAX reagent. After 48 hours, cells were lysed, and EPCR expression was analyzed by western blotting using a human EPCR–specific antibody. (B) Scrambled RNA and EPCR siRNA-transfected cells were treated with control vehicle or FVIIa (100 nM). Twenty-four hours later, cell supernatants were collected, and EVs were isolated and quantified by NTA NanoSight. (C) HUVECs were preincubated with EPCR blocking antibody (EPCR B-mAb; JRK1494; 100 µg/mL) or nonblocking control antibody (EPCR NB-mAb; JRK1500; 100 µg/mL) for 1 hour prior to the addition of FVIIa (100 nM). EVs released from these cells after 24 hours were quantified by NTA NanoSight. (D) HUVECs were transfected with scrambled siRNA (100 nM) or PAR1-specific siRNA (PAR1 siRNA; 100 nM) in a similar approach as mentioned in panel A, and PAR1 knockdown was verified by western blotting with human PAR1–specific antibody. (E) Control and PAR1 siRNA-transfected cells were treated with FVIIa (100 nM) or control vehicle for 24 hours. EVs isolated from cell supernatants were quantified using NTA NanoSight. (F) HUVECs were pretreated with PAR1-specific blocking antibodies (PAR1 mAb; ATAP2; 25 µg/mL) or control immunoglobulin G (IgG; 25 µg/mL) for 1 hour before the addition of FVIIa (100 nM) or control vehicle. After 24 hours, EVs in cell supernatants were quantified by NTA NanoSight. (G) HUVECs were treated with control IgG (Con IgG; 10 µg/mL) or TF neutralizing antibody (αTF; 10 µg/mL) for 1 hour followed by FVIIa (100 nM) or a control vehicle. After 24 hours, EVs released into cell supernatant were quantified by NTA NanoSight. (H) HUVECs were transfected with scrambled siRNA (100 nM) or PAR2-specific siRNA (PAR2 siRNA, 100 nM). After 48 hours of transfection, PAR2 expression in cells was analyzed by western blotting. (I) PAR2 siRNA or scrambled RNA–transfected or control cells were treated with control vehicle or FVIIa (100 nM) for 24 hours, and EVs released in the supernatant culture medium were quantified by NTA NanoSight. (J) Murine brain endothelial cells were isolated from WT C57BL/6J (WT), EPCR knockout (EPCR-KO), EPCR-overexpressing (EPCR-OX), PAR1-R41Q mutant (PAR1-R41Q), and PAR1-R46Q–mutant (PAR1-R46Q) mice as mentioned in “Materials and methods.” Confluent monolayers of cells were treated with FVIIa (100 nM) or a control vehicle for 24 hours. EVs, isolated from the cell supernatants, were quantified by NTA NanoSight. Data are presented as mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001.

FVIIa-mediated EV generation from HUVECs is dependent on the EPCR-PAR1 signaling axis. (A) HUVECs were transfected with scrambled RNA (Scr RNA; 200 nM) or EPCR-specific siRNA (EPCR siRNA; 200 nM) using Lipofectamine RNAiMAX reagent. After 48 hours, cells were lysed, and EPCR expression was analyzed by western blotting using a human EPCR–specific antibody. (B) Scrambled RNA and EPCR siRNA-transfected cells were treated with control vehicle or FVIIa (100 nM). Twenty-four hours later, cell supernatants were collected, and EVs were isolated and quantified by NTA NanoSight. (C) HUVECs were preincubated with EPCR blocking antibody (EPCR B-mAb; JRK1494; 100 µg/mL) or nonblocking control antibody (EPCR NB-mAb; JRK1500; 100 µg/mL) for 1 hour prior to the addition of FVIIa (100 nM). EVs released from these cells after 24 hours were quantified by NTA NanoSight. (D) HUVECs were transfected with scrambled siRNA (100 nM) or PAR1-specific siRNA (PAR1 siRNA; 100 nM) in a similar approach as mentioned in panel A, and PAR1 knockdown was verified by western blotting with human PAR1–specific antibody. (E) Control and PAR1 siRNA-transfected cells were treated with FVIIa (100 nM) or control vehicle for 24 hours. EVs isolated from cell supernatants were quantified using NTA NanoSight. (F) HUVECs were pretreated with PAR1-specific blocking antibodies (PAR1 mAb; ATAP2; 25 µg/mL) or control immunoglobulin G (IgG; 25 µg/mL) for 1 hour before the addition of FVIIa (100 nM) or control vehicle. After 24 hours, EVs in cell supernatants were quantified by NTA NanoSight. (G) HUVECs were treated with control IgG (Con IgG; 10 µg/mL) or TF neutralizing antibody (αTF; 10 µg/mL) for 1 hour followed by FVIIa (100 nM) or a control vehicle. After 24 hours, EVs released into cell supernatant were quantified by NTA NanoSight. (H) HUVECs were transfected with scrambled siRNA (100 nM) or PAR2-specific siRNA (PAR2 siRNA, 100 nM). After 48 hours of transfection, PAR2 expression in cells was analyzed by western blotting. (I) PAR2 siRNA or scrambled RNA–transfected or control cells were treated with control vehicle or FVIIa (100 nM) for 24 hours, and EVs released in the supernatant culture medium were quantified by NTA NanoSight. (J) Murine brain endothelial cells were isolated from WT C57BL/6J (WT), EPCR knockout (EPCR-KO), EPCR-overexpressing (EPCR-OX), PAR1-R41Q mutant (PAR1-R41Q), and PAR1-R46Q–mutant (PAR1-R46Q) mice as mentioned in “Materials and methods.” Confluent monolayers of cells were treated with FVIIa (100 nM) or a control vehicle for 24 hours. EVs, isolated from the cell supernatants, were quantified by NTA NanoSight. Data are presented as mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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