Figure 2.
Figure 2. The FVIIa-mediated anti-inflammatory effect requires its protease activity, EPCR, PAR1, and β-arrestin-1. (A-C) FVIIa protease activity requirement. Serum-starved HUVECs were treated with FVIIa or active-site–inhibited FVIIa or FVIIaAS (100 nM) for 1 hour and then left unperturbed (NS) or stimulated with TNF-α (5 ng/ml) for 6 hours. ICAM-1 (A), VCAM-1 (B), and IL-6 (C) mRNA levels were measured by quantitative RT-PCR. (D-F) EPCR-blocking or PAR1 antibodies attenuate the anti-inflammatory effect of FVIIa. HUVECs were incubated with EPCR blocking (bl.) or nonblocking (nbl.) mAb (25 µg/mL) or a mixture of PAR1 mAb (ATAP-2, 25 µg/mL, and WEDE15, 20 µg/mL) for 1 hour prior to the addition of FVIIa (100 nM). Following FVIIa treatment for 1 hour, cells were stimulated with TNF-α (5 ng/ml) for 6 hours and the relative levels of ICAM-1 (D), VCAM-1 (E), and IL-6 (F) mRNA were measured by quantitative RT-PCR. (G). Silencing of EPCR, PAR1, or PAR2. HUVECs were transfected with control or EPCR-, PAR1-, or PAR2-specific siRNAs (200 nM) using Lipofectamine RNA Max reagent in serum-free medium. After 72 hours, cell lysates were made and analyzed for the expression of EPCR, PAR1, or PAR2 by western blot analysis using specific antibodies against these proteins. (H-I). HUVECs transfected with scrambled siRNA (scRNA), EPCR, PAR1, or PAR2 siRNA as described in panel G were treated with FVIIa (100 nM) for 1 hour and then stimulated with TNF-α (5 ng/ml) for 6 hours. ICAM-1 (H) and VCAM-1 (I) mRNA levels were measured in quantitative RT-PCR. (K-L) HUVECs were transfected with control siRNA or siRNA specific for β-arrestin-1 (β-AR-1), β-arrestin-2 (β-AR-2), or an equimolar mixture of both β-arrestin isoforms (β-AR-1/2). Forty-eight hours after transfection, cell lysates were made from the transfected cells probed for the expression of β-arrestin-1 (J) or β-arrestin-2 (K) by western blot analysis. (L) HUVECs transfected with control siRNA or siRNA specific for β-arrestin-1, β-arrestin-2, or β-arrestin-1/2 were treated with FVIIa (100 nM) and then stimulated with TNF-α (5 ng/ml). Cell lysates were probed for VCAM-1 levels by immunoblot analysis. Data are the mean ± SEM of 3 to 4 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.

The FVIIa-mediated anti-inflammatory effect requires its protease activity, EPCR, PAR1, and β-arrestin-1. (A-C) FVIIa protease activity requirement. Serum-starved HUVECs were treated with FVIIa or active-site–inhibited FVIIa or FVIIaAS (100 nM) for 1 hour and then left unperturbed (NS) or stimulated with TNF-α (5 ng/ml) for 6 hours. ICAM-1 (A), VCAM-1 (B), and IL-6 (C) mRNA levels were measured by quantitative RT-PCR. (D-F) EPCR-blocking or PAR1 antibodies attenuate the anti-inflammatory effect of FVIIa. HUVECs were incubated with EPCR blocking (bl.) or nonblocking (nbl.) mAb (25 µg/mL) or a mixture of PAR1 mAb (ATAP-2, 25 µg/mL, and WEDE15, 20 µg/mL) for 1 hour prior to the addition of FVIIa (100 nM). Following FVIIa treatment for 1 hour, cells were stimulated with TNF-α (5 ng/ml) for 6 hours and the relative levels of ICAM-1 (D), VCAM-1 (E), and IL-6 (F) mRNA were measured by quantitative RT-PCR. (G). Silencing of EPCR, PAR1, or PAR2. HUVECs were transfected with control or EPCR-, PAR1-, or PAR2-specific siRNAs (200 nM) using Lipofectamine RNA Max reagent in serum-free medium. After 72 hours, cell lysates were made and analyzed for the expression of EPCR, PAR1, or PAR2 by western blot analysis using specific antibodies against these proteins. (H-I). HUVECs transfected with scrambled siRNA (scRNA), EPCR, PAR1, or PAR2 siRNA as described in panel G were treated with FVIIa (100 nM) for 1 hour and then stimulated with TNF-α (5 ng/ml) for 6 hours. ICAM-1 (H) and VCAM-1 (I) mRNA levels were measured in quantitative RT-PCR. (K-L) HUVECs were transfected with control siRNA or siRNA specific for β-arrestin-1 (β-AR-1), β-arrestin-2 (β-AR-2), or an equimolar mixture of both β-arrestin isoforms (β-AR-1/2). Forty-eight hours after transfection, cell lysates were made from the transfected cells probed for the expression of β-arrestin-1 (J) or β-arrestin-2 (K) by western blot analysis. (L) HUVECs transfected with control siRNA or siRNA specific for β-arrestin-1, β-arrestin-2, or β-arrestin-1/2 were treated with FVIIa (100 nM) and then stimulated with TNF-α (5 ng/ml). Cell lysates were probed for VCAM-1 levels by immunoblot analysis. Data are the mean ± SEM of 3 to 4 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.

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