Enhanced inhibition of ternary complex PAR1 signaling by rTFPI-1 in HUVECs coexpressing PAR2. HUVECs were cotransduced with virus encoding TF (50 particles/cell) and PAR2 or cleavage-insensitive PAR2/P1 mutant (100 particles/cell) for 3 hours, then grown for 2 days, rendered quiescent in serum-free medium for 5 hours, and pretreated with anti-PAR1 (WEDE-15, 20 μg/mL; ATAP-2, 10 μg/mL), the indicated concentrations of rTFPI-1, or 50 μg/mL anti-TF 5G9 prior to stimulation with VIIa (10 nM) and X (100 nM) for 10 minutes. Thrombin signaling was excluded by the routine addition of 200 nM hirudin prior to stimulation with proteases. (Left) Xa activity in the supernatant at 10 minutes. (Right) Xa was quenched with 1 μM NAP5 at 10 minutes, and TR3 mRNA levels at 90 minutes were determined by real-time PCR. (A) Mean ± SD (n = 3); significantly different based on t test. (B) Mean ± SD (n = 4). ^*PAR2-transduced statistically different (t test) from non–PAR2-transduced (2.5 nM rTFPI-1, P < .005; 5 nM rTFPI-1, P < .05) or PAR2/P1-transduced (2.5 nM rTFPI-1, P < .05). □ indicates TF + PAR2; ▦, TF + PAR2/P1; ▪, TF only.