rTFPI-1 is a poor inhibitor of TF-dependent PAR1 signaling in endothelial cells. (A-B) HUVECs were transduced with TF (50 particles/cell) for 3 hours, followed by growth for 2 days. Confluent cells were rendered quiescent in serum-free medium for 5 hours in the presence of TNF-α prior to stimulation with VIIa (10 nM) and X (100 nM) without (□) or with 5 nM rTFPI-1 (○)or50 μg/mL 5G9 anti-TF antibody (^*). Alternatively, purified Xa was added at 1, 3, 5, 7, and 9 minutes (5, 2, 1, 1, and 1 nM, respectively; □) to mimic the kinetics of endogenous Xa generation. (A) Xa activity was determined at the indicated times by chromogenic assay. (B) Xa activity was quenched with 1 μM NAP5 at 10 minutes, and mRNA levels of TR3 were determined at 90 minutes by real-time PCR; mean ± SD (n = 6). (C) HUVECs were transduced with virus encoding TF (50 particles/cell) for 3 hours, then grown for 2 days and incubated in serum-free medium for 5 hours. After treatment with anti–PAR-1 or rTFPI-1 (or both) for 10 minutes, cells were stimulated with VIIa (10 nM) and X (100 nM). (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. Mean ± SD (n = 4); asterisk indicates the reduction in TR3 induction on anti-PAR1 addition was statistically significant (P < .01, t test) for each of the rTFPI-1 concentrations.