Inhibition of TF-dependent PAR2 signaling by rTFPI-1 in HUVECs transduced with TF and PAR2. HUVECs were cotransduced with virus encoding TF (50 particles/cell) and PAR2 (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), polyclonal rabbit anti-PAR2 (200 μg/mL), the indicated concentrations of rTFPI-1, or 50 μg/mL anti-TF exosite antibody 5G9 prior to stimulation with VIIa (10 nM) and X (100 nM) or with Xa added at 1, 3, 5, 7, and 9 minutes (5, 2, 1, 1, and 1 nM, respectively) for 10 minutes. (A) Representative Western blot and densitometric quantitation of inhibition of VIIa/X-induced ERK1/2 phosphorylation by rTFPI-1 or 5G9 antibody. The left panel shows Xa activity in the supernatant at 10 minutes and the right panel the fold induction of ERK1/2 phosphorylation; mean ± SD (n = 3). (B) Preincubation with anti-PAR1 or anti-PAR2 demonstrates that TF initiation phase-mediated ERK1/2 phosphorylation is PAR2 dependent. (C) TF cytoplasmic domain (TFCD) phosphorylation was determined in cells transduced under identical conditions as in panel A with a palmitoylation-deficient mutant of TF (Cys²⁴⁵-Ser TF) and treated as described. Representative Western blot for dose-dependent inhibition of TF cytoplasmic domain phosphorylation by rTFPI-1. In the bottom graph, the left panel indicates Xa activity by chromogenic assay at 10 minutes; the right panel, densitometric quantification of TF phosphorylation given as fold increase over control; mean ± SD (n = 3).