Inhibition of proteolytic PAR2 signaling by anti-TF antibodies on MDA-MB-231 human breast cancer cells. (A) Mab-10H10 and Mab-5G9 inhibit TF-VIIa signaling in MDA-MB-231 cells. Cells were preincubated for 15 minutes with 50 μg/mL Mab-10H10 or Mab-5G9 or 100 μg/mL polyclonal PAR2-cleavage-blocking antibody, followed by addition of 10 nmol/L VIIa, ternary complex (0.5 nmol/L VIIa, 100 nmol/L X, and 200 nmol/L NAPc2) or 10 nmol/L thrombin (IIa). TR3 and IL-8 mRNA induction over control after 90 minutes was determined by quantitative PCR. (B) TF and PAR2 expression in parental MDA-MB-231 and MDA-MD-231mfp cells by Western blotting using polyclonal anti-TF and anti-PAR2 with loading controls of abundant cellular proteins, PDI and actin. (C) Comparative FACS analysis for integrin expression by parental and mfp MDA-MB-231 cells. Averages for 2 experiments are shown. (D) Comparison of Xa generation by parental and mfp cells on monolayers seeded at equal cell density. (E) Matrix-dependent TF-VIIa signaling. MDA-MB-231mfp cells were grown on vitronectin or laminin 5-containing matrix. Cells were pretreated with 50 μg/mL anti-β1 AIIB2, Mab-5G9, or Mab-10H10 for 15 minutes before stimulation with VIIa (10 nmol/L) for 90 minutes.