TF-FVIIa–induced IL-8 mRNA upregulation does not require matriptase activity. (A) Representative western blots of matriptase (ST-14) in cell lysates and cell-free supernatant from HaCaT cells stimulated with FVIIa (25 nM), signaling deficient FVIIa mutant Q40A (25 nM), FVIIa (1 nM)/FX (50 nM), and catalytic domain of recombinant human matriptase (rhST-14, 5 nM) for 30 and 60 minutes. Uncleaved matriptase has an apparent molecular weight of 72 kDa, whereas the protease domain is detected at 26 kDa. (B) HaCaT cell surface FXa generation with 10 nM FVIIa in the presence of indicated concentrations of protease inhibitors camostat and aprotinin. (C) IL-8 mRNA in HaCaT cells stimulated for 90 minutes with recombinant matriptase (rhST-14, 5 nM), FVIIa (10 nM), or FVIIa (1 nM)/FX (50 nM) in the absence or presence of serine protease inhibitors camostatin (1 μM) and aprotinin (5 μM) (n = 3; one-way analysis of variance (ANOVA) and Dunnett’s test. (D) Representative western blot of matriptase (ST-14) in cells and cleared cell supernatant of A7 melanoma cells stimulated with FVIIa (25 nM), FVIIa (1 nM)/FX (50 nM), and recombinant human catalytic domain of matriptase (rhST-14, 5 nM) for 30 and 60 minutes, respectively. Because of their low inherent PAR2 signaling activity (PAR2 agonist SLIGRL induced IL-8 by 2.2- ± 1.0-fold, and FVIIa showed no induction of IL-8 [1.0- ± 0.3-fold]), A7 cells were transduced to express PAR2 and TF using adenoviral vectors. (E) Quantification of IL-8 mRNA in A7 melanoma cells stimulated for 90 minutes with recombinant matriptase (rhST-14, 5 nM), FVIIa (10 nM), or FVIIa (0.5 nM)/FX (50 nM) in the absence or presence of serine protease inhibitor aprotinin (5 μM) (n = 4). *P < .05, Student t test.