Coagulation proteases transactivate latent matriptase to enhance epithelial PAR2 cleavage. (A) HaCaT cells were incubated for the indicated time with prostasin (25 nM), FVIIa (25 nM), or FXa (50 nM) cell lysates, and concentrated supernatants were denatured, size-separated, blotted, and probed with an anti-matriptase antibody. Note the consumption of cell-associated matriptase and corresponding accumulation of the extracellular (by FVIIa and FXa only) and catalytic (by all 3 proteases) domains in supernatants. The blot is representative of 4 independent experiments (quantification in supplemental Figure 5A). (B) HaCaT cells were incubated for 1 hour with prostasin (2.5 or 25 nM), hepsin (30 nM), FXa (50 nM), PAR1-AP (50 µM), or PAR2-AP (50 µM), either alone or supplemented with marimastat (MM, 1 µM). Where indicated, cell-free supernatants of FXa-treated cells were incubated for an additional hour with no further addition of protease or after supplementation of prostasin (25 nM) or hepsin (30 nM). Samples were denatured, size-separated, blotted, probed with an anti-matriptase antibody, and revealed. Note that the full-length extracellular domain released by Xa can be further matured in solution by prostasin and that shedding is independent of zinc metalloproteases and does not require engagement of PARs. (C) Activation of serine proteases with matriptase-like specificity was studied by stimulation of HaCaT cells with prostasin (5 nM) or FVIIa (20 nM) in the presence of the chromogenic substrate S2765. OD405 values were normalized to prestimulation values. Note that prostasin and FVIIa both stimulate matriptase-like activity in HaCaT, which also shows constitutive matriptase-like activity. All were inhibited by the MASP inhibitor ecotinRR (20 nM). The graph (mean ± SEM) is representative of 3 separate experiments performed in triplicate. (D) HeLa cells transfected with zymogens of wild-type matriptase, catalytically inactive matriptase, or prostasin were used to determine the cell surface activity of serine proteases against the chromogenic substrate S2765 upon stimulation with soluble prostasin. Note that only transfection of wild-type pro-matriptase yields a net increase in OD405 after stimulation with prostasin for 3.5 hours. (E) HeLa (left) and KOLF (right) cells were transfected to express TF, SEAP-PAR2, or SEAP-PAR2_G35K together with candidate protease zymogen, as indicated, and stimulated with FVIIa (20 nM), FXa (40 nM), or prostasin (5 nM) for 1 hour, followed by a trypsin strip for determination of receptor cleavage. Only wild-type matriptase zymogen permitted (FVIIa, prostasin) or enhanced (FXa) receptor cleavage. Pooled data ± SD of 4 independent experiments performed in duplicate.