MASP transactivation facilitates PAR2 cleavage by coagulation proteases. (A) MCF7 cells transfected to express TF and SEAP-PAR1, SEAP-PAR2, or SEAP-PAR2G35K were stimulated for 1 hour with either FVIIa (20 nM) or FXa (40 nM), with or without serine protease inhibitors aprotinin (1 µM), camostat (1 µM), or ecotinRR (20 nM), or the matriptase active-site blocking antibody fragment FabE2 (500 nM). Receptor cleavage was determined after a trypsin strip, unstimulated control subtracted, and uninhibited stimulation set to 100% to facilitate comparison. SEAP-PAR1 cleavage suggests FVIIa is resistant to all inhibitors and FXa to all but ecotinRR. The contrasting sensitivity to the same inhibitors for cleavage of SEAP-PAR2 variants suggests the engagement of an intermediate serine protease, probably matriptase. (B) MCF7 (left) or KOLF (right) cells transfected to express TF and SEAP-PAR2G35K with (right) or without (left) pro-matriptase zymogen were stimulated for 1 hour with matriptase (3 nM), hepsin (10 nM), or FVIIa (20 nM) with or without FabE2 (500 nM) and cleavage inhibition determined as in A. Note that despite potent inhibition of PAR2G35K cleavage by the soluble catalytic domain of recombinant matriptase, FabE2 does not fully inhibit PAR2G35K cleavage by membrane-associated matriptase elicited by FVIIa or hepsin in either a natural (left) or a fully reconstituted (right) system. Statistical analysis was by 1-way ANOVA followed by Newman-Keuls multiple comparison test. ##P < .01; ###P < .001 relative to uninhibited.
