PAR2G35K is selectively cleaved by matriptase over coagulation proteases in a cell type-dependent manner. (A) KOLF cells transfected to express TF alone (left) or together with PAR2 (right) were loaded with the fluorescent calcium dye Fura2-AM and then stimulated with PAR2-AP (100 μM), matriptase (3 nM), FVIIa (10 nM), or FXa (20 nM), followed by trypsin (10 nM). Changes in intracellular calcium were calculated as described in “Methods” and normalized to the fluorescence ratio prestimulation. Note the potency of matriptase. The graph (mean ± SEM) is representative of 3 separate experiments performed in duplicate. (B) KOLFs transfected to express G35-point mutants of SEAP-PAR2 were stimulated for 30 min with matriptase (1 nM) and percentage cleavage determined as detailed in “Methods,” after a trypsin strip of remaining surface SEAP moieties. Of the 3 coagulation factor-resistant PAR2 mutants tested, only PAR2G35K remained a good substrate for recombinant matriptase. (C) Evaluation of the expression levels of selected MASPs and coagulation cofactors in selected cell lines relative to glyceraldehyde-3-phosphate dehydrogenase by quantitative reverse transcription polymerase chain reaction with species-specific primers. Note that KOLF quantification does not include EPCR. (D) HeLa, MCF7, KOLF, and PC3 cells transfected to express TF and SEAP-PAR1, SEAP-PAR2, SEAP-PAR2G35K, or SEAP-PAR2R36G were stimulated with matriptase (1 nM), prostasin (5 nM), thrombin (3 nM), FVIIa (20 nM), or FXa (40 nM) for 30 min and percentage receptor cleavage assessed. Note that SEAP-PAR2G35K is cleaved by matriptase but resistant to coagulation proteases and to prostasin, a known activator of pro-matriptase, in HeLa and KOLF cells, but not MCF7 and PC3 cells. This cleavage reflects canonical activation, as indicated by the lack of processing of SEAP-PAR2R36G. Thrombin did not cleave any PAR2 variant, but efficiently processed SEAP-PAR1. Statistical analysis was by a Student t test in B and a 1-way ANOVA followed by Newman-Keuls multiple comparison test in D. *P < .05; **P < .01; ***P < .001, relative to vehicle control.
