Figure 7.

Tryptase-6 triggers calcium signals in PAR2 knockout HEK-293 cells. The calcium signaling assay [fluorescence emission at 530 nm, as a percentage of the signal generated by the calcium ionophore A23187 (%)] was used to determine whether tryptase-6 can trigger calcium signaling via PAR2-independent pathways. Tracing (A): The absence of a response to the potent PAR2-selective agonist, 2fLI (open triangle) but presence of a response to the selective PAR1 peptide agonist, TFLLR-amide (open square) confirmed the absence of functional PAR2 and the presence of functional PAR1 in the HEK-293-PAR2-null cells. Tracing (B): Volume equivalent to 5.8 FU/min/ng of PsA SF-derived tryptase-6 (solid circle) elicited a robust calcium response (dark circle), that was desensitized by the second consecutive exposure to tryptase-6 (second solid circle, Tracing B). These tryptase-6-treated cells no longer responded to the PAR1 activator, thrombin (0.5 U/ml, open circle, first tracing panel B). Cells not treated with tryptase-6 showed a strong calcium signal caused by thrombin (right-hand tracing, panel B).