INTERACTIONS OF MAST-CELL TRYPTASE WITH THROMBIN RECEPTORS AND PAR-2

Tryptase is a serine protease secreted by mast cells that is able to a ctivate other cells. In the present studies we have tested whether the se responses could be mediated by thrombin receptors or PAR-2, two G-p rotein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleav ed the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombi n receptor peptide at the activating site but less efficiently. When a dded to COS-1 cells expressing either receptor, tryptase stimulated ph osphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC 366, or by antibodies to tryptase and PAR-2. When added to human endot helial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase i n cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neithe r aggregation nor increased Ca2+, These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) fo r PAR-2, this potential is realized, although cleavage at secondary si tes may Limit activation, particularly at higher tryptase concentratio ns; and 3) in contrast, although tryptase clearly activates thrombin r eceptors in COS-1 cells, it does not appear to cleave endogenous throm bin receptors in platelets or CHRF-288 cells. These distinctions corre late with the observed differences in the rate of cleavage of the PAR- 2 and thrombin receptor peptides by tryptase. Tryptase is the first pr otease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR -2 is expressed but trypsin is unlikely to reach.