Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Abstract

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-proteincoupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved 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 thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca 2؉. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca 2؉ . These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease 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.

Glycosylation and the activation of proteinase‐activated receptor 2 (PAR2) by human mast cell tryptase

“…. ., were in accord with a previous study by Molino et al (1997). Our results indicated that tryptase was as e cient or more so than trypsin in hydrolyzing the P20 peptide to release SLIGRL, again in agreement with Molino et al (1997).…”

“…However, some reports (Alm et al, 2000;Corvera et al, 1999;Molino et al, 1997;Schechter et al, 1998), but not others (Corvera et al, 1997;Steinho et al, 2000), have indicated that tryptase appears to behave as a partial agonist compared to trypsin for activating PAR 2 , implying that PAR 2 activation by tryptase may be in¯uenced by other factors. Interestingly, Huang et al (2001) demonstrated that recombinant bI-tryptase was unable to activate any of the PARs cloned to date, including PAR 2 expressed on HEK293 cells.…”

“…However, the mechanism whereby tryptase activates cells remains unclear. Although it has recently been suggested that tryptase can a ect target cells via activation of proteaseactivated receptor 2 (PAR 2 ) (Corvera et al, 1997;Mirza et al, 1997;Molino et al, 1997), others using recombinant tryptase have failed to detect PAR 2 activation by tryptase (Huang et al, 2001). PAR 2 is a member of the novel family of G-protein coupled receptors activated by proteolytic cleavage (Dery et al, 1998).…”

“…Tryptase activation of PAR 2 has been demonstrated in a number of cell types including cultured human endothelial cells (HUVEC), rat colonic myocytes, keratinocytes and guinea-pig myenteric neurons (Corvera et al, 1997;1999;Molino et al, 1997;Schechter et al, 1998). However, some reports (Alm et al, 2000;Corvera et al, 1999;Molino et al, 1997;Schechter et al, 1998), but not others (Corvera et al, 1997;Steinho et al, 2000), have indicated that tryptase appears to behave as a partial agonist compared to trypsin for activating PAR 2 , implying that PAR 2 activation by tryptase may be in¯uenced by other factors.…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…Thus, increased expression of serine proteinases capable of activating PARs (Figure 1) has the potential to exacerbate inflammation. For example, PAR-2 can be activated by trypsin, mast cell tryptase (163), tissue factor-factor VIIa and factor Xa (15), some kallikreins (164), PR3 (165), and matriptase (166).…”