Mechanism by which heparin proteoglycan modulates mast cell chymase activity

Chymases are highly basic chymotrypsin-like serine proteases expressed excl usively by mast cells. Large amounts of chymases complexed with heparin pro teoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the prote ase toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA ( S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates, Incubation of thrombin with oligonucleotide s that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants w ith defects in their heparin-binding regions were less efficiently inactiva ted by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-cat alyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptid e substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, wherea s the cleavage of uncharged substrates was not affected by the presence of heparin PG, On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrate s.