Enzymatic investigation for delayed-type hypersensitivity reaction. Assay for thrombin and plasmin activities in tuberculin reaction sites.

Induration is a prominent feature of delayed hypersensitivity reaction (DHR) and is associated with fibrin deposition. To determine whether thrombin and plasmin mediate the development of induration, we examined guinea pig skin extracts of tuberculin reaction sites for the protease activities. To measure their low activities without inactivation by the inhibitors in the extracts, fluorogenic peptide substrates specific to each of the proteases were used. Because thiol proteases in the extracts hydrolyzed the substrates, the two serine protease activities were selected using a serine protease inhibitor. The extracts contained alpha 2-macroglobulin (alpha 2-MG)-trapped thrombin that hydrolyzes the substrate, but no alpha 2-MG-trapped plasmin. To exclude alpha 2-MG-trapped thrombin activity, the extract treated with 0.2 M methylamine was incubated for two hours and the residual activity was excluded from the initial thrombin activity. Thrombin activity paralleled increasing intensity of induration, and plasmin activity was associated with the reduction of induration. Neither induration nor an increase of protease activity was observed at control sites. The results show that thrombin and plasmin mediate the development of induration probably by regulating fibrin deposition in DHR sites and that the present method can measure the protease activities in biological fluids and tissue extracts.