Heparin has been shown to inhibit such proteases as trypsin (1-3), pepsin (4), thrombin ( 5 ) , human plasma kallikrein and Pf/di12 (6). The latter two enzymes were able to be distinguished from each other by the relative degree of inhibition by heparin. The release of heparin in experimental shock states associated with protease activation (7) and the use of heparin as an anticoagulant in studies involving the assay of kininforming enzymes, provoked this present investigation. The effect of heparin on the in vitro kinin-forming activity of the following proteases was studied: crystalline trypsin, streptokinase-activated human plasmin, human plasma kallikrein, dog plasma kallikrein, and dog salivary kallikrein.Materials and Methods. Crystalline bovine trypsin was obtained from Worthington Laboratories, Freehold, New Jersey. Streptokinase-activated human plasmin was a product of Parke Davis and Co. (Fibrinolysin) with an activity of approximately 10 RPM13 plasmin units/mg protein. Human and dog plasma kallikrein were prepared by precipitation with 10 times the volume of 20 % acetone according to a previously published method (8). The precipitated plasma solution was permitted to stand overnight a t 4O, centrifuged a t 2500 rpm, the precipitate collected and lyophilized. Dog salivary kallikrein was prepared from parotid saliva collected by direct duct cannulation technique. The saliva was filtered through a Whatman No. 1 filter paper, treated with three times the volume of 1 Supported by U. S. Public Health Service Grants HE-11492 and HE-00784 fmm the National Heart Institute. 2 Permeability factor. 3 Roswell Park Memorial Institute.20 % acetone, permitted to stand overnight at 4O, centrifuged a t 3500 rpm for 30 min, and lyophilized. Heparin was avaliable from Abbott Laboratories (Pan-Heparin) at a concentration of 50,000 units/ml. Other inhibitors used included Trasylol (product of Farbenfabriken Bayer AG, Germany), soya bean trypsin inhibitor, and ovomucoid obtained from Worthington Laboratories, Freehold, New Jersey.Human kininogen substrate was prepared from -4CD plasma heated to 80' and placed immediately on ice. The precipitated protein was homogenized in a Waring Blendor, centrifuged at 3500 rpm, and the supernatant collected. Approximately 50 ml of plasma made 20 ml of heated plasma substrate which contained 35 % of the kinin-yielding activity of the original plasma. The kininogen substrate was stable when stored frozen in a 0.6 % protein concentration solution. The substrate did not have any kinin-forming or kinin-destroying activity. PfJdil was unable to form kinin from the heated substrate (6). Incubation of 0.005 pg trypsin with 1.2 mg of substrate yielded 0.14 pg of kinin.The kinin-forming activity generated by acetone treatment of heparinized or citrated dog and human plasma first was studied. Plasma was placed into 10 times the volume of 20 % acetone, and the precipitate, collected after overnight incubation a t 4O, was reconstituted to the original volume of plasma with 0.1 M Tris buffer, p H 7.8. ...