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Peptide-Generating Protease (Fibrinolytic/Neutrophil Enzymes) BRUCE U

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Peptide-Generating Protease (Fibrinolytic/Neutrophil Enzymes) BRUCE U Proc. Nati. Acad. Sci. USA Vol. 77, No. 9, pp. 5448-5452, September 1980 Medical Sciences Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease (fibrinolytic/neutrophil enzymes) BRUCE U. WINTROUB*tI, JONATHAN S. COBLYNt§, CAROL E. KAEMPFERt, AND K. FRANK AUSTENt§ Departments of *Dermatology and §Medicine, Harvard Medical School; and the tDepartment of Rheumatology and Immunology, and the *Division of Dermatology, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 Contributed by K. Frank Austen, June 2, 1980 ABSTRACT The human neutrophil neutral peptide-gener- MATERIALS AND METHODS ating protease, which generates a low molecular weight vaso- active peptide from a plasma protein substrate, is directly fi- Materials. The following were obtained as noted: aprotinin brinolytic and cleaves human fibrinogen in a manner distinct (Boehringer Mannheim); human fibrinogen, fraction I, B grade from plasmin. Fibrinogen was reduced from 340,000 Mr to de- (Calbiochem); thrombin, bovine topical (Parke, Davis, St. Louis, rivatives of 270,000-325,000 Mr during interaction with the MO); streptokinase (Hyland, Costa Mesa, CA); urokinase protease at enzyme-to-substrate ratios of 0.3 or 1.0 yg/1.0 mg. (Sigma); and trypsin and soybean trypsin inhibitor (Worth- The 310,000-325,000 M, cleavage fragments exhibited pro- longed thrombin-induced clotting activity but were able to be ington). Human thrombin, purified to homogeneity (12), was coagulated, whereas the 270,000-290,000 Mr fragments were a gift of Robert Rosenberg. not able to be coagulated. Anticoagulants were not generated Neutral peptide-generating protease, obtained from purified at either enzyme dose. As analyzed by sodium dodecyl sul- human neutrophils, was purified to homogeneity (8) as assessed fate/polyacrylamide gel electrophoresis in 4-30% gradient gels by NaDodSO4/polyacrylamide gel electrophoresis in 10% and 10% gels stained for protein and carbohydrate, the dimi- (wt/wt) acrylamide gels (13). Plasminogen was purified from nution to 310,000-325,000 Mr and the prolongation of throm- bin-induced clotting time resulted from cleavage of the fibrin- normal human plasma by the method of Deutsch and Mertz ogen Aa chain. The further decrease in size to 270,000-290,000 (14). Fibrinogen was purified from commercial human fi- Mr was associated with Bkchain and y-chain cleavage and an brinogen (15) and rendered plasminogen free as described (14). inability to form 'y-'y dimers. The neutral peptide-generating This preparation was more than 95% coagulable by thrombin protease, a distinct human neutrophil neutral protease with fi- [5 National Institutes of Health units/mg of fibrinogen]. The brinolytic and fibrinogenolytic activities comparable to those preparation was devoid of plasminogen; it was not digested of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, during 24-hr treatment with urokinase [10 Committee on and leukocyte granule extracts. It may be that the concerted Thrombolytic Agents (CTA) units/mg of fibrinogen] at action of this neutrophil protease to generate a vasoactive 370C. peptide and to digest fibrinogen and fibrin facilitates neutrophil Functional Assay of Fibrinolytic and Fibrinogenolytic movement through vascular and extravascular sites. Activities. Fibrinolytic activity was assayed on human plas- Neutrophils in mixed leukocyte suspensions were visually ob- minogen-free fibrin agarose plates (16). Fibrinogen was io- served to degrade fibrin clots (1, 2), and neutrophils have been dinated and 125I-labeled fibrinogen was prepared in Linbrow identified within intravascular and extravascular thrombi (3). plates (Falcon) as described (15). Total available radioactivity Neutrophil-mediated clot lysis may proceed by at least three was defined as that amount of 125I solubilized when 8 ,ug of mechanisms. The finding of degradation products of fibrin trypsin in 200 ,g of 10 mM Tris-HCl, pH 7.4/0.15 M NaCl was within the neutrophil (4, 5) is consistent with fibrin dissolution added to a well and the plate was incubated for 1 hr at 370C. during phagocytosis. Because phagocytosis is associated with Radioactivity in a 100-,ul sample from the supernatant of each exocytosis of lysosomes, clot lysis may also involve release of well was measured in a gamma counter (Searle Model 1185, lysosomal enzymes such as leukocyte elastase and cathepsin G, Chicago, IL) and corrected for radioactivity solubilized by which have direct fibrinolytic activity (6). Finally, the neu- buffer alone. trophil, when activated by the lectin concanavalin A, syn- Assessment of Fibrin and Fibrinogen Degradation Prod- thesizes and secretes an activator of plasminogen that converts ucts by NaDodSO4/Polyacrylamide Gel Electrophoresis. The plasminogen to the fibrinolytic enzyme plasmin (7). electrophoresis was carried out in 5% and 10% acrylamide gels Recently, a human neutrophil neutral protease was isolated by a modification of a described procedure (13). Electrophoresis and distinguished from leukocyte granule elastase, cathepsin in gradient acrylamide gels (gradient electrophoresis) was G, and collagenase (8). The protease, designated the neutral performed with 4-30% gradient polyacrylamide gels (Phar- peptide-generating protease, cleaves a-N-carbobenzoxy-L- macia) in 40 mM Tris-HCl, pH 7.4/20 mM sodium acetate/2 lysine-p-nitrophenyl ester (8), a synthetic substrate of plasmin mM EDTA/0.2% NaDodSO4. Preelectrophoresis of gradient (9); this activity suggested that the protease may also have the gels was carried out at 70 V for 1 hr; after application of the capacity to degrade fibrin and fibrinogen directly. The neutral samples, electrophoresis was performed at 300 V for 10 min and peptide-generating protease (10, 11) has fibrinogenolytic ac- then at 150 V for 2.5 hr. Gels were stained for protein with 0.1% tivity comparable to plasmin on a weight basis, but cleaves the Coomassie brilliant blue in 45% (vol/vol) methanol/10% chains of fibrinogen at sites different from those cleaved by (vol/vol) acetic acid overnight, destained for 18-24 hr at room plasmin so as to generate a different set of reaction products. temperature in 10% acetic acid, and stored in that solution. Alternatively, gels were stained for carbohydrate with the pe- The publication costs of this article were defrayed in part by page riodic acid-Schiff base reagent (PAS) according to published charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: DFP, di[iisopropyltluorophosphate; PAS, periodic this fact. acid-Schiff base reagent. 5448 Downloaded by guest on October 1, 2021 Medical Sciences: Wintroub et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5449 methods (17). The molecular weights of the Aa, BO3, and y chains of fibrinogen, determined by NaDodSO4/gradient polyacrylamide gel electrophoresis, were estimated from the -! 12 values obtained for reduced protein standards run indepen- dently. Functional Assessment of Fibrinogen Degradation ;80 - Products. Fibrinogen (3.0 mg/ml in 10 mM Tris-HCl, pH rz4 7.4/0.15 M NaCl) was treated with various enzymes or buffer 8 I.-,I alone and the reaction was stopped by addition of soybean trypsin inhibitor to a final concentration of 10 mM. The func- v 1 2 3 4 5 tional effects of fibrinogen degradation were studied by de- Protein, pg termination of the thrombin-induced clotting time, the presence of to FIG. 1. Fibrinolytic activity of neutral peptide-generating pro- anticoagulants, and the capacity form crosslinked fibrin. tease (@) and streptokinase-activated human plasminogen (0) as- For determination of thrombin-induced clotting time, 10 MAl of sessed on plasminogen-free fibrin plates. a solution containing purified thrombin (50 National Institutes of Health units/ml) and CaCl2 (10 mM) in 10 mM Tris-HCl, pH 7.4/0.15 M NaCl (thrombin/calcium solution) was added generating protease gave a linear dose-related solubilization to 100,ul of the reaction mixture, and the time at which fibrin of radioactivity from 1.0 to 4.0,Mg, with the highest dose of each formed was measured visually at room temperature. The ref- enzyme having an effect comparable to 8 ,g of trypsin. Solu- erence thrombin-induced clotting time was 25 sec. Based upon bilization of radioactivity in streptokinase or buffer alone at 1 dose-response studies with normal fibrinogen, a 50% reduction hr was less than 10% of that achieved with the enzymes. To in functional fibrinogen extended the thrombin-induced assess the kinetics of this reaction, we incubated 0.5 and 1.0 Mg clotting time to 35 sec. Failure to form fibrin in less than 180 of protease and 2.0 Mg of streptokinase-activated plasminogen sec indicated that at most only 10% of the functional fibrinogen in quadruplicate under the same reaction conditions. Single remained. To detect anticoagulant activity, we mixed 100-,Ml reaction mixtures were assessed at 10, 20, 40, and 60 min. So- samples of fibrinogen digests with 100,Ml of fibrinogen (3.0 lubilization of radioactivity increased with time in a linear mg/ml in 10 mM Tris-HCl, pH 7.4/0.15 M NaCl) and added manner for both enzymes (Fig. 2). 10 ,ul of thrombin/calcium solution. The thrombin-induced Structural Assessment of Degradation of Fibrinogen. In clotting time was measured, and anticoagulant activity was order to determine the ratio of soluble fibrinogen to neutral assessed by prolongation of the clotting time. To assess the ca- peptide-generating protease to be used in detailed degradation pacity of fibrinogen digests to form crosslinked fibrin, we added studies, 1.0-mg portions of fibrinogen in 330 Ml of 10 niM 10 ,l of thrombin/calcium solution to 100-,l samples of fi- Tris1HCI, pH 7.4/0.15 M NaCl were incubated with 0.3, 1.0, brinogen digests and incubated the mixtures for 3 hr at room 5.0, and 20.0 Mug of neutral peptide-generating protease at 370C temperature.
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