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. Fibrin was solubilized by addition of 300,ul of a for 1 hr. To stop the reaction, we made each mixture 1.0 mM solution containing 9% urea, 3% NaDodSO4, and 3% 2-mer- in diisopropylfluorophosphate (DFP) and held it overnight at captoethanol (all wt/vol). Samples were subjected to gradient 40C. Twenty-microliter samples of each mixture were subjected electrophoresis and the gels were stained for protein and de- to gradient electrophoresis without reduction. Fibrinogen in- stained as described above. Under these conditions, protein cubated with buffer alone gave a major band of 340,000 Mr and bands were detected that corresponded in size to fibrin a a minor, higher molecular weight band. The major band was (66,000-69,000 Mr), (53,000 Mr) and y (45,000 Mr) chains. not apparent in the reaction mixtures incubated with increasing A reduction in -y-chain staining was associated with the ap- concentrations of neutral peptide-generating protease. Cleavage pearance of a 100,000 Mr band representing y-'y dimers. Factor fragments of 310,000-325,000 Mr, 280,000-290,000 Mr, and XIII was a trace contaminant of the fibrinogen preparation and 260,000-270,000 Mr were generated by 0.3, 1.0, and 5.0,Mg of was used as the source of crosslinking enzyme (18). protease, respectively. With 20 Mg of protease, derivatives of 300,000-260,000 Mr, 185,000 Mr, 150,000 Mr, 100,000 Mr, RESULTS 70,000 Mr, 58,0Q0 Mr, 16,000 Mr, and less than 10,000 Mr were Fibrinogenolytic and Fibrinolytic Activities of the Neutral present. Peptide-Generating Protease. The fibrinolytic activity of In order to study the sequential degradation of fibrinogen, neutral peptide-generating protease was compared with that we incubated 0.9 ug of neutral peptide-generating protease of streptokinase-activated human plasminogen by use of plas- minogen-free fibrin plates. One- to 4-pig portions of each en- zyme were applied in 15 ,l of 10 mM Tris-HCl, pH 7.4/0.15 Protease, 1.0 pgg M NaCl. No lysis was produced by plasminogen, streptokinase, 800800 Plasmin, or either of the buffers. Increasing the concentrations of the 2.0,ug neutral peptide-generating protease resulted in a linear increase 0 in the zone of lysis (Fig. 1). When compared on a weight basis 600 t-* with streptokinase-activated plasminogen, the neutral pep- 0-0 400- tide-generating protease was approximately half as active. 0 .' ~ __Pro~tease, 0.5 p~g The fibrinogenolytic activity of neutral peptide-generating 0~~~~ protease was compared with that of streptokinase-activated 200 human plasminogen by use of l25I-labeled fibrinogen plates. A 1- to portion 8.0-1ug of each enzyme was added to each well 0 10 20 30 40 50 60 in 20 ,ul of 10 mM Tris-HCl, pH 7.4/0.15 M NaCl, and the Incubation, min plates were incubated for 1 hr at 37°C. Trypsin gave a linear FIG. 2. Time-dependent solubilization ofradioactivity from in- dose-response solubilization of radioactivity from 1.0 to 8.0,Mg. soluble l25-Ilabeled fibrinogen by neutral peptide-generating protease Streptokinase-activated plasminogen and neutral peptide- and streptokinase-activated human plasminogen. Downloaded by guest on October 1, 2021 5450 Medical Sciences: Wintroub et al. Pioc. Natl. Acad. sci. USA 77 (1980) with 3.0 mg of fibrinogen (0.3 ,.g of neutral peptide-generating protease per mg of fibrinogen) in a final volume of 1.0 ml of 10 .a ,, . ; ri ,, _ mM Tris-HCI, pH 7.4/0.15 M NaCl for varying periods of time up to 24 hr. Sixty-microliter samples were removed from the reaction mixture at each time point and made 0.1 mM in DFP for subsequent analysis of structural changes. A 5-,1l portion (7.5 Mg of fibrinogen) of the DFP-treated sample from each time point was subjected to gradient electrophoresis without re- duction, and a 7.5-,Ml (10.5 ,tg of fibrinogen) DFP-treated h H sample from each time point was analyzed by gradient elec- I, trophoresis after reduction. Analysis of unreduced samples revealed fibrinogen of 340,000 Mr at time zero and the ap- pearance of a 325,000 Mr fibrinogen fragment at 5 min (Fig. 3). Fibrinogen was progressively reduced in size such that the major derivatives were 310,000-325,000 Mr by 60 min and 270,Q00-280,000 Mr by 24 hr (Fig. 3). To detect low molecular weight fragments, we subjected a 2-,ul portion (30 ,g of fi- it!- brinogen) of each DFP-treated sample to gradient electro-

phoresis without reduction. Fragments of less than 10,000 Mr I were identified at 5 min and at each subsequent time. Gradient electrophoresis of reduced material from the mixture revealed two proteins in the Aa-chain region (70,000 and 67,000 Mr), the B13-chain region (58,000 Mr), and the y-chain region (45,000 Mr) at time zero (Fig. 4). After 5 min of digestion, the FIG. 4. Time-dependent degradation of soluble fibrinogen by 0.3 Aa chain was markedly diminished in the 67,000-70,000 Mr ,g of protease per mg of fibrinogen as assessed by gradient electro- region and protein bands of 55,000 Mr and 42,000 Mr appeared phoresis of reduced substrate stained for protein. (Fig. 4). The 55,000 Mr band is clearly distinguished from the BB chain in the original gels; however, this fragment can be seen throughout the 24-hr digestion but decreased in intensity at 4 in the photographs only as a widening of the stained band in hr with the appearance of a 51,000 Mr PAS-positive protein. the BB region. At 4 hr, a protein band of 51,000 Mr and protein The PAS-positive -y chain decreased in intensity at 2 hr and was bands of 32,000-38,000 Mr were detected, and the staining of not apparent after 4 hr. Because the 32,000-38,000 Mr frag- these fragments increased in intensity between 4 and 24 hr. The ments were not defined in the PAS-stained gradient gel, 30,ul BB chain (58,000 Mr) was present during the entire 24-hr di- (45 Mg of fibrinogen) of each reduced sample was subjected to gestion, and the y chain (45,000 Mr) was not detectable after electrophoresis in 10% acrylamide gels and stained with PAS; 8 hr. the 32,000-38,000 Mr protein bands were PAS positive. Because only the BB and -y chains of fibrinogen are known Functional and Structural Assessment of Degradation of to stain with PAS (19), a 7.5-,Ml portion (10.5 Mig of fibrinogen) Fibrinogen. The functional consequences of fibrinogen of each reduced DFP-treated sample was subjected to gradient cleavage by neutral peptide-generating protease were assessed electrophoresis and stained with PAS to determine the most by incubation of 1.8 Mug and 6.0 Mug of neutral peptide-gener- likely source of the 51,000 Mr and the 32,000-38,000 Mr ating protease with 6.0 mg of fibrinogen for various periods of fragments recognized in Coomassie blue-stained gels. The band time up to 4 hr. After 0, 5, 10, 30, 60, 120, and 240 min, 250-MI corresponding to the chain stained with PAS and was present samples of each reaction mixture were removed and made 10 Bo3 mM in soybean trypsin inhibitor. Each sample was evaluated for the molecular weight of unreduced and reduced fibrinogen degradation products, thrombin-induced clotting activity, capacity to form crosslinked fibrin, and presence of antico- agulant activity. To characterize the molecular weight of fi- brinogen degradation products, we subjected duplicate 7.5-,Ml (10.5 Mg of fibrinogen) portions of each sample to gradient electrophoresis with and without reduction. To determine thrombin-induced clotting activity and the capacity to form crosslinked fibrin, we added 10 Ml of thrombin/calcium solution to 100-Ml portions of each sample; each portion was assessed for thrombin-induced clotting time and then incubated for 3 hr |iI;i:--~ ~ ~ at room temperature before analysis for y-'y dimers by gradient electrophoresis of solubilized samples. A separate 100-Ml portion of each sample was analyzed for the presence of anticoagulant activity. As analyzed by gradient electrophoresis of unreduced and reduced samples, neutral peptide-generating protease digestion at a ratio of 0.3 Mug of protease/1.0 mg of fibrinogen reduced fibrinogen to derivatives of 325,000-340,000 Mr, 320,000- 330,000 Mr, 310,000-325,000 Mr, and 290,000- ,000 Mr by 5, 10, 60, and 240 min, respectively. The Aa chain (67,000- FIG. 3. Time-dependent degradation of soluble fibrinogen by 0.3 70,000 Mr) was progressively cleaved to its 42,000 Mr deriva- Ag of protease per mg of fibrinogen as assessed by gradient electro- tive, and this process was complete at 60 min. Digestion of the phoresis of unreduced substrate stained for protein. BO3 chain (58,000 Mr) to a 51,000 Mr fragment and of the y Downloaded by guest on October 1, 2021 Medical Sciences: Wintroub et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5451 chain (45,000 Mr) to 32,000-38,000 Mr fragments was detected account for some of the divergent results obtained when the at 60 min, but the majority of both chains remained intact at cleavage of soluble fibrinogen by purified leukocyte elastase 4 hr. During the first 5 min of digestion there was no loss of (20) is compared with the results observed with crude leukocyte thrombin-induced clotting activity. After 5 min of fibrinogen extracts (21, 22). digestion there occurred a time-dependent prolongation of the Neutral peptide-generating protease cleaved soluble fi- clotting time so that by 30 min the clotting time was greater brinogen in a time-dependent manner into progressively than 3 min (Fig. 5). Thrombin-induced clotting resulted in smaller fragments that differed from those derived during formation of fibrin that was normal in appearance through the plasmin degradation. After 5 min of digestion with 0.3 ,g of initial 30 min of digestion but was less well formed after 60 min neutral peptide-generating protease per mg of fibrinogen, a of digestion. Gradient electrophoresis of solubilized thrombin major cleavage fragment of 325,000 Mr was recognized by clots disclosed y-,y dimers at each time point. However, the y-'y NaDodSO4/polyacrylamide gel electrophoresis of the unre- dimers progressively diminished in staining intensity in samples duced protein, and there was a gradual and progressive re- obtained at 60 min and after. Anticoagulant activity was not duction in size of this fragment to derivatives of 310,000- detected at any time during fibrinogen digestion. 325,000 Mr at 60 min and 270,000-280,000 Mr by 24 hr (Fig. Neutral peptide-generating protease digestion at a ratio of 3). The only other distinct fragments observed at each time 1.0 Asg of protease/1.0 mg of fibrinogen reduced fibrinogen to point in the unreduced gels had Mr less than 10,000. Anarysis derivatives of 300,000-310,000 Mr and 270,000-280,000 Mr of the degradation products after reduction and alkylation re- after 10 min and 120 min, respectively. The Aa chain vealed loss of the 67,000-70,000 Mr bands corresponding to the (67,000-70,000 Mr) was completely cleaved to its 42,000 Mr Aa chain and the appearance of bands with 55,000 Mr and derivative at 10 min. Digestion of the Bf3 chain (58,000 Mr) to 42,000 Mr by 5 min. At this time, the 58,000 Mr B/3 chain and a 51,000 Mr fragment was detectable at 10 min and progressed the 45,000 Mr 'Y chain remained intact. Cleavage of the Bf3 until no intact BB chain was detectable at 240 min. Digestion chain with the appearance of a PAS-positive 51,000 Mr frag- of the Y chain (45,000 Mr) to 32,000-38,000 Mr derivatives was ment was seen at 4 hr in the gradient gels (Fig. 4). The y chain evident at 30 min and was progressive, although a small amount disappeared coincidently with the appearance of PAS-positive of intact y chain was detected after 240 min. The thrombin- 32,000-38,000 Mr bands at 4 hr (Fig. 4). Although each of the induced clotting time was greater than 3 min after only a 10- three chains of fibrinogen is cleaved by the neutral peptide- min incubation. Thrombin treatment of 10-, 30-, and 60-min generating protease, the order of polypeptide chain digestion digests resulted in poorly formed fibrin, and no fibrin was de- is most consistent with Aa before Bf3 and oy. tectable after 120 min of digestion. The 'y-'y dimers dinished Cleavage of fibrinogen by neutral peptide-generating pro- in quantity through the first 60 min of digestion and were not tease at a ratio of 0.3 and 1.0,Mg of protease/1.0 mg of fibrin- detectable after 120 min. Anticoagulant activity was not de- ogen resulted in 310,000-325,000 Mr derivatives that were tected at any time. slowly clottable and 270,000-280,000 Mr fragments that were not clottable. The prolongation of the thrombin-induced DISCUSSION clotting time (Fig. 5) observed with the 310,000-325,000 Mr The human neutrophil contains a neutral protease, previously derivatives resulted from the Aa-chain cleavage (Fig. 4), but designated the neutrophil neutral peptide-generating protease the derivatives' capacity to form crosslinked fibrin was retained, (10, 11), that cleaves fibrin and fibrinogen with an activity as shown by detection of y-'y dimers. The loss of coagulability comparable on a weight basis to that of plasmin (Figs. 1 and 2). that characterized the 270,000-280,000 Mr fragments was as- The neutral peptide-generating protease is a single polypeptide sociated with the appearance of the 51,000 Mr Bf3-chain frag- chain of 29,000-30,000 Mr with an isoelectric point of pH ment, the 32,000-38,000 Mr y-chain fragments, and inability 7.7-8.3. It is distinguished from previously recognized neutral to form -y-y dimers. Because anticoagulants were not detected, proteases of the human neutrophil, elastase and cathepsin G, the initial prolongation of thrombin-induced clotting time is by its failure to cleave their synthetic and natural substrates (8). attributed to the extensive Aa-chain damage, whereas the A third neutrophil neutral protease, collagenase, is at least twice subsequent loss of coagulability is due to Bfl- or y-chain di- as large as these three neutrophil proteases (19). It seems likely gestion (or both). that the activity of neutral peptide-generating protease would The functional results and pattern of fibrinogen cleavage by the neutral peptide-generating protease differ from those re- sulting from digestion by plasmin, leukocyte elastase, and leukocyte extracts. Plasmin-limited proteolytic digestion of 180 - fibrinogen is characterized by a number of cleavages in the carboxy-terminal portion of the Aa chain (23,24) and removal of about 40 residues from the amino-terminal portion of the B3 chain, including fibrinopeptide B (25), to yield fragment X (260,000-300,000 Mr), which exhibits prolonged thrombin- induced clotting activity (26). Although the 310,000-325,000 20 Mr fragment obtained during neutral peptide-generating protease cleavage of fibrinogen was able to be coagulated, the 0 270,000-280,000 Mr derivative was not. Fragment X is split asymmetrically by y-chain cleavage and by further a- and 3-chain cleavage to yield the anticoagulant fragments, fragment Y (153,000 Mr) and fragment D (85,000 Mr) (27). At no time 100 20 30 40 50 during degradation by neutral peptide-generating protease at Incubation time, min enzyme/substrate ratios of 0.3,Mg or 1.0 Mg of protease/1.0 mg FIG. 5. Time-dependent alterations of thrombin-induced clotting of fibrinogen was anticoagulant activity detected. The a, 3, and time of fibrinogen during interaction with 0.3 ,gg of neutral peptide- y chains of fragment Y are further cleaved to yield another D geherating protease per mg of fibrinogen. fragment and an E fragment of 50,000 Mr (23). Core fragments Downloaded by guest on October 1, 2021 5452 Medical Sciences: Wintroub et al. Proc. Nati. Acad. Sci. USA 77 (1980) of 50,000 Mr were not detected during neutral peptide-gen- 4. Barnhart, M. I. (1965) Fed. Proc. Fed. Am. Soc. Exp. Biol. 24, erating protease digestion of fibrinogen at ratios of 0.3 jig and 846-853. 1.0 ,ug of protease/1.0 mg of fibrinogen. However, 70,000 Mr 5. Riddle, J. M. & Barnhart, M. I. (1964) Am. J. Pathol. 45,805- and 58,000 Mr fragments were recognized at a dose of 20.0 ug 823. of protease. Unlike plasmin-derived D and E fragments, the 6. Schmidt, W. & Havemann, K. (1974) Hoppe-Seyler's Z. Physiol. neutral peptide-generating protease-derived fragments were Chem. 355, 1077-1082. arise 7. Granelli-Piperno, A., Vassali, J. & Reich, E. (1977) J. Exp. Med. transient degradation products and did not from limited 146, 1693-1706. proteolysis. 8. Coblyn, J. S., Austen, K. F. & Wintroub, B. U. (1979) J. Clin. The proteolytic activity of a leukocyte granule extract for Invest. 63, 998-1005. fibrinogen was distinguished from that of plasmin by the gen- 9. Silverstein, R. M. (1973) Anal. Biochem. 65,500-506. eration-of a high molecular weight fragment composed of 10. Wintroub, B. U., Goetzl, E. J. & Austen, K. F. (1974) J. Exp. Med. cleaved Aa, B(3, and y chains and by the continued degradation 140,812-824. to smaller fragments unlike those seen from the limited pro- 11. Wintroub, B. U., Goetzl, E. J. & Austen, K. F. (1977) Immunology teolytic cleavage by plasmin (21, 22). Because these latter 33,41-49. studies were carried out with fractions enriched for the neu- 12. Rosenberg, R. D. & Damus, P. S. (1973) J. Biol. Chem. 248, trophil granules, the activities observed cannot be attributed 6490-6505. to a single protease; it is likely that elastase and cathepsin G are 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- present, both of which have fibrinogenolytic activity (6). 4412. Low-dose cleavage of fibrinogen by leukocyte elastase (0.3 ag 14. Deutsch, D. & Mertz, E. (1970) Science 170, 1095-1096. of elastase per mg of fibrinogen) cleaved the Aa chain with little 15. Gordon, S., Werb, Z. & Cohn, Z. A. (1976) in In Vitro Methods effect"on the By3 and y chains, whereas higher concentrations in Cell-Mediated and Tumor Immunity, eds. Bloom, B. & cleaved all three chains and resulted in the appearance of David, J. (Academic, New York), pp. 343-352. 16. Binder, B. R., Spragg, J. & Austen, K. F. (1979) J. Biol. Chem. fragments having the antigenic characteristics of plasmin 254, 1998-2003. fragments D and E (20). In addition, digestion with elastase was 17. Zacharius, R. M., Zell, T. E., Morrison, H. & Woodlock, J. J. (1969) awsciated with the development of anticoagulant activity (20). Anal. Biochem. 30,148-152. Because the purified neutral peptide-generating protease is 18. Doolittle, R. (1975) in The Plasma Proteins: Structure, Function devoid of elastase activity and cleaves fibrinogen in a manner and Genetic Control, ed. Putnam, F. (Academic, New York), distinctly different from that of plasmin, it represents a neu- pp. 110-162. trophil peutral protease with fibrinolytic and fibrinogenolytic 19. Ohlsson, K. & Olsson, I. (1973) Eur. J. Biochem. 36,473-481. activities. It is tempting to speculate that the function of this 20. Gramse, M., Bingenheimer, C., Schmidt, W., Egbring, R. & protease is to facilitate neutrophil movement through vascular Havemann, K. (1978) J. Clin. Invest. 61,1027-1033. and extravascular sites by generating a vasoactive peptide and 21. Plow, E. F. & Edgington, T. S. (1975) J. Clin. Invest. 56,30- controlling local fibrin deposition. 38. 22. Bilezikian, S. & Nossel, H. (1977) Blood 5,21-28. This work was supported by Grants AI-07722, AI-10356, AM-05577, 23. Pizzo, S., Schwartz, M., Hill, R. & McKee, P. (1972) J. Biol. Chem. HL-17382, HL-19777, and RR-05669 from the National Institutes of 247,636-645. Health. J.S.C. was a postdoctoral trainee supported by Training Grant 24. Fullan, M. & Beck, E. A. (1972) Biochim. Blophys. Acta 263, AM.07031 from the National Institutes of Health. B.U.W. is the re- 631-644. cipient of a Clinical Investigator Award (AM-00430) from the National 25. Lahiri, B. & Shainoff, J. R. (1973) Biochim. Biophys. Acta 303, Institutes of Health. 161-170. 1. Rulot, H. (1904) Arch. Int. Physiol. 1, 152-158. 26. Marder, V. J., Shulman, N. R. & Caroll, W. R. (1969) J. Blol. 2. Opie, E. L. (1907) J. Exp. Med. 9,391-413. Chem. 244,2111-2119. 3. Welch, W. H. (1887) Trans. Pathol. Soc. (Philadelphia) 13, 27. Marder, V. J. & Shulman, R. (1969) J. Biol. Chem. 244,2120- 281-300. 2124. Downloaded by guest on October 1, 2021