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Development of an Assay for in Vivo Human Neutrophil Elastase Activity

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Development of an Assay for in Vivo Human Neutrophil Elastase Activity Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1- proteinase inhibitor deficiency. J I Weitz, … , S Birken, F J Morgan J Clin Invest. 1986;78(1):155-162. https://doi.org/10.1172/JCI112545. Research Article Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A- containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less […] Find the latest version: https://jci.me/112545/pdf Development of an Assay for In Vivo Human Neutrophil Elastase Activity Increased Elastase Activity in Patients with a1-Proteinase Inhibitor Deficiency Jeffrey 1. Weitz, Sharon L. Landman, Kathryn A. Crowley, Steven Birken, and Francis J. Morgan Department ofMedicine, Columbia University College ofPhysicians & Surgeons, New York 10032 Abstract It has been suggested that leukocyte proteinases function in the of fibrinogen and fibrin, both normally and in Leukocyte extracts contain enzymes that digest fibrinogen and degradation release a fibrinopeptide A-containing fragment. This study was disease states (4-1 1). Electron microscopy studies indicate that can fibrin and alter undertaken to identify the responsible proteinase and to char- polymorphonuclear leukocytes (PMN) ingest (7). PMN accumulate within acterize the fibrinopeptide A-containing fragment so that it could its morphologic appearance thrombi (8) and can readily penetrate preformed blood clots (9). be used as an index of enzyme activity. Both the fibrinogenolytic Infusion of isolated leukocyte enzymes into primates results in and of the fibrinopeptide A-containing frag- activity the release and elevated levels of fibrin(ogen) deg- ment mediated by the leukocyte extracts were shown to be due fibrinogen proteolysis radation products (10). Increased levels of PMN elastase-related to human neutrophil elastase (HNE) by the following criteria: antigen (11) and inhibitor complexes (5, was completely blocked by a specific HNE inhibitor or elastase-al-proteinase activity 12) have been detected in the blood of patients with acute leu- by HNE from the extracts with a monospecific anti- adsorbing kemia and septicemia. Finally, PMN release elastase-related an- body and reconstitution with purified HNE restored the ability in association with blood which may be the to A-containing fragment. This frag- tigen coagulation, release the fibrinopeptide of leukocyte enzyme-mediated fibri- a variety of other proteinases or by mechanism for activation ment was not released by in vivo of fibrinogen (or HNE-inhibitor complexes indicating that, at least with respect nogenolysis (4). However, proteolysis other potential substrates) by leukocyte proteinases is likely to to the it is a product of HNE and its enzymes tested, specific the balance between the enzymes and proteinase inhibitors release requires the free enzyme. By separating the products of reflect such as inhibitor or (13). There- HNE digestion of fibrinogen using high performance liquid aI-proteinase a2-macroglobulin fore, a specific probe of leukocyte enzyme activity is needed to chromatography, identifying the immunoreactive fractions and assess the contributions of these enzymes to in vivo proteolysis. them to amino acid analysis, the fragment was iden- subjecting The leukocyte extract-mediated cleavage of the HNE site at the pattern of tified as Aal-21, indicating an cleavage of fibrinogen in vitro is distinct from that bond. The mean plasma Aal-21 level was NH2-terminal regions Val(Aa21)-Glu(Aa22) Thrombin converts to markedly higher in patients with a1-proteinase inhibitor defi- produced by thrombin (14). fibrinogen fibrin by releasing fibrinopeptide A (FPA or Aal-16)1 and fi- ciency as compared to healthy controls (0.2 nM vs. 7.9 nM; P brinopeptide B (FPB or Bfll- 14) from the NH2-terminal regions < 0.0001), consistent with increased in vivo HNE activity in of the Aa and In contrast, leukocyte these individuals. Bf3-chains, respectively (15). extracts release FPA and FPB-containing fragments (14). Ad- Introduction ditionally, the fibrinogen degradation products produced by leu- kocyte proteinases are structurally (6, 14, 16), immunochemically Human leukocyte proteinases have been implicated in the (17), and functionally (16) distinct from plasmin-derived cleavage pathogenesis of a variety of diseases (1). The activity of these products. enzymes in blood or body fluids has been difficult to measure Quantitatively, elastase (EC 3.4.21.1 1) and cathepsin G (EC because they rapidly interact with their substrates or bind to 3.4.21.37) are the most abundant of the neutral proteinases proteinase inhibitors. The enzyme-inhibitor complexes are then present in PMN. Of these, elastase may be physiologically more cleared from the circulation and/or degraded in situ (2, 3). An- important since it is released from the PMN azurophilic granules tigenic determinations (4) or measurements of enzyme-inhibitor during phagocytosis or when the cells are activated by stimuli complexes (5) fail to discriminate between free (i.e., active) pro- such as soluble immune complexes, C5a or endotoxin, while teinase and enzyme bound to an inhibitor. These limitations cathepsin G remains within the cell (18). In vitro both enzymes have hampered investigations into the role of leukocyte pro- can digest a variety of protein substrates including fibrinogen teinases in disease. (19-21). Since plasma levels of a specific fibrinogen cleavage product can provide an index of in vivo enzyme activity, we set Presented in part at the Xth International Congress on Thrombosis and out to determine whether a single proteinase was responsible for Hemostasis, San Diego, CA (1985. Thromb. Hemostasis. 54:232 and the fibrinogenolytic activity ofleukocyte extracts and the cleavage 276a. Abstr.) and the 25th Annual Meeting of the American Society of of an FPA-containing fragment, to characterize the FPA-con- Hematology, New Orleans, LA (1985. Blood. 62:1086a. Abstr.) taining fragment, and to measure the fragment in human plasma Address reprint requests to Dr. Weitz. samples. Receivedfor publication 22 January 1986. J. Clin. Invest. 1. Abbreviations used in this paper: EACA, epsilon-amino caproic acid; © The American Society for Clinical Investigation, Inc. FPA, B, fibrinopeptide A (Aa1-16) or B (B,B1-14); HNE, human neu- 0021-9738/86/07/0155/08 $1.00 trophil elastase; TBS, Tris-buffered saline; TIFPA, thrombin increasable Volume 78, July 1986, 155-162 fibrinopeptide A. Assay for In Vivo Neutrophil Elastase Activity 155 Methods has previously been shown to be specific for FPA and to crossreact poorly with fibrinogen or FPA-containing fragments from the NH2-terminal Reagents. Purified human neutrophil elastase (HNE) (22), a specific region of the Aa-chain of fibrinogen (28, 29). Samples were assayed for polyclonal rabbit antibody to this enzyme (5) and a2-macroglobulin (23) FPA immunoreactivity before and after treatment with 2 U/ml of human were generous gifts of Drs. P. Harpel and M. Brower, Cornell Medical a-thrombin for 60 min at 37°C. Thrombin treatment of synthetic Aal- Center. Specific peptide chloromethyl ketone inhibitors of HNE and 21 caused a 1,000-fold increase in FPA immunoreactivity (thrombin cathepsin G, MeO-Suc-Ala2-Pro-ValCH2Cl and Z-Gly-Leu-PheCH2Cl, increasable FPA immunoreactivity or TIFPA). respectively, were from Enzyme Systems Products, Livermore, CA. Hu- Cleavage of the NHrterminal Aar chain offibrinogen by leukocyte man a-thrombin was from Dr. J. Fenton, III. A purified fraction of extracts or purified enzymes. Experiments were performed at 37°C in reptilase (venom from Bothrops atrox) was a gift of Dr. B. Blomback. TBS. 1-ml samples of purified fibrinogen (1.2 or 0.6 mg/ml) were in- Trypsin (TRTPCK; 235 U/mg), chymotrypsin (67 U/mg), porcine pan- cubated at 37°C with 20 ,l of a 1/40 (final) dilution of the leukocyte creas elastase (ESFF: 8 U/mg), and carboxypeptidases A (COAPMS: 36 extracts or of purified proteinases at intervals from 5 to 120 min. Control U/mg) and B (COBPMS: 68 U/mg) were from Worthington Biochem- samples were incubated with buffer in place of enzyme or with leukocyte icals, Malvern, PA. The activity ofcarboxypeptidases A and B was tested extracts that had been preabsorbed with immobilized control or anti- immediately before use by measuring hydrolysis of hippuryl-L-phenyl- HNE IgG. At each time point, 100-,d aliquots were removed and the alanine and hippuryl-L-arginine, respectively (Sigma Chemical Co., St. reaction was stopped and the fibrinogen precipitated by the addition of Louis, MO). Plasmin was from Kabi Diagnostica, Stockholm, Sweden, 300 Ml ethanol followed by centrifugation at 4,000 g at 4°C for 20 min. a1-proteinase was from Calbiochem, San Diego, CA, and aprotinin was The ethanol supernatants were evaporated to dryness in a Speed Vac from FBA Pharmaceuticals, New York.
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