34 1
Clinical Science (1991) 81,341-346
Heparin and heparan sulphate are inhibitors of human leucocyte elastase R. L. WALSH, T. J. DILLON, R. SCICCHITANO" AND G. McLENNAN" Division of Clinical Chemistry, Institute of Medical and Veterinary Science, Adelaide, and *Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia
(Received 14 August 1990/3 April 1991; accepted 8 April 1991)
SUMMARY
1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(L-alanine),-pnitroanilide (50% inhibition of 250 pl of 10 pg of human leucocyte elastase/ml was obtained with 80 pl of 2.8 pg of heparin/ml and 8 p g of heparan sulphate/ml).Less significant inhibition at the same concentrations was seen with the other glycosaminoglycans tested: hyaluronic acid and chondroitin sulphates A-C. 2. Heparin and heparan sulphate also strongly inhibited human leucocyte elastase activity towards insoluble human lung elastin, as determined by an e.1.i.s.a. for soluble elastin-derived peptides released by elastolytic activity on the elastin. This inhibition was shown not to be due to a direct interference of the glycosaminoglycans in the e.1.i.s.a. nor to the inhibition causing a change in the size of the elastin-derived peptides. However, unlike the chromogenic assay with n-succinyl-(L-alanine),-p-nitroanilide as substrate, where heparin was the more effective inhibitor, in this assay system heparan sulphate was the more effective inhibitor (50% inhibition of 100 pl of 50 ng of human leucocyte elastase/ml was obtained with 100 pl of 4.5 pg of heparin/ml and 0.8 pg of heparan sulphate/ml). These results suggest that heparin and heparan sulphate, as components of cellular and basement membranes, are likely to have a role in protecting structural proteins, such as elastin, from the proteolytic activity of human leucocyte elastase. 3. The degree of inhibition by heparin was independent of pH within the range of pH studied (pH 6-9) and was almost immediate in the automated chromogenic assay system where a 10 s preincubation step was used. The inhibitory effect of heparin could be prevented by the Dr G. McLennan, Department of Thoracic Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia.
addition of protamine sulphate or by the removal of heparin by an ion-exchange resin. 4. Low M , preparations of heparin were also found to inhibit human leucocyte elastase, although preparations with M , of 2000 or less did not inhibit the enzyme to the same extent as commercial preparations. 5. Heparin at the same concentrations did not inhibit other leucocyte enzymes, such as cathepsin G and myeloperoxidase, nor the pancreatic enzymes, pancreatic elastase, trypsin and chymotrypsin. Key words: elastase, elastin, glycosaminoglycans, heparin, low-M, heparin. Abbreviations: CSL, Commonwealth Serum Laboratories; DBL, David Bull Laboratories; GAG, glycosaminoglycan; HIE, human leucocyte elastase; SLAPN, n-succinyl-(L-alanine),-p-nitroanilide.
INTRODUCTION The glycosaminoglycans (GAGs), heparin and the structurally similar heparan sulphate, as components of proteoglycans are found both in mast cells [l,21 and as part of the luminal surface of cell membranes [3], especially the vascular endothelium [4]. It has been suggested that tissue GAGs inhibit proteases and have a role in tissue injury and repair mechanisms [5, 61. Acute tissue inflammation is associated with the influx and activation of polymorphonuclear neutrophils. These cells gain access to the interstitial space by a process which initially is associated with increased adherence to the vascular endothelium [7]. Once activated, the human neutrophil is able to cause tissue damage by a number of mechanisms including the release of a powerful serine protease, human leucocyte elastase (HLE), which is capable of degrading several macromolecular constituents of connective tissue [8].
342
R. L. Walsh et al.
Normal tissue components are protected in part against HLE by a ,-anti-trypsin, the predominant endogenous regulator of HLE activity [9]. However, during acute tissue inflammation activated neutrophils release oxidants which can inactivate a,-anti-trypsin and thus prevent it from binding to HLE [lo]. Therefore in this situation, and when leucocytes adhere to membrane surfaces, another inhibitor of HLE may become important. In addition, it is reasonable to propose that in the healing phase the synthesized matrix elastin needs to be preserved against further elastase digestion. We propose that heparin and heparan sulphate, or their respective proteoglycans, can fulfil this role in part. Heparin is commonly used as an anticoagulant. It is prepared commercially from animal tissue (usually intestine and lung) and consists of a number of chemically related sulphated mucopolysaccharides. The high anticoagulant potency of heparin is primarily due to a unique pentasaccharide binding site for the coagulation factor, anti-thrombin [ll-131. This sequence is only found on 35% of heparin molecules. However, the physiological function of heparin has not been defined. Heparin cannot be detected in normal human blood [14, 151, which suggests that physiologically it functions within or close to membranes, intracellularly or in the interstitial tissue spaces. Here we report that heparin from different biologics1 sources, heparan sulphate and low-molecular-mass heparin fractions strongly inhibit HLE activity towards a chromogenic substrate and that heparin and heparan sulphate strongly inhibit HLE activity towards human elastin.
3.4.2 1.20; Elastin Products Co., Owensville, MO, U.S.A.) and myeloperoxidase (EC 1.11.1.7; Calbiochem Corp, San Diego, CA, U.S.A.). Pancreatic enzymes used were elastase (EC 3.4.21.36; porcine; Calbiochem), trypsin (EC 3.4.21.4; porcine type IX;Sigma) and chymotrypsin (EC 3.4.21.1; bovine type 11; Sigma).
MATERIALS AND METHODS
Porcine pancreatic elastase assay
GAGs
This assay was the same as the HLE chromogenic assay, except that the assay was monitored for 10 min.
Heparin was obtained from the following sources: porcine-derived [David Bull Laboratories (DBL), Mulgarve, Victoria, Australia, and Fisons, Wrexham, Clwyd, U.K.], ovine-derived (DBL), bovine-derived (H5765; Sigma Chemical Co., St Louis, MO, U.S.A.) and source not stated [Commonwealth Serum Laboratories (CSL), Melbourne, Victoria, Australia]. The low-M, heparin preparations used were as follows: OP 381/2, M , 850-1000; O P 381/1, M , 2000; OP 2123, M , 4500 (gifts from Opocrin S.P.A., Modena, Italy) and Kabi 2165, M , 4000-6000 (a gift from Kabi Vitrum AB, Stockholm, Sweden). Sources of other GAGs were as follows: hyaluronic acid, sodium salt from rooster combs (Pharmacia, AB, Uppsala, Sweden); chondroitin sulphate A from bovine trachea (Sigma), chrondroitin sulphate B (dermatan sulphate) from pig skin and chondroitin sulphate C from shark cartilage (Seikagaku Kogyo, Tokyo, Japan); heparan sulphate from bovine kidney (Sigma).
HLE assays Chromogenic assay. This assay was performed on a Cobas-Bio centrifugal analyser (Hoffman-LaRoche, Basle, Switzerland) as follows. Eighty microlitres of sample (e.g. heparin) were mixed with 250 pl of 0.05 mol/l Tris/HCI, 0.05 mol/l.NaCI, pH 8.0 and 0.01% Brij 35, containing 2.5 pg of HLE. After incubation for 60 s at 25"C, 20 p1 of 5 mmol/l of the chromogenic substrate n-succinyl-(L-alanine),-p-nitroanilide (SLAPN, Calbiochem) in the Tris/HCl buffer was added. The rate of hydrolysis was monitored at 405 nm for 30 min. HLE was demonstrated to be at least 90% active against SLAPN using previously published kinetic constants [ 161. Elastin degradation assay. Human lung elastin (Elastin Products Co., catalogue no. HS395) was added to each well of a microtitre tray as a 5 p g suspension (particle size
Clinical Science (1991) 81,341-346
Heparin and heparan sulphate are inhibitors of human leucocyte elastase R. L. WALSH, T. J. DILLON, R. SCICCHITANO" AND G. McLENNAN" Division of Clinical Chemistry, Institute of Medical and Veterinary Science, Adelaide, and *Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia
(Received 14 August 1990/3 April 1991; accepted 8 April 1991)
SUMMARY
1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(L-alanine),-pnitroanilide (50% inhibition of 250 pl of 10 pg of human leucocyte elastase/ml was obtained with 80 pl of 2.8 pg of heparin/ml and 8 p g of heparan sulphate/ml).Less significant inhibition at the same concentrations was seen with the other glycosaminoglycans tested: hyaluronic acid and chondroitin sulphates A-C. 2. Heparin and heparan sulphate also strongly inhibited human leucocyte elastase activity towards insoluble human lung elastin, as determined by an e.1.i.s.a. for soluble elastin-derived peptides released by elastolytic activity on the elastin. This inhibition was shown not to be due to a direct interference of the glycosaminoglycans in the e.1.i.s.a. nor to the inhibition causing a change in the size of the elastin-derived peptides. However, unlike the chromogenic assay with n-succinyl-(L-alanine),-p-nitroanilide as substrate, where heparin was the more effective inhibitor, in this assay system heparan sulphate was the more effective inhibitor (50% inhibition of 100 pl of 50 ng of human leucocyte elastase/ml was obtained with 100 pl of 4.5 pg of heparin/ml and 0.8 pg of heparan sulphate/ml). These results suggest that heparin and heparan sulphate, as components of cellular and basement membranes, are likely to have a role in protecting structural proteins, such as elastin, from the proteolytic activity of human leucocyte elastase. 3. The degree of inhibition by heparin was independent of pH within the range of pH studied (pH 6-9) and was almost immediate in the automated chromogenic assay system where a 10 s preincubation step was used. The inhibitory effect of heparin could be prevented by the Dr G. McLennan, Department of Thoracic Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia.
addition of protamine sulphate or by the removal of heparin by an ion-exchange resin. 4. Low M , preparations of heparin were also found to inhibit human leucocyte elastase, although preparations with M , of 2000 or less did not inhibit the enzyme to the same extent as commercial preparations. 5. Heparin at the same concentrations did not inhibit other leucocyte enzymes, such as cathepsin G and myeloperoxidase, nor the pancreatic enzymes, pancreatic elastase, trypsin and chymotrypsin. Key words: elastase, elastin, glycosaminoglycans, heparin, low-M, heparin. Abbreviations: CSL, Commonwealth Serum Laboratories; DBL, David Bull Laboratories; GAG, glycosaminoglycan; HIE, human leucocyte elastase; SLAPN, n-succinyl-(L-alanine),-p-nitroanilide.
INTRODUCTION The glycosaminoglycans (GAGs), heparin and the structurally similar heparan sulphate, as components of proteoglycans are found both in mast cells [l,21 and as part of the luminal surface of cell membranes [3], especially the vascular endothelium [4]. It has been suggested that tissue GAGs inhibit proteases and have a role in tissue injury and repair mechanisms [5, 61. Acute tissue inflammation is associated with the influx and activation of polymorphonuclear neutrophils. These cells gain access to the interstitial space by a process which initially is associated with increased adherence to the vascular endothelium [7]. Once activated, the human neutrophil is able to cause tissue damage by a number of mechanisms including the release of a powerful serine protease, human leucocyte elastase (HLE), which is capable of degrading several macromolecular constituents of connective tissue [8].
342
R. L. Walsh et al.
Normal tissue components are protected in part against HLE by a ,-anti-trypsin, the predominant endogenous regulator of HLE activity [9]. However, during acute tissue inflammation activated neutrophils release oxidants which can inactivate a,-anti-trypsin and thus prevent it from binding to HLE [lo]. Therefore in this situation, and when leucocytes adhere to membrane surfaces, another inhibitor of HLE may become important. In addition, it is reasonable to propose that in the healing phase the synthesized matrix elastin needs to be preserved against further elastase digestion. We propose that heparin and heparan sulphate, or their respective proteoglycans, can fulfil this role in part. Heparin is commonly used as an anticoagulant. It is prepared commercially from animal tissue (usually intestine and lung) and consists of a number of chemically related sulphated mucopolysaccharides. The high anticoagulant potency of heparin is primarily due to a unique pentasaccharide binding site for the coagulation factor, anti-thrombin [ll-131. This sequence is only found on 35% of heparin molecules. However, the physiological function of heparin has not been defined. Heparin cannot be detected in normal human blood [14, 151, which suggests that physiologically it functions within or close to membranes, intracellularly or in the interstitial tissue spaces. Here we report that heparin from different biologics1 sources, heparan sulphate and low-molecular-mass heparin fractions strongly inhibit HLE activity towards a chromogenic substrate and that heparin and heparan sulphate strongly inhibit HLE activity towards human elastin.
3.4.2 1.20; Elastin Products Co., Owensville, MO, U.S.A.) and myeloperoxidase (EC 1.11.1.7; Calbiochem Corp, San Diego, CA, U.S.A.). Pancreatic enzymes used were elastase (EC 3.4.21.36; porcine; Calbiochem), trypsin (EC 3.4.21.4; porcine type IX;Sigma) and chymotrypsin (EC 3.4.21.1; bovine type 11; Sigma).
MATERIALS AND METHODS
Porcine pancreatic elastase assay
GAGs
This assay was the same as the HLE chromogenic assay, except that the assay was monitored for 10 min.
Heparin was obtained from the following sources: porcine-derived [David Bull Laboratories (DBL), Mulgarve, Victoria, Australia, and Fisons, Wrexham, Clwyd, U.K.], ovine-derived (DBL), bovine-derived (H5765; Sigma Chemical Co., St Louis, MO, U.S.A.) and source not stated [Commonwealth Serum Laboratories (CSL), Melbourne, Victoria, Australia]. The low-M, heparin preparations used were as follows: OP 381/2, M , 850-1000; O P 381/1, M , 2000; OP 2123, M , 4500 (gifts from Opocrin S.P.A., Modena, Italy) and Kabi 2165, M , 4000-6000 (a gift from Kabi Vitrum AB, Stockholm, Sweden). Sources of other GAGs were as follows: hyaluronic acid, sodium salt from rooster combs (Pharmacia, AB, Uppsala, Sweden); chondroitin sulphate A from bovine trachea (Sigma), chrondroitin sulphate B (dermatan sulphate) from pig skin and chondroitin sulphate C from shark cartilage (Seikagaku Kogyo, Tokyo, Japan); heparan sulphate from bovine kidney (Sigma).
HLE assays Chromogenic assay. This assay was performed on a Cobas-Bio centrifugal analyser (Hoffman-LaRoche, Basle, Switzerland) as follows. Eighty microlitres of sample (e.g. heparin) were mixed with 250 pl of 0.05 mol/l Tris/HCI, 0.05 mol/l.NaCI, pH 8.0 and 0.01% Brij 35, containing 2.5 pg of HLE. After incubation for 60 s at 25"C, 20 p1 of 5 mmol/l of the chromogenic substrate n-succinyl-(L-alanine),-p-nitroanilide (SLAPN, Calbiochem) in the Tris/HCl buffer was added. The rate of hydrolysis was monitored at 405 nm for 30 min. HLE was demonstrated to be at least 90% active against SLAPN using previously published kinetic constants [ 161. Elastin degradation assay. Human lung elastin (Elastin Products Co., catalogue no. HS395) was added to each well of a microtitre tray as a 5 p g suspension (particle size
