HEPARIN AND HEPARAN-SULFATE ARE INHIBITORS OF HUMAN-LEUKOCYTE ELASTASE

1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(L-alanine)3-p-nitroanilide (50% inhibition of 250-mu-l of 10-mu-g of human leucocyte elastase/ml was obtained with 80-mu-l of 2.8-mu-g of heparin/ml and 8-mu-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.l.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.l.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)3-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-mu-l of 50 ng of human leucocyte elastase/md was obtained with 100-mu-l of 4.5-mu-g of heparin/ml and 0.8-mu-g 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 addition of protamine sulphate or by the removal of heparin by an ion-exchange resin. 4. Low M(r) preparations of heparin were also found to inhibit human leucocyte elastase, although preparations With M(r) 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.