Neutrophil proteinase 3 (Pr3) cleaves elastin and other matrix proteins, and is thought to cause lung tissue destruction in emphysema and cystic fibrosis. Its deleterious action is theoretically prevented by alpha1-antitrypsin, a serpin present in lung secretions. We have evaluated the anti-Pr3 activity of this inhibitor to decide whether it may play a physiologic proteolysis-preventing function in vivo. We show that (i). the oxidized inhibitor does not inhibit Pr3; (ii). the inhibitor competes favorably with elastin for the binding of Pr3, but is less efficient for inhibiting elastin-bound proteinase than for complexing free enzyme; and (iii). the inhibition takes place in at least two steps: the enzyme and the inhibitor first form a high-affinity reversible inhibitory complex EI* with an equilibrium dissociation constant K*i of 38 nM; EI* subsequently transforms into an irreversible complex EI with a first-order rate constant k2 of 0.04 s-1. Because the alpha1-antitrypsin concentration in the epithelial lining fluid is much higher than K*i, any Pr3 molecule released from neutrophils will be taken up as an EI* complex within much less than 1 s, indicating very efficient inhibition in vivo.

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a K i of 2 ؋ 10 ؊7 M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The K i for the complexes with cathepsin G and proteinase 3 are 8 ؋ 10 ؊8 and 5 ؋ 10 ؊7 M, respectively. Ionic strength increases the K i of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.

Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha 1-antichymotrypsin (ACT) and alpha 1-proteinase inhibitor (alpha 1PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a detailed kinetic investigation of their reaction with cat G. The kinetics of inhibition of cat G in the presence of inhibitor and substrate evidenced a two-step inhibition mechanism: E + I EI EI. The cat G/ACT interaction is described by Ki = 6.2 x 10(-)8 M and k2 = 2.8 x 10(-)2 s-1, while the cat G/alpha 1PI association is governed by Ki = 8.1 x 10(-)7 M and k2 = 5.5 x 10(-)2 s-1. The reliability of these kinetic constants was checked using a number of experiments which all gave consistent results: (i) both EI complexes were found to be enzymatically inactive, (ii) the Ki values were determined directly using initial velocity experiments of cat G-catalyzed hydrolysis of substrate in the presence of inhibitor, (iii) the second-order rate constants k2/Ki were measured using second-order inhibition experiments in the absence of substrate, and (iv) the ratio of the two second-order rate constants was determined by measuring the partition of cat G between the two fluorescently labeled serpins. Since the plasma concentrations of ACT and alpha 1PI are much higher than their Ki values, cat G released from neutrophils will be fully taken up as rapidly forming EI complexes, that is, 70% with ACT and 30% with alpha 1PI. Both ACT and alpha 1PI are thus physiological cat G inhibitors whose inhibitory potential does not depend on the formation of the stable inhibitory species EI characteristic of serpins. Such an in vivo inhibition mechanism might take place with other serpin/proteinase systems.

Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymotrypsin, alpha1-proteinase inhibitor and eglin c, three proteins which do not bind heparin [Ermolieff J., Boudier C., Laine A., Meyer B. and Bieth J.G. (1994) J. Biol. Chem. 269, 29502-29508]. Here we show that heparin no longer protects cathepsin G from inhibition when the enzyme is reacted with mucus proteinase inhibitor (MPI), a heparin-binding protein. Heparin fragments of Mr=4500 and 8100 and O-butyrylated heparin of Mr=8000 form tight complexes with cathepsin G (Kd=0.5-2.2 nM) and MPI (Kd=0. 4-0.8 muM) and accelerate the MPI-promoted inhibition of cathepsin G by a factor of 17-26. They also accelerate the inhibition of neutrophil elastase and pancreatic chymotrypsin. The rate acceleration is due to the binding of heparin to MPI. Butyrylation of heparin slightly decreases its affinity for cathepsin G and MPI but sharply decreases the ionic interactions between the positively charged proteins and the negatively charged polyanion. The butyrylated heparin derivative is the best rate accelerator: it increases the rate constant for the MPI-induced inhibition of cathepsin G and elastase by factors of 26 and 23, respectively. This, together with the fact that it has a good bioavailability and a very low anticoagulant activity, suggests that it might be an adjuvant of MPI-based therapy of cystic fibrosis.