INHIBITION BY PROTEASE INHIBITORS OF CHEMOTAXIS INDUCED BY ELASTIN-DERIVED PEPTIDES

We studied the effect of two inhibitors of human neutrophil proteases on neutrophil chemotaxis induced by the hexapeptide Val-Gly-Val-Ala-Pr o-Gly (VGVAPG), a recurring sequence in the elastin molecule. The inhi bitors were tosyl-Phe chloromethyl ketone (TFCK) and N-methoxysuccinyl -Ala-Ala-Pro-Val chloromethyl ketone (MAAPVCK). We assayed chemotactic activity by the double-membrane technique in a modified Boyden chambe r, after incubating the cells for 1 hr with varying concentrations of inhibitor. We observed a concentration-dependent inhibitory effect. We also measured the potency of the two chloromethyl ketones as protease inhibitors. The more potent protease inhibitor, MAAPVCK, was also the more effective in inhibiting VGVAPG-induced chemotaxis; its inhibitor dissociation constant was K-I = 28 nM with elastase and K-I = 33 nM w ith cathepsin G. For TFCK the corresponding K-I values were 37 mu M an d 200 mu M. The incubating concentration required to lower chemotaxis by half its uninhibited value was C-0.5 = 0.64 mu M for MAAPVCK, compa red to C-0.5 = 3.4 mu M for TFCK. A third peptide, triglycinate (gly(3 )), which did not inhibit the proteolytic activity of either elastase or cathepsin a, did not inhibit chemotaxis. Chemotaxis induced by form yl Met-Leu-Phe (fMLP) was weakly inhibited by both chloromethyl ketone s with TFCK being somewhat more effective than MAAPVCK. We concluded t hat inhibition of VGVAPG-induced chemotaxis is in part specific, recep tor mediated. We suggest that proteolytic inhibitors protect the extra cellular matrix from degradation by inhibiting chemotaxis, Comparing t he inhibitor concentrations required to half proteolytic activity with the concentration required to half chemotactic activity, we further s uggest that the two functions may be of comparable significance. (C) 1 996 Academic Press, Inc.