Use of Inhibitory (Anti-Catalytic) Antibodies to study extracellular Proteolysis

Abstract

The specific role of proteolytic enzymes in the degradation by live cells of fibrillar model matrices (fibrin, collagen) was studied using monoclonal and polyclonal inhibitory (anti-catalytic) antibodies. Dissolution of fibrin by plasminogen-supplemented human HT-1080 cells was blocked by (1) omission of plasminogen, (2) inhibitory anti-plasmin antibody, and (3) inhibitory anti-u-PA antibody but not by non-inhibitory control antibodies. Using a similar approach, it was shown that the dissolution of reconstituted type I collagen fibrils by trypsin-supplemented live human skin fibroblasts was blocked by inhibitory antibodies to fibroblast-type procollagenase but not by non-inhibitory control antibodies. These findings permit us to deduce that, at least in culture, the dissolution of fibrin by plasminogen-supplemented HT-1080 cells was mediated by plasminogen-assisted proteolysis which entailed the extracellular conversion of plasminogen to plasmin by cell-derived u-PA, and that the dissolution of collagen fibrils by trypsin-supplemented skin fibroblasts was mediated by a collagenase-dependent pathway.