GENERATION OF ANGIOSTATIN BY REDUCTION AND PROTEOLYSIS OF PLASMIN - CATALYSIS BY A PLASMIN REDUCTASE SECRETED BY CULTURED-CELLS

Extracellular manipulation of protein disulfide bonds has been implied in diverse biological processes, including penetration of viruses and endotoxin into cells and activation of certain cytokine receptors, We now demonstrate reduction of one or more disulfide bonds in the serin e proteinase, plasmin, by a reductase secreted by Chinese hamster ovar y or HT1080 cells, Reduction of plasmin disulfide bond(s) triggered pr oteolysis of the enzyme, generating fragments with the domain structur e of the angiogenesis inhibitor, angiostatin. Two of the known reducta ses secreted by cultured cells are protein disulfide isomerase and thi oredoxin, and incubation of plasmin with these purified reductases res ulted in angiostatin fragments comparable with those generated from pl asmin in cell culture. Thioredoxin-derived angiostatin inhibited proli feration of human dermal microvascular endothelial cells with half-max imal effect at approximately 0.2 mu g/ml, Angiostatin made by cells an d by purified reductases contained free sulfhydryl group(s), and S-car bamidomethylation of these thiol group(s) ablated biological activity, Neither protein disulfide isomerase nor thioredoxin were the reductas es used by cultured cells, because immunodepletion of conditioned medi um of these proteins did not affect angiostatin generating activity, T he plasmin reductase secreted by HT1080 cells required a small cofacto r for activity, and physiologically relevant concentrations of reduced glutathione fulfilled this role, These results have consequences for plasmin activity and angiogenesis, particularly in the context of tumo r growth and metastasis, Moreover, this is the first demonstration of extracellular reduction of a protein disulfide bond, which has general implications for cell biology.