Angiostatin formation involves disulfide bond reduction and proteolysis inkringle 5 of plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibit or, angiostatin, and microplasmin, Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that form ation of angiostatin/microplasmin involves reduction of plasmin by a plasmi n reductase followed by proteolysis of the reduced enzyme. We present evide nce that the Cys(461)-Cys(540) and Cys(511)-Cys(535) disulfide bonds in kri ngle 5 of plasmin were reduced by plasmin reductase, Plasmin reductase acti vity was secreted by HT1080 and Chinese hamster ovary cells and the human m ammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte /macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood mo nocyte/ macrophages, nor macrovascular or microvascular endothelial cells s ecreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plas min reductase. Reduction of the kringle 5 disulfide bonds triggered cleavag e at either Arg(529)-Lys(530) Or two other positions C-terminal of Cys(461) in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT 1080 conditioned medium. Three serine proteinases with apparent M-r of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of whi ch could contribute to proteolysis of reduced plasmin.