Purification and characterization of A₆₁: An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors

Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000) Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys⁷⁸-Lys⁴⁶⁸. To avoid confusion with the plasmin(ogen) fragment, angiostatin® (Lys⁷⁸-Ala ⁴⁴⁰), we have named this protein A₆₁. Unlike angiostatin, A₆₁ was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A₆₁ bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A₆₁ was composed of 21% β-strand, β-turn, 18% 3₁-helix and 8% 3₁₀-helix. A₆₁ was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A₆₁ by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A₆₁ was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a non-pathological setting.