Purification and characterization of A(61) - 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 t he 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, Ri,, Fogg, D, K,, and Waisman, D, M, (2 000)Bio chemistry 39, 1921-1028), In the present communication we have iden tified the 61-kDa plasmin fragment as a novel four kringle-containing prote in consisting of the amino acid sequence Lys(78)-Lys(468). To avoid Confusi on with the plasmin(ogen) fragment, angiostatin(R) (Lys(78)- Ala(440)), we have named this protein A(61) Unlike angiostatin A(61) was produced in vitr o from plasmin autodigestion: in the absence of sulfhydryl donors, A(61) bo und to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanec arboxylic acid. Circular dichroism suggested that A(61), was composed of 21 % beta -strand, 14% beta -turn, 18% 3(1)-helix and 8% 3(10)-heIix. A(61) wa s an anti-angiogenic protein as indicated by the inhibition of bovine capil lary endothelial cell proliferation. Plasminogen was converted to A(61) by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasmin ogen fragment similar to A(61) was present in the serum of humans as well a s normal and tumor-bearing mice. These results establish that plasmin turno ver can generate anti-angiogenic plasmin fragments in a nonpathological set ting.