THROMBOSIS AND HEMOSTASIS Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen

When Glu-plasminogen binds to cells, its activation to plasmin is markedly en-hanced compared with the reaction in solu-tion, suggesting that Glu-plasminogen on cell surfaces adopts a conformation dis-tinct from that in solution. However, di-rect evidence for such conformational changes has not been obtained. There-fore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially rec-ognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-RKT, and to fibrin, plasmin-treated fi-brinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatogra-phy, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that bind-ing of Glu-plasminogen to cells induces a conformational change in Glu-plasmino-gen distinct from that of Lys-Pg. (Blood