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Identification of a conformational epitope induced when plasminogen binds to fibrin
Author(s) -
Kim KeeHwan,
Baik Nagyung,
Parmer Robert J,
Miles Lindsey A
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.951.1
Subject(s) - epitope , fibrin , chemistry , plasmin , monoclonal antibody , blot , trypsin , conformational epitope , conformational change , microbiology and biotechnology , fibrinolysis , epitope mapping , biochemistry , antibody , medicine , enzyme , biology , immunology , gene
A key step in thrombolysis is the interaction of plasminogen with the substrate, fibrin. This results in marked enhancement of plasminogen activation. To study this mechanism we developed a monoclonal antibody (mAb 109) that reacts with fibrin‐bound plasminogen in the presence of a ≥ 50‐fold molar excess of soluble plasminogen. The objective of our study was to define the epitope recognized by mAb 109 that reports a conformational change in plasminogen upon binding to fibrin. Plasminogen was denatured in urea and reduced and alkylated followed by digestion with trypsin. Using western blotting and a specific mAb 109‐based ELISA for plasminogen, we established the concentration of trypsin that resulted in maximal digestion of plasminogen, while retaining reactivity with mAb 109. The plasminogen digest was applied to a mAb 109 immunoaffinity column. The eluate was subjected to LC‐MS‐MS. Two peaks with masses of 1298 AMU and 1030 AMU corresponding to a nonlinear epitope contained within H 494 ‐R 504 within the kringle 5 domain and L 652 ‐K 661 in the latent protease domain, respectively, were obtained. These results were corroborated in western blotting of plasminogen domains. Our data suggest that this domain becomes accessible to mAb 109 when plasminogen interacts with fibrin. Thus, the conformational change detected by mAb 109 is likely to enhance the ability of plasminogen to be activated on the fibrin surface. Supported by a Recovery Act Supplement to NIH HL081046 for Summer Research Experiences for Students and Science Educators and by the Department of Veterans Affairs.