Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides

Muehlenweg, B.; Guthaus, E.; de Prada, N.A.; Schmitt, M.; Schmiedeberg, N.; Kotzsch, M.; Creutzburg, S.; Kramer, M.D.; Kessler, H.; Wilhelm, O.G.; Magdolen, V.

Thrombosis Research 98(1): 73-81

2000


ISSN/ISBN: 0049-3848
PMID: 10706935
DOI: 10.1016/s0049-3848(99)00238-8
Accession: 010599913

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Abstract
Plasminogen activator inhibitor type-1 is a key regulatory protein of the fibrinolytic system that is involved in a variety of physiological and patho-physiological processes. A panel of 14 monoclonal antibodies directed against plasminogen activator inhibitor type-1 was analyzed regarding epitope specificity on plasminogen activator inhibitor type-1. For this purpose, chimera consisting of plasminogen activator inhibitor type-1 and another plasminogen activator inhibitor, plasminogen activator inhibitor type-2, with different portions of the respective wild-type proteins, were generated and plasminogen activator inhibitor type-1-derived 20-mer and 10-mer linear peptides were synthesized. Nine of the 14 monoclonal antibodies recognized an epitope located in the region between amino acid 76-188 of plasminogen activator inhibitor type-1, which encompasses the binding sites for vitronectin, heparin, and part of the fibrin binding region. Of these nine monoclonal antibodies, six reacted with a quadruple plasminogen activator inhibitor type-1 mutant (N152H, K156T, Q321L, M356I), and seven detected a plasminogen activator inhibitor type-1 deletion mutant (DELTAF111-H114). Two of the remaining five monoclonal antibodies recognized epitopes located between amino acid 209-227 and amino acid 352-371, respectively, while the other three antibodies reacted with wild-type plasminogen activator inhibitor type-1, only. Additional experiments revealed that two of the 14 mAbs neutralized and one monoclonal antibodies increased plasminogen activator inhibitor type-1 activity toward urokinase-type plasminogen activator, one of its target proteases.