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Thromb Haemost 2000; 83(05): 742-751
DOI: 10.1055/s-0037-1613903
Review Article
Schattauer GmbH

Vitronectin and Substitution of a β-Strand 5A Lysine Residue Potentiate Activity-neutralization of PA Inhibitor-1 by Monoclonal Antibodies against α-Helix F

Susanne L. Schousboe
1   From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark
,
Rikke Egelund
1   From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark
,
Tove Kirkegaard
1   From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark
,
Klaus T. Preissner
2   Department of Biochemistry, Justus-Liebig University, Giessen, Germany
,
Kees W. Rodenburg   3
1   From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark
,
Peter A. Andreasen
1   From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark
› Author AffiliationsThe excellent technical assistance of Anni Christensen, Line V. Nielsen, and Lissy Nielsen is gratefully acknowledged. Dr. Ditlev Brodersen is thanked for help with the Molscript program. The coordinate file for the model of active PAI-1 was kindly provided by Dr. P. J. Declerck. Dr. W. Günzler is thanked for the gift of reagents. This work was supported by grants from The Danish Cancer Society, the Danish Medical Research Council, the Novo-Nordisk Foundation, the Danish Heart Foundation, and the Danish Biotechnology Programme.
Further Information

Publication History

Received 28 July 1999

Accepted after resubmission 03 January 2000

Publication Date:
08 December 2017 (online)

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Summary

Some monoclonal antibodies against plasminogen activator inhibitor-1 (PAI-1) are able to inhibit its reaction with its target proteinases. We have characterized the effect on PAI-1 of two monoclonal antibodies, Mab-2 and Mab-6, with overlapping epitopes in a sequence encompassing α-strand 1A, α-helix F, and the loop connecting α-helix F and β-strand 3A (the hF/s3A loop). Mab-2 reduced the inhibitory activity of wild type PAI-1 and almost totally abolished the inhibitory activity of a PAI-1 variant harboring an Ala substitution of Lys 325 (335 in the α1-proteinase inhibitor template residue numbering) in α-strand 5A. In both cases, the neutralizing effect of the antibody was strongly potentiated by vitronectin. Mab-6 had no effect on wild type PAI-1, but reduced the inhibitory activity of the K325A variant. The effect of Mab-6 was not potentiated by vitronectin. With both Mab-2 and Mab-6, the neutralization of PAI-1 activity was associated with PAI-1 substrate behaviour. Mab-2, but not Mab-6, prevented vitronectin from rescuing PAI-1 from cold-induced substrate behaviour. We propose that the antibodies act by weakening the anchoring of α-helix F to the adjacent structures, resulting in an increased flexibility of α-strand 5A and the hF/s3A loop and a changed conformational response to the binding of vitronectin in the α-helix E region. The potentiating effect of vitronectin on neutralization of PAI-1 by antibodies is a novel concept in the development of compounds for neutralizing PAI-1 in vivo.

Key words

Serpins - serine proteinases - thrombosis - cancer

3 Current address: Dr. K. W. Rodenburg, Biochemical Physiology, Department of Biology, Utrecht University, The Netherlands.


 
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