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; Rikke Egelund; Tove Kirkegaard; Klaus T. Preissner; Kees W. Rodenburg; Peter A. Andreasen

doi:10.1055/s-0037-1613903

Thrombosis and Haemostasis, Table of Contents

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

¹From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark

,

Rikke Egelund

¹From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark

,

Tove Kirkegaard

¹From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark

,

Klaus T. Preissner

²Department of Biochemistry, Justus-Liebig University, Giessen, Germany

,

Kees W. Rodenburg³

¹From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark

,

Peter A. Andreasen

¹From the Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark

› Author Affiliations

Abstract

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

Full Text

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