A Novel Regulatory Epitope Defined by a Murine Monoclonal Antibody to the Platelet GPIIb-IIIa Complex (αIIbβ3 Integrin)

Michihide Tokuhira; Makoto Handa; Tetsuji Kamata; Atsushi Oda; Masahiko Katayama; Yoshiaki Tomiyama; Mitsuru Murata; Yohko Kawai; Kiyoaki Watanabe; Yasuo Ikeda

doi:10.1055/s-0038-1650704

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 76(06): 1038-1046
DOI: 10.1055/s-0038-1650704

Original Article

Schattauer GmbH Stuttgart

A Novel Regulatory Epitope Defined by a Murine Monoclonal Antibody to the Platelet GPIIb-IIIa Complex (αIIbβ3 Integrin)

Michihide Tokuhira

¹The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Makoto Handa

²The Department of Blood Center, School of Medicine, Keio University, Tokyo, Japan

,

Tetsuji Kamata

¹The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Atsushi Oda

¹The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Masahiko Katayama

³The Department of Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Japan

,

Yoshiaki Tomiyama

⁴The Second Department of Internal Medicine, Osaka University Medical School, Osaka, Japan

,

Mitsuru Murata

¹The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Yohko Kawai

⁵The Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Kiyoaki Watanabe

⁵The Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan

,

Yasuo Ikeda

¹The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

› Author Affiliations

Abstract

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Summary

We characterized a murine monoclonal antibody, PT25-2 (IgG₁), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced ¹²⁵I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the αIIbβ3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37°C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody’s epitope depends upon a conformational structure maintained by αβ subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (K_d = 40 nM, B_max = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the αIIbβ3 integrin on unstimulated, quiescent platelets.

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References

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