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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
1   The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Makoto Handa
2   The Department of Blood Center, School of Medicine, Keio University, Tokyo, Japan
,
Tetsuji Kamata
1   The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Atsushi Oda
1   The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Masahiko Katayama
3   The Department of Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Japan
,
Yoshiaki Tomiyama
4   The Second Department of Internal Medicine, Osaka University Medical School, Osaka, Japan
,
Mitsuru Murata
1   The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Yohko Kawai
5   The Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Kiyoaki Watanabe
5   The Department of Laboratory Medicine, School of Medicine, Keio University, Tokyo, Japan
,
Yasuo Ikeda
1   The Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
› Author Affiliations
Further Information

Publication History

Received 18 March 1996

Accepted after resubmission 05 August 1996

Publication Date:
11 July 2018 (online)

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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 (Kd = 40 nM, Bmax = 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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