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J. Biol. Chem., Vol. 280, Issue 27, 25403-25408, July 8, 2005

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A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complex of Integrin _IIb3 Inhibits Platelet Aggregation and Adhesion^*

Ping Chen, Chong-Xiu Sun, and Jian-Ning Liu

From the Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China

Activation or ligand binding induces conformational changes in _IIb3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated _IIb3 mutant (CHO _IIb3717) as the immunogen. This IgG_2b named 3C7 was specific for the complex of _IIb3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another _IIb3 mutant (₃Y178A) suggesting that the Cys¹⁷⁷–Cys¹⁸⁴ loop of ₃ was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC₅₀s of 5.6 and 0.05 µg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin _IIb3 and a potential anti-thrombotic agent.

Received for publication, January 13, 2005 , and in revised form, May 12, 2005.

^* This work was supported by Nanjing University (985-FZS, 20021201, and 20020284025) and by Jiangsu Kejiting (BG2000001 and BK2002082). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this paper.

To whom correspondence should be addressed: Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Rd., Nanjing 210093, China. Tel.: 86-25-8359-5678; Fax: 86-25-8326-0284; E-mail: jnliu{at}verizon.net.

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