A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (alpha IIb beta 3 integrin).
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 alpha IIb beta 3 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 degrees 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 alpha beta 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 alpha IIb beta 3 integrin on unstimulated, quiescent platelets.
Tokuhira
M
M
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Handa
M
M
Kamata
T
T
Oda
A
A
Katayama
M
M
Tomiyama
Y
Y
Murata
M
M
Kawai
Y
Y
Watanabe
K
K
Ikeda
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D001792:Blood Platelets / Q000276:immunology*; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology*
D000911:Antibodies, Monoclonal; D016056:Immunodominant Epitopes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex