Characterisation, cloning and sequencing of a conformation-dependent monoclonal antibody to the alpha(IIb)beta(3) integrin: interest for use in thrombus detection

The detection of newly formed thrombi is of primary importance in clinical medicine. The activated platelet is a potential target for the localization of thrombotic lesions in arteries. The integrin alpha (IIb)beta (3) in the platelet membrane changes conformation upon activation. A novel anti-alpha (IIb)beta (3) monoclonal antibody (MAb), XIIF9, is described which recogni zes an epitope whose expression was enhanced by activation. Radioiodinated XIIF9 bound to a single class of sites on the beta (3) subunit, with 13600 +/- 2000 molecules bound per unstimulated platelet and a K-d of 34.5 nM. Pl atelets stimulated with 0.5 U/ml of thrombin bound 66000 +/- 4000 molecules /cell (K-d = 51.6 nM). Moreover, XIIF9 binding to unstimulated platelets co uld be increased 4-fold by treatment of the alpha (IIb)beta (3) complex wit h 5 mM EDTA. Thus, XIIF9 recognized an epitope on the beta (3) subunit whos e accessibility was increased upon thrombin activation or EDTA treatment. S equence analysis of the gene segment encoding the XIIF9 heavy chain reveale d interesting motifs shared with cyclic CX6-7C anti-alpha (IIb)beta (3) pep tides or with AC7, a published MAb specific for activated alpha (IIb)beta ( 3). In vivo experiments in atherosclerotic rabbits followed by immunohistol ogical analysis, revealed a specific binding of XIIF9 on platelets engaged in thrombus formation, demonstrating real clinical potential for such MAbs in imaging.