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

Abstract

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) membrane changes conformation upon activation. A novel anti-alpha(IIb)beta(3) monoclonal antibody (MAb), XIIF9, is described which recognizes 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. Platelets stimulated with 0.5 U/ml of thrombin bound 66000 +/- 4000 molecules/cell (K(d) = 51.6 nM). Moreover, XIIF9 binding to unstimulated platelets could be increased 4-fold by treatment of the alpha(IIb)beta(3) complex with 5 mM EDTA. Thus, XIIF9 recognized an epitope on the beta(3) subunit whose accessibility was increased upon thrombin activation or EDTA treatment. Sequence analysis of the gene segment encoding the XIIF9 heavy chain revealed interesting motifs shared with cyclic CX6-7C anti-alpha(IIb)beta(3) peptides or with AC7, a published MAb specific for activated alpha(IIb)beta(3). In vivo experiments in atherosclerotic rabbits followed by immunohistological analysis, revealed a specific binding of XIIF9 on platelets engaged in thrombus formation, demonstrating real clinical potential for such MAbs in imaging.

Laminin deposition in the extracellular matrix: a complex picture emerges

“…The development of conformation-dependent monoclonal antibodies against laminins might allow testing of such a possibility, as conformation-dependent antibodies have proved invaluable in dissecting structural and functional modifications in a variety of proteins, including the tau protein and aIIbb3 integrin (Dabadie et al, 2001;Wolozin et al, 1986). …”

Development of a Platform of Antibody-Presenting Liposomes

“…For labelling activated platelets from atherosclerotic plaques, we used the murine monoclonal antibody (mAb) XIIF9 directed against the b3 subunit of the aIIbb3 integrin [26]. Magnetoliposomes made of DOPC/DOPS (70/30, mole ratio) were functionalized with complexes between Anx5ZZ and XIIF9 mAbs.…”

“…The antibody used for liposome functionalization, called XIIF9 mAb, is directed against the b3 subunit of aIIbb3 integrin, which is expressed by activated platelets present in thrombi observed in ruptured atherosclerotic plaques [26]. In order to detect liposomes in TEM, we used magnetoliposomes loaded with colloidal iron oxide particles [24], which give a characteristic signal in TEM images (Fig.…”

Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin

“…XIIF9 (a well characterized mouse IgG anti␣IIb␤3) was used as a positive control (Fig. 6a) [27]. TRAP activated and non-activated human platelets were labeled with scFv[EBB3] issuing from the different purification processes (soluble and secreted scFv, soluble and unsecreted scFv or inclusions bodies).…”