title: Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin.
abstract: The RGD (Arg-Gly-Asp) binding integrins α(v)β(3) and α(IIb)β(3) are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to α(v)β(3) and α(IIb)β(3) integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between α(IIb)β(3) integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound α(IIb)β(3) receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor-ligand interactions such as nondissociable interactions and varying receptor activation states.
authors.affiliation: ANSTO LifeSciences, Australian Nuclear Science and Technology Organisation, Sydney 2234, Australia. alexander.szabo@ansto.gov.au
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Szabo
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Howell
authors.forename: Paul
authors.initials: P
authors.lastname: Pellegrini
authors.forename: Ivan
authors.initials: I
authors.lastname: Greguric
authors.forename: Andrew
authors.initials: A
authors.lastname: Katsifis
mesh_terms: D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D019921:Biotinylation; D045744:Cell Line, Tumor; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007553:Isotope Labeling; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results
chemical_list: D039302:Integrin alphaVbeta3; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C057986:echistatin; C047981:arginyl-glycyl-aspartic acid