The adherence of the major platelet integrin GPIIb-IIIa (alphaIIb-beta3) to specific proteins of the subendothelial matrix, to other platelets, and to other cells plays a fundamental role in normal hemostasis. The importance of these platelet adhesive interactions is evidenced by the several pathologies (ranging from severe bleeding disorders to organ failure due to thrombotic occlusion of vessels) that arise from abnormalities in these adhesive interactions. The objective of this proposal is to determine the molecular basis of ligand binding to alphaIIb-beta3. Elucidation of the mechanism of ligand recognition is central to the understanding of receptor function, providing insights into modulation of platelet function and thus, into the development of novel antithrombotic therapies. Several lines of evidence indicate the presence of multiple ligand contact points in alphaIIb, most likely located in the amino terminal portions of alphaIIb and beta3. Thus, using random mutagenesis, we propose to identify individual amino acid residues on both the alphaIIb and beta3 subunits that are essential for ligand binding function, concentrating on the first 490 residues of alphaIIb and the first 450 residues of beta3. CHO cells will be transfected with both a mutagenized subunit and a corresponding wild type subunit. Selection of mutant cell lines will be done by flow cytometry using two color fluorescence to sort single cells. The criteria for selection will be for those cells that bind to a panel of complex-specific anti-alphaIIb-beta3 antibodies (thus, express the receptor on the cell surface), but do not bind to the ligand mimetic antibody PAC1. Mutant receptors will be characterized for their ability to bind several adhesive ligands such as Fg, Fn and vWF. Specific binding affinities of these ligands will be determined using a direct 125-I ligand binding assay. Manipulation of potential differences in binding affinities could lead to the production of novel therapeutic agents.
| Liu, Chun; Cao, Ying; Malhotra, Atul et al. (2011) Sleep fragmentation attenuates the hypercapnic (but not hypoxic) ventilatory responses via adenosine A1 receptors in awake rats. Respir Physiol Neurobiol 175:29-36 |
| Tozer, E C; Baker, E K; Ginsberg, M H et al. (1999) A mutation in the alpha subunit of the platelet integrin alphaIIbbeta3 identifies a novel region important for ligand binding. Blood 93:918-24 |
| Tozer, E C; Hughes, P E; Loftus, J C (1996) Ligand binding and affinity modulation of integrins. Biochem Cell Biol 74:785-98 |
