title: Flow cytometric measurement of kinetic and equilibrium binding parameters of arginine-glycine-aspartic acid ligands in binding to glycoprotein IIb/IIIa on platelets.
abstract: Antagonists of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) represent a new therapeutic approach in inhibiting platelet aggregation, thus providing a powerful form of antithrombotic therapy. The measurement of binding of arginine-glycine-aspartic acid (RGD) peptidomimetics to GPIIb/IIIa on platelets is a key for the further understanding of ligand-receptor interactions and, thus, the design of new antagonists. The flow cytometric measurement of dynamic and equilibrium binding parameters of two new potent RGD peptidomimetics, L-762,745 and L-769,434, containing a fluorescein moiety is described in this paper. Kinetic binding measurements with these fluorescent ligands indicate a two-step binding mechanism that involves a conformational rearrangement of the receptor-ligand complex. The overall second-order binding constants are for both fluorescent ligands several orders of magnitude slower than for diffusion-controlled processes. The values of k(-1) and K(D) obtained by fitting the kinetic binding data in a two-step model are in good agreement with directly detected values of k(off)(L-762,745) = (1.9 +/- 0.6) 10(-3) s(-1), k(off)(L-769,434) = (5.1 +/- 0.7) 10(-3) s(-1), KD(L-762,745) = 12 +/- 0.5 nM, and K(D)(L-769,434) = 8 +/- 0.3 nM. Equilibrium binding measurements of fluorescent ligands with an orally active nonfluorescent antagonist, L-738,167, provided apparent dissociation binding constant K(D) of this ligand in the range from 0.1 to 0.2 nM. The kinetic dissociation measurement of L-738,167 using the binding of the fluorescent ligand L-762,745 as a reporting method yielded a k(off) for L-738,167 of (4.1 +/- 0.1) x 10(-4) s(-1) (t1/2 = 28 min).
authors.affiliation: Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. bohumil_bednar@merck.com
authors.forename: B
authors.initials: B
authors.lastname: Bednar
authors.forename: M E
authors.initials: ME
authors.lastname: Cunningham
authors.forename: P A
authors.initials: PA
authors.lastname: McQueney
authors.forename: M S
authors.initials: MS
authors.lastname: Egbertson
authors.forename: B C
authors.initials: BC
authors.lastname: Askew
authors.forename: R A
authors.initials: RA
authors.lastname: Bednar
authors.forename: G D
authors.initials: GD
authors.lastname: Hartman
authors.forename: R J
authors.initials: RJ
authors.lastname: Gould
mesh_terms: D001667:Binding, Competitive; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009842:Oligopeptides / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011971:Receptors, Immunologic; C047981:arginyl-glycyl-aspartic acid