FLOW CYTOMETRIC MEASUREMENT OF KINETIC AND EQUILIBRIUM BINDING PARAMETERS OF ARGININE-GLYCINE-ASPARTIC ACID LIGANDS IN BINDING TO GLYCOPROTEIN IIB IIIA ON PLATELETS/

Antagonists of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) represent a new therapeutic approach in inhibiting platelet aggregation, thus pro viding a powerful form of antithrombotic therapy. The measurement of b inding of arginine-glycine-aspartic acid (RGD) peptidomimetics to GPII b/IIIa on platelets is a key for the further understanding of ligand-r eceptor interactions and, thus, the design of new antagonists, The flo w cytometric measurement of dynamic and equilibrium binding parameters of two new potent RGD peptidomimetics, L-762,745 and L-769,434, conta ining a fluorescein moiety is described in this paper, Kinetic binding measurements with these fluorescent ligands indicate a two-step bindi ng mechanism that involves a conformational rearrangement of the recep tor-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 b y fitting the kinetic binding data in a two-step model are in good agr eement 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), K-D( L-762,745) = 12 +/- 0.5 nM, and K-D(L-769,434) = 8 +/- 0.3 nM, Equilib rium 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, T he 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) (t(1/2) = 28 min). (C) 1997 Wiley-Liss, Inc.