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 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 KD obtained by fitting the kinetic binding data in a two‐step model are in good agreement with directly detected values of koff(L‐762,745) = (1.9 ± 0.6) 10−3 s−1, koff(L‐769,434) = (5.1 ± 0.7) 10−3 s‐1, KD(L‐762,745) = 12 ± 0.5 nM, and KD(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 KD 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 koff for L‐738,167 of (4.1 ± 0.1) × 10−4 s−1 (t½ = 28 min). Cytometry 28: 58–65, 1997. © 1997 Wiley‐Liss, Inc.