Vol. 285, Issue 1, 228-235, April 1998

Binding of [³H]-SK&F 107260 and [³H]-SB 214857 to Purified Integrin _IIb3: Evidence for a Common Binding Site for Cyclic Arginyl-Glycinyl-Aspartic Acid Peptides and Nonpeptides

Angela Wong, Shing Mei Hwang, Kyung Johanson, James Samanen, Donald Bennett, Scott W. Landvatter, Wenting Chen, J. Richard Heys, Fadia E. Ali, Thomas W. Ku, William Bondinell, Andrew J. Nichols, David A. Powers and Jeffrey M. Stadel

Departments of Cellular Biochemistry (A. W., S. M. H.), Protein Biochemistry (K. J.), Macromolecular Sciences (D. B.), Synthetic Chemistry (S. W. L., W. C., J. R. H.), Medicinal Chemistry (J. S., F. A., T. W. K., W. B.), and Pharmacology (A. J. N., D. A. P., J. M. S.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania

The aggregation of activated platelets is mediated by the binding of fibrinogen to its cell surface receptor, the integrin _IIb3. The recognition of fibrinogen by _IIb3 depends, in part, on the tripeptide sequence Arg-Gly-Asp (RGD) in the adhesive protein. The interactions of a cyclic RGD-containing pentapeptide, [³H]-SK&F-107260, and a 1,4-benzodiazepine-based nonpeptide [³H]-SB-214857, with purified _IIb3 have been investigated. Both compounds potently inhibit platelet aggregation at submicromolar concentrations. Binding of both [³H]-SK&F-107260 (K_d = 1.19 nM) and [³H]-SB-214857 (K_d = 1.85 nM) to _IIb3 is of high affinity and fully reversible. The binding is monophasic, indicating a single class of noncooperative binding sites. The two radioligands exhibited similar values in binding to _IIb3 purified on an RGD-affinity column (B_max = 0.2 mol/mol _IIb3) or to _IIb3 purified over a lentil lectin column (B_max = 0.03 mol/mol _IIb3), suggesting that SK&F-107260 and SB-214857 interact with the same population of receptors. Binding of [³H]-SK&F-107260 and [³H]-SB-214857 to _IIb3 require divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺ are able to support binding, with Mn⁺⁺ being the most effective. Thirteen _IIb3 antagonists, including four linear and three cyclic RGD peptides, five peptidomimetics, the fibrinogen -chain dodecapeptide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [³H]-SK&F-107260 or [³H]-SB-214857 binding to _IIb3. The affinity constants (K_i) of these compounds, determined by the two radioligand binding assays, are similar. Furthermore, these compounds exhibit the same rank order of potency in inhibiting biotinylated-fibrinogen binding to _IIb3. Scatchard plot analyses of the [³H]-SK&F-107260 binding isotherms in the presence of unlabeled SB-214857 and -chain dodecapeptide reveal competitive-type antagonism, indicating that SB-214857, -chain dodecapeptide and SK&F-107260 interact with mutually exclusive binding sites on _IIb3.