Abstract

The aggregation of activated platelets is mediated by the binding of fibrinogen to its cell surface receptor, the integrin α_IIbβ₃. The recognition of fibrinogen by α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ purified on an RGD-affinity column (B_max = 0.2 mol/mol α_IIbβ₃) or to α_IIbβ₃ purified over a lentil lectin column (B_max = 0.03 mol/mol α_IIbβ₃), 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 α_IIbβ₃require divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺ are able to support binding, with Mn⁺⁺being the most effective. Thirteen α_IIbβ₃antagonists, including four linear and three cyclic RGD peptides, five peptidomimetics, the fibrinogen γ-chain dodecapeptide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [³H]-SK&F-107260 or [³H]-SB-214857 binding to α_IIbβ₃. 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 α_IIbβ₃. Scatchard plot analyses of the [³H]-SK&F-107260 binding isotherms in the presence of unlabeled SB-214857 and γ-chain dodecapeptide reveal competitive-type antagonism, indicating that SB-214857, γ-chain dodecapeptide and SK&F-107260 interact with mutually exclusive binding sites on α_IIbβ₃.