BINDING OF [H-3]-SK-AND-F-107260 AND [H-3]-SB-214857 TO PURIFIED INTEGRIN ALPHA(IIB)BETA-3 - EVIDENCE FOR A COMMON BINDING-SITE FOR CYCLIC ARGINYL-GLYCINYL-ASPARTIC ACID PEPTIDES AND NONPEPTIDES

The aggregation of activated platelets is mediated by the binding of f ibrinogen to its cell surface receptor, the integrin alpha(IIb)beta(3) . The recognition of fibrinogen by alpha(IIb)beta(3) depends, in part, on the tripeptide sequence Arg-Gly-Asp (RGD) in the adhesive protein. The interactions of a cyclic RGD-containing pentapeptide, [H-3]-SK&F- 107260, and a 1,4-benzodiazepine-based nonpeptide [H-3]-SB-214857, wit h purified alpha(IIb)beta(3) have been investigated. Both compounds po tently inhibit platelet aggregation at submicromolar concentrations. B inding of both [H-3]-SK&F-107260 (K-d= 1.19 nM) and [H-3]-SB-214857 (K -d= 1.85 nM) to alpha(IIb)beta(3) is of high affinity and fully revers ible. The binding is monophasic, indicating a single class of noncoope rative binding sites. The two radioligands exhibited similar values in binding to alpha(IIb)beta(3) purified on an RGD-affinity column (B-ma x = 0.2 mol/mol alpha(IIb)beta(3)) or to alpha(IIb)beta(3) purified ov er a lentil lectin column (B-max = 0.03 mol/mol alpha(IIb)beta(3)), su ggesting that SK&F-107260 and SB-214857 interact with the same populat ion of receptors. Binding of [H-3]-SK&F-107260 and [H-3]-SB-214857 to alpha(IIb)beta(3) require divalent cations, Mg++, Ca++ and Mn++ are ab le to support binding, with Mn++ being the most effective. Thirteen al pha(IIb)beta(3) antagonists, including four linear and three cyclic RG D peptides, five peptidomimetics, the fibrinogen gamma-chain dodecapep tide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [H-3]-SK&F-107260 or [H-3]-SB-214857 binding to alpha(IIb)beta(3). The affinity constants (K-i) of these compounds, determined by the tw o radioligand binding assays, are similar. Furthermore, these compound s exhibit the same rank order of potency in inhibiting biotinylated-fi brinogen binding to alpha(IIb)beta(3). Scatchard plot analyses of the [H-3]-SK&F-107260 binding isotherms in the presence of unlabeled SB-21 4857 and gamma-chain dodecapeptide reveal competitive-type antagonism, indicating that SB-214857, gamma-chain dodecapeptide and SK&F-107260 interact with mutually exclusive binding sites on alpha(IIb)beta(3).