Binding of (3H)-SK&F 107260 and (3H)-SB 214857 to purified integrin alphaIIbbeta3: evidence for a common binding site for cyclic arginyl-glycinyl-aspartic acid peptides and nonpeptides.

摘要

The aggregation of activated platelets is mediated by the binding of fibrinogen to its cell surface receptor, the integrin alphaIIbbeta3. The recognition of fibrinogen by alphaIIbbeta3 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 alphaIIbbeta3 have been investigated. Both compounds potently inhibit platelet aggregation at submicromolar concentrations. Binding of both [3H]-SK&F-107260 (Kd = 1.19 nM) and [3H]-SB-214857 (Kd = 1.85 nM) to alphaIIbbeta3 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 alphaIIbbeta3 purified on an RGD-affinity column (Bmax = 0.2 mol/mol alphaIIbbeta3) or to alphaIIbbeta3 purified over a lentil lectin column (Bmax = 0.03 mol/mol alphaIIbbeta3), 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 alphaIIbbeta3 require divalent cations, Mg++, Ca++ and Mn++ are able to support binding, with Mn++ being the most effective. Thirteen alphaIIbbeta3 antagonists, including four linear and three cyclic RGD peptides, five peptidomimetics, the fibrinogen gamma-chain dodecapeptide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [3H]-SK&F-107260 or [3H]-SB-214857 binding to alphaIIbbeta3. The affinity constants (Ki) 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 alphaIIbbeta3. Scatchard plot analyses of the [3H]-SK&F-107260 binding isotherms in the presence of unlabeled SB-214857 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 alphaIIbbeta3.