Wong A, Hwang S M, Johanson K, Samanen J, Bennett D, Landvatter S W, Chen W, Heys J R, Ali F E, Ku T W, Bondinell W, Nichols A J, Powers D A, Stadel J M
Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
J Pharmacol Exp Ther. 1998 Apr;285(1):228-35.
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.
活化血小板的聚集是由纤维蛋白原与其细胞表面受体整合素αIIbβ3的结合介导的。αIIbβ3对纤维蛋白原的识别部分取决于粘附蛋白中的三肽序列精氨酸 - 甘氨酸 - 天冬氨酸（RGD）。已经研究了含环RGD的五肽[3H]-SK&F-107260和基于1,4 - 苯二氮䓬的非肽[3H]-SB-214857与纯化的αIIbβ3的相互作用。两种化合物在亚微摩尔浓度下均能有效抑制血小板聚集。[3H]-SK&F-107260（Kd = 1.19 nM）和[3H]-SB-214857（Kd = 1.85 nM）与αIIbβ3的结合具有高亲和力且完全可逆。结合是单相的，表明存在单一类别的非协同结合位点。两种放射性配体与在RGD亲和柱上纯化的αIIbβ3（Bmax = 0.2 mol/mol αIIbβ3）或在扁豆凝集素柱上纯化的αIIbβ3（Bmax = 0.03 mol/mol αIIbβ3）结合时表现出相似的值，这表明SK&F-107260和SB-214857与相同的受体群体相互作用。[3H]-SK&F-107260和[3H]-SB-214857与αIIbβ3的结合需要二价阳离子，Mg++、Ca++和Mn++都能够支持结合，其中Mn++最有效。包括四种线性和三种环RGD肽、五种拟肽、纤维蛋白原γ链十二肽（HHLGGAKQAGDV）和蛇毒蛋白echistatin在内的13种αIIbβ3拮抗剂竞争[3H]-SK&F-107260或[3H]-SB-214857与αIIbβ3的结合。通过两种放射性配体结合测定确定的这些化合物的亲和常数（Ki）相似。此外，这些化合物在抑制生物素化纤维蛋白原与αIIbβ3结合方面表现出相同的效价顺序。在未标记的SB-214857和γ链十二肽存在下对[3H]-SK&F-107260结合等温线的Scatchard图分析显示出竞争性拮抗作用，表明SB-214857、γ链十二肽和SK&F-107260与αIIbβ3上相互排斥的结合位点相互作用。
