STRUCTURE AND FUNCTION OF BLOOD PLATELET GPIIB-IIIA

血小板聚集和血小板粘附于 细胞外基质是止血和血栓形成的控制事件， 是通过一组粘附蛋白的结合来调节的， 纤维蛋白原、纤连蛋白和血管性血友病因子都含有Arg- Gly-Asp（RGD）序列。 GPIIb-IIIa是一种常见受体的成分， 并与含有RGD的肽结合 顺序 此外，GPIIb-IIIa是整合素粘附的成员， 受体超家族，并与某些 其他整合素。 不同的α和β亚基是同源的 蛋白质，并拥有几个高度保守的序列。 的结合 这些受体的粘附蛋白是二价阳离子依赖性的， 氨基酸序列的推定的二价阳离子结合位点， 整合素α亚基是高度保守的，并且含有结合RGD的α亚基是高度保守的。 GPIIb-IIA的配体引起二价阳离子调节的表达， GPIIb和GPIIIa上的抗原位点。 这项提案将考验 假设这些假定的钙结合位点中的一个或多个 参与RGD结合。 此外，我们还将检验以下假设： 结合RGD肽的GPIIIa的高度保守区域可以是 化学交联也有助于RGD结合。 我们将首先 分析GPIIb和GPIIIa cDNA中这些区域的序列， 来自GPIIb-IIIa异二聚体缺乏RGD结合的天然突变体 功能 其次，我们将评估重组GPIIb-IIIa在 利用新的细胞计数分析、亲和性 色谱法和化学交联。 第三，在那些 缺失消除了RGD结合，我们将鉴定那些单独的氨基酸 通过饱和诱变识别RGD所必需的氨基酸。最后， 除了这些特定区域，我们还将确定 GPIIb-IIIa，其结构完整性对于普通粘合剂至关重要 通过随机核苷酸插入突变的蛋白质结合功能 在GPIIb和GPIIIa中。 这些研究将提供基本的 关于GPIIb-IIIa的那些区域的信息， 血小板粘附和聚集。

Platelet aggregation and platelet adhesion to components of the extracellular matrix are control events in hemostasis and thrombosis and are regulated through the binding of a set of adhesive proteins including fibrinogen, fibronectin and von Willebrand factor all which contain Arg- Gly-Asp (RGD) sequences. GPIIb-IIIa is a component of a common receptor for these adhesive proteins and binds to peptides containing the RGD sequence. Moreover, GPIIb-IIIa is a member of the Integrin adhesion receptor superfamily and shares the RGD recognition function with certain other integrins. The various alpha and beta subunits are homologous proteins and possess several highly conserved sequences. The binding of adhesive proteins to these receptors is divalent cation dependent, the amino acid sequences of the putative divalent cation binding sites in the Integrin alpha subunits is highly conserved, and the binding RGD containing ligands to GPIIb-IIA causes the expression of divalent cation regulated antigenic sites on GPIIb and GPIIIa. This proposal will test the hypothesis that one or more of these putative calcium binding sites participate in RGD binding. In addition, we will test the hypothesis that the highly conserved region of GPIIIa to which bound RGD peptides may be chemically crosslinked also contributes to RGD binding. We will first analyze the sequence of these regions in GPIIb and GPIIIa cDNAs derived from a natural mutant in which the GPIIb-IIIa hetrodimer lacks RGD binding function. Secondly, we will asses the effects of recombinant GPIIb-IIIa in a transient expression system utilizing a novel cytometric assay, affinity chromatography, and chemical crosslinking. Third, in those regions whose deletion abolishes RGD binding,we will identify those individual amino acids essential for the RGD recognition by saturation mutagenesis. Finally, in addition to those specific regions we will identify those regions of GPIIb-IIIa whose structural integrity is essential for normal adhesive protein binding function through random nucleotide insertion mutations throughout both GPIIb and GPIIIa. These studies will provide fundamental information about those regions of GPIIb-IIIa which are essential for the platelet adhesion and aggregation.