STRUCTURE AND FUNCTION OF BLOOD PLATELET GPIIB-IIIA

血小板聚集性和血小板粘附性的研究 细胞外基质是止血和血栓形成的控制事件， 是通过结合一系列黏附蛋白来调节的，包括 纤维蛋白原、纤维连接蛋白和von Willebrand因子均含有Arg- Gly-Asp(RGD)序列。GPIIb-IIIa是常见受体的一种成分 用于这些黏附蛋白并与含有RGD的多肽结合 序列。此外，GPIIb-IIIa是整合素黏附的成员 受体超家族，与某些人分享RGD识别功能 其他整合素。各种α和β亚基是同源的。 蛋白质，并拥有几个高度保守的序列。的约束性 这些受体上的黏附蛋白是二价阳离子依赖的， 双价阳离子结合位点的氨基酸序列 整合素α亚基高度保守，结合的RGD包含 GPIIb-IIA的配体调节二价阳离子的表达 GPIIb和GPIIa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绑定。此外，我们还将检验以下假设 结合RGD多肽的GPIIIa的高度保守区 化学上的交联性也有助于RGD结合。我们将首先 分析GPIIb和GPIIIa cDNAs中这些区域的序列 来自一个天然突变体，其中GPIIb-IIIa杂二聚体缺乏RGD结合 功能。其次，我们将评估重组GPIIb-IIIa在 一种利用一种新的细胞分析方法-亲和力的瞬时表达系统 层析和化学交联法。第三，在那些地区， 缺失取消RGD结合，我们将确定这些单独的氨基 饱和诱变识别RGD所必需的酸。最后， 除了这些特定的区域外，我们还将确定 GPIIb-IIIa，其结构完整性是正常粘合剂所必需的 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的哪些区域对于 血小板黏附和聚集。

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.