STRUCTURE AND FUNCTION OF BLOOD PLATELET GPIIB-IIIA

血小板 GPIIB-IIIA 的结构和功能

• 批准号: 3472698
• 负责人: JOSEPH C LOFTUS
• 金额: $ 11.8万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3472698
• 关键词: affinity chromatography; aminoacid; arginine; binding proteins; biological polymorphism; calcium binding protein; complementary DNA; crosslink; extracellular matrix; fibrinogen; fibronectins; gene expression; glycoproteins; hemostatics; in situ hybridization; integrins; ligands; membrane lipids; membrane proteins; messenger RNA; platelet aggregation; platelets; polymerase chain reaction; receptor; thrombosis; transfection; von Willebrand's disease

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.

血小板聚集和血小板对成分的粘附 细胞外基质是止血和血栓形成的控制事件 通过一组粘附蛋白的结合进行调节，包括 纤维蛋白原、纤连蛋白和血管性血友病因子均含有 Arg- 甘氨酸-天冬氨酸 (RGD) 序列。 GPIIb-IIIa 是共同受体的组成部分 对于这些粘附蛋白并与含有 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，与某些受体共享 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。 的绑定 这些受体的粘附蛋白是二价阳离子依赖性的， 推定的二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RGD含有 GPIIb-IIA 配体引起二价阳离子调节的表达 GPIIb 和 GPIIIa 上的抗原位点。 该提案将测试 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，我们将检验以下假设： GPIIIa 的高度保守区域，RGD 肽可能与其结合 化学交联也有助于 RGD 结合。 我们首先会 分析 GPIIb 和 GPIIIa cDNA 中这些区域的序列 来自天然突变体，其中 GPIIb-IIIa 异二聚体缺乏 RGD 结合 功能。 其次，我们将评估重组 GPIIb-IIIa 在 利用新型细胞分析法、亲和力的瞬时表达系统 色谱法、化学交联法。 第三，在那些地区 删除消除了 RGD 结合，我们将识别那些单独的氨基 通过饱和诱变进行 RGD 识别所必需的酸。最后， 除了这些特定区域之外，我们还将确定以下区域 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实现蛋白质结合功能 贯穿 GPIIb 和 GPIIIa。 这些研究将提供基础 有关 GPIIb-IIIa 区域的信息，这些信息对于 血小板粘附和聚集。