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粘附于成分 细胞外基质是止血和血栓形成中的控制事件， 通过一组粘合蛋白（包括）的结合来调节 纤维蛋白原，纤连蛋白和von Willebrand因子都包含grug- gly-asp（RGD）序列。 GPIIB-IIIA是公共受体的组成部分 对于这些粘附蛋白，并与含有RGD的肽结合 顺序。 此外，GPIIB-IIIA是整联蛋白粘附的成员 受体超家族，并与一定的RGD识别功能共享 其他整合素。 各种alpha和beta亚基是同源的 蛋白质并具有多个高度保守的序列。 结合 这些受体的粘合蛋白是依赖于二价阳离子的， 推定的二价阳离子结合位点的氨基酸序列 整合素α亚基是高度保守的，结合RGD包含 GPIIB-IIA的配体导致调节二价阳离子的表达 GPIIB和GPIIIA上的抗原位点。 该建议将测试 假设其中一个或多个这些假定的钙结合位点 参加RGD绑定。 此外，我们将检验以下假设 GPIIIA的高度保守区域可能是该区域的RGD肽可能是 化学交联还有助于RGD结合。 我们将首先 分析这些区域在GPIIB和GPIIIA CDNA中的序列 从天然突变体中，gpiib-iiia hetrodimer缺乏RGD结合 功能。 其次，我们将评估重组GPIIB-IIIA在 使用新型的细胞术测定，亲和力的瞬态表达系统 色谱和化学交联。 第三，在那些地区 删除废除了RGD绑定，我们将确定那些个体的氨基 通过饱和诱变对RGD识别必不可少的酸。最后， 除了这些特定地区，我们还将确定 GPIIB-IIIA的结构完整性对于正常粘合剂至关重要 蛋白质结合功能通过随机核苷酸插入突变 在gpiib和gpiiia中。 这些研究将提供基本 有关GPIIB-IIIA地区的信息，这对于 血小板粘附和聚集。