STRUCTURE AND FUNCTION OF THE PLATELET MEMBRANE GLYCOPROTEIN IIB-IIIA COMPLEX

血小板膜糖蛋白 IIB-IIIA 复合物的结构和功能

• 批准号: 6573406
• 负责人: Joel S. Bennett
• 金额: $ 19.72万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6573406
• 关键词: calcium binding protein; crosslink; fibrinogen; glycoprotein structure; human subject; integrins; membrane proteins; membrane structure; oligopeptides; phlebotomy; platelets; protein folding; protein structure function; recombinant proteins; site directed mutagenesis; thrombasthenia; tissue /cell culture; vascular endothelium

The objective of this project is to correlate the structure and function of the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex. GPIIb-IIIa is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and von Willebrand factor is exposed by platelet activation. Because ligand binding to GPIIb-IIIa is directly responsible for platelet aggregation, regulation of ligand binding is a critical step in platelet function. The mechanism by which agonists convert GPIIb-IIIa to an active conformation and the features of the folded conformation of GPIIb-IIIa that underlay its function are unresolved questions. Specific Aim 1 will address the nature of the signaling pathways involved in GPIIb-IIIa activation. Because it has been difficult to study the mechanism by which agonists activate GPIIb-IIIa using platelets, we have expressed GPIIb-IIIa in B lymphocytes. We found that GPIIb-IIIa in these cells can be induced to interact with fibrinogen using phorbol myristate acetate (PMA) and recently, by stimulating cells expressing the formyl peptide receptor with the peptide formyl-Met-Leu- Phe. We will examine the role of the actin cytoskeleton, small GTP-binding proteins, phosphoinositide 3-kinase isoenzymes, pleckstrin, and signals initiated by G protein-coupled receptors in GPIIb-IIIa activation using the B lymphocyte system. Specific Aim 2 will continue studies of study of structure-function relationships in the GPIIb-IIIa heterodimer. Although x-ray or solution structures for all or parts of the GPIIb-IIIa molecule are not available, important features have been determined by examining the consequences of naturally-occurring and site-directed mutations of GPIIb or GPIIIa. We will continue studies of GPIIb-IIIa folding and intracellular transport, with emphasis on the effects of naturally occurring mutations responsible for Glanzmann thrombasthenia and on the role of intracellular chaperones in the expression of the thrombasthenic phenotype. Because our previous work demonstrated the importance of the putative calcium binding region of GPIIb in overall GPIIb-IIIa folding, the influence of divalent cations on the secondary structure of this region of GPIIb will be examined using biophysical techniques such as circular dichroism and nuclear magnetic resonance spectroscopy.

本项目的目标是将结构和功能联系起来 血小板膜糖蛋白IIb-IIIa（GPIIb-IIIa）复合物。 GPIIb-IIIa是一种钙依赖性异源二聚体， 配体如纤维蛋白原和血管性血友病因子暴露于 血小板活化因为配体与GPIIb-IIIa的结合是直接 负责血小板聚集，配体结合的调节是一个重要的机制。 血小板功能的关键步骤。激动剂的作用机制 将GPIIb-IIIa转化为活性构象， GPIIb-IIIa的折叠构象，其功能是 悬而未决的问题具体目标1将解决 参与GPIIb-IIIa激活的信号通路。因为它已被 难以研究激动剂激活GPIIb-IIIa的机制 使用血小板，我们在B淋巴细胞中表达了GPIIb-IIIa。我们发现 这些细胞中的GPIIb-IIIa可以被诱导与纤维蛋白原相互作用， 使用佛波醇肉豆蔻酸酯乙酸酯（PMA），最近，通过刺激细胞 用肽甲酰基-Met-Leu-表达甲酰基肽受体， 菲。我们将研究肌动蛋白细胞骨架的作用，小GTP结合 蛋白质、磷酸肌醇3-激酶同工酶、普列克底物蛋白和信号 在GPIIb-IIIa激活中由G蛋白偶联受体启动， B淋巴细胞系统。具体目标2将继续研究 GPIIb-IIIa异二聚体的结构-功能关系。虽然 全部或部分GPIIb-IIIa分子的X射线或溶液结构 没有可用的，重要的功能已经确定，通过检查 自然发生的和定点突变的后果， GPIIb或GPIIIa。我们将继续研究GPIIb-IIIa折叠， 细胞内运输，重点是自然的影响， 导致血小板无力症的突变， 细胞内伴侣在血小板生成素表达中的作用 表型因为我们之前的工作证明了 在整个GPIIb-IIIa折叠中GPIIb的推定钙结合区， 二价阳离子对这种二级结构的影响 GPIIb区域将使用生物物理技术进行检查，例如 圆二色性和核磁共振光谱。