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

这个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)复合体。 GPIIb-IIIa是一种钙依赖的异二聚体，其结合部位与 纤维蛋白原和血管性血友病因子等配体暴露于 血小板被激活。因为与GPIIb-IIIa的配体结合是直接的 负责血小板聚集的配体结合的调节是一种 血小板功能的关键一步。激动剂的作用机制 将GPIIb-IIIa转换为活性构象以及 支持其功能的GPIIb-IIIa的折叠构象是 悬而未决的问题。具体目标1将阐述 参与GPIIb-IIIa激活的信号通路。因为它一直是 难以研究激动剂激活GPIIb-IIIa的机制 利用血小板，我们已经在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 使用佛波酯(PMA)和最近通过刺激细胞 用甲酰-蛋氨酸-亮氨酸多肽表达甲酰肽受体 Phe.我们将研究肌动蛋白细胞骨架的作用，即与小GTP结合 蛋白质、磷脂酰肌醇3-激酶同工酶、Pleckstrin和信号 G蛋白偶联受体在GPIIb-IIIa激活中的启动作用 B淋巴细胞系统。具体目标2将继续研究 GPIIb-IIIa异源二聚体的结构-功能关系虽然 全部或部分GPIIb-IIIa分子的X射线或溶液结构 都不可用，已通过检查确定了重要功能 自然发生的和定点突变的后果 GPIIb或GPIIa。我们将继续研究GPIIb-IIIa折叠和 细胞内运输，重点是自然的影响 导致Glanzmann血小板减少症的发生突变 细胞内伴侣蛋白在凝血酶原激活剂表达中的作用 表型。因为我们之前的工作证明了 在整个GPIIb-IIIa折叠中推测的GPIIb的钙结合区， 二价阳离子对其二级结构的影响 将使用生物物理技术检查GPIIb区域 圆二色谱和核磁共振波谱。

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.