STRUCTURAL STUDIES OF BLOOD CLOTTING PROTEINS

凝血蛋白的结构研究

• 批准号: 2216738
• 负责人: JOHN W WEISEL
• 金额: $ 22.51万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2216738
• 关键词: blood coagulation; charge coupled device camera; chemical models; computer simulation; conformation; crosslink; cryoscopy; digital imaging; fibrin; fibrin stabilizing factor; fibrinogen; fibrinopeptide; glycoproteins; high voltage electron microscopy; human tissue; image processing; intermolecular interaction; mutant; polymerization; protein structure function; scanning electron microscopy; scanning transmission electron microscopy; structural biology; transmission electron microscopy

The major goals of this research project are the elucidation of aspects of the molecular mechanisms of assembly of the fibrin clot, as well as interactions of fibrinogen with GPIIb/IIIa and of high molecular weight kininogen with several proteins. Various methods of structural molecular biology, including transmission, scanning and intermediate voltage electron microscopy and computer image processing will be employed in these studies. Most of the required specimen preparation procedures and applications of the appropriate methods have already been developed in this laboratory. Aspects of the mechanism of assembly of the fibrin clot will be studied through analysis of differences in the structures formed by highly selected variant fibrinogens that have specific molecular defects. Electron microscopy of unstained, frozen-hydrated microcrystals of fibrinogen and fibrin will be used to obtain structural data and define intermolecular interactions in fibrin fibers. We will determine whether the Factor XIIIa-induced intermolecular cross-linking between gamma chains occurs between fibrin molecules that are interacting in an end-to-end or half- staggered manner. The alphaC domains of fibrin in protofibrils will be localized by electron microscopy of protofibrils with antibody bound to this region, and their interactions visualized in polymers of alphaC fragments and in complexes of alphaC fragment and fibrin. The functions of this part of the molecule will be further studied by analysis of oligomers made from desA fibrin monomer missing the alphaC domains. Protofibrils formed by cleavage of fibrinopeptide B with venzyme will be examined by electron microscopy. Structural studies of whole clots are necessary for observation and analysis of fiber branch points to understand the mechanisms involved in branching. Specifically designed software will be used to reconstruct and analyze quantitatively networks from stereo images of clots obtained with an intermediate voltage electron microscope. The nature of the inter- actions between GPIIb/IIIa and fibrinogen will be further investigated, using electron microscopy of several complexes as one approach to developing a more detailed molecular model for adhesive interactions between various fibrinogen domains and GPIIb/IIIa. The shape of high molecular weight kininogen will be determined and its structural and functional domains will be localized, providing insight into its mechanisms of action. One of the long-term goals of this research is to relate the results of these structural studies to biochemical, physiological and clinical work in the same area. The complex clotting/fibrinolytic system is unbalanced in many pathological conditions; to develop more effective and specific methods to control these processes, it is necessary to understand the molecular structures and interactions of the proteins involved.

该研究项目的主要目标是阐明以下方面 纤维蛋白凝块组装的分子机制，以及 纤维蛋白原与 GPIIb/IIIa 和高分子量的相互作用 激肽原与几种蛋白质。 多种结构方法 分子生物学，包括传输、扫描和中间体 电压电子显微镜和计算机图像处理 受雇于这些研究。 大部分所需的标本制备 适当方法的程序和应用已经 是在这个实验室开发的。 机制方面 将通过分析来研究纤维蛋白凝块的组装 精心选择的变体形成的结构差异 具有特定分子缺陷的纤维蛋白原。 电子显微镜 未染色、冷冻水合的纤维蛋白原和纤维蛋白微晶 将用于获取结构数据并定义分子间 纤维蛋白纤维中的相互作用。 我们将确定该因素是否 XIIIa 诱导的 γ 链之间发生分子间交联 以端对端或半端相互作用的纤维蛋白分子之间 交错的方式。 原纤维中纤维蛋白的 alphaC 结构域将 通过电子显微镜对结合有抗体的原纤维进行定位 到这个区域，以及它们在 alphaC 聚合物中的相互作用 片段以及αC片段和纤维蛋白的复合物。 这 该部分分子的功能将进一步研究 分析由缺失 alphaC 的 desA 纤维蛋白单体制成的寡聚物 域。 纤维蛋白肽 B 裂解形成的原纤维 venzyme 将通过电子显微镜检查。 结构研究 整个凝块对于纤维的观察和分析是必要的 分支点以了解分支所涉及的机制。 专门设计的软件将用于重建和分析 从通过获得的凝块立体图像定量网络 中压电子显微镜。 间的性质 将进一步研究 GPIIb/IIIa 和纤维蛋白原之间的作用， 使用几种复合物的电子显微镜作为一种方法 开发更详细的粘合剂相互作用分子模型 各种纤维蛋白原结构域和 GPIIb/IIIa 之间。 形状高 将测定激肽原的分子量及其结构和 功能域将被本地化，从而提供对其的深入了解 行动机制。 这项研究的长期目标之一是 将这些结构研究的结果与生化联系起来， 生理和临床工作在同一领域。 综合体 许多病理状态下凝血/纤溶系统不平衡 状况;制定更有效、更具体的控制方法 这些过程，有必要了解分子结构 以及相关蛋白质的相互作用。