STRUCTURAL STUDIES OF BLOOD CLOTTING PROTEINS

凝血蛋白的结构研究

• 批准号: 2468992
• 负责人: JOHN W WEISEL
• 金额: $ 30.02万
• 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 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2468992
• 关键词: blood coagulation; blood viscosity; crosslink; fibrin; fibrinogen; fibrinolysis; glycosylation; hemostasis; human tissue; image processing; intermolecular interaction; plasmin; polymerization; protein structure function; recombinant proteins; scanning electron microscopy; scanning transmission electron microscopy; structural biology; thrombin; thromboplastin; thrombosis

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.

本研究项目的主要目标是阐明 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 含有几种蛋白质的激肽原。 各种结构方法 分子生物学，包括传输，扫描和中间 电压电子显微镜和计算机图像处理将是 在这些研究中使用。 大多数所需的标本制备 适当方法的程序和应用已经 是在这个实验室里开发出来的。 机制的各个方面 纤维蛋白凝块的组装将通过分析 高度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 纤维蛋白原和纤维蛋白的未染色的冷冻水化微晶 将用于获得结构数据和定义分子间 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链之间的分子间交联发生 纤维蛋白分子之间的相互作用是端对端或半- 交错的方式。 原纤维中纤维蛋白的α C结构域将 通过电子显微镜观察抗体结合的原纤维进行定位 这一区域，以及它们在alphaC聚合物中的相互作用 片段以及α C片段和纤维蛋白的复合物。 的 这部分分子的功能将进一步研究， 由缺失α C的德萨纤维蛋白单体制备的寡聚体的分析 域. 纤维蛋白肽B与 通过电子显微镜检查venzyme。 结构研究 为了观察和分析纤维， 分支点了解分支中涉及的机制。 专门设计的软件将用于重建和分析 从凝块的立体图像定量网络， 中压电子显微镜 国际组织的性质- GPIIb/IIIa和纤维蛋白原之间的作用将进一步研究， 使用电子显微镜的几个复合物作为一种方法， 开发更详细的粘附相互作用的分子模型 不同的纤维蛋白原结构域和GPIIb/IIIa之间。 高的形状 分子量激肽原将被确定，其结构和 功能域将被本地化，提供洞察其 作用机制。 这项研究的长期目标之一是 将这些结构研究的结果与生物化学联系起来， 生理和临床工作在同一领域。 复杂 凝血/纤溶系统在许多病理性 条件;制定更有效和具体的方法来控制 这些过程中，有必要了解分子结构 以及相关蛋白质的相互作用。