STRUCTURAL STUDIES OF BLOOD CLOTTING PROTEINS

本研究项目的主要目的是阐明各方面。 纤维蛋白凝块组装的分子机制，以及 纤维蛋白原与高分子量GPIIb/IIIa的相互作用 激肽原和几种蛋白质。结构设计的各种方法 分子生物学，包括传递、扫描和中间体 电压电子显微镜和计算机图像处理将是 受雇于这些研究。所需的大部分样品准备工作 程序和应用的适当方法已经 是在这个实验室里开发出来的。机制的几个方面 纤维蛋白凝块的组装将通过分析 高度选择变异体形成的结构差异 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 将被用来获得结构数据和定义分子间 纤维蛋白纤维中的相互作用。我们将确定这一因素是否 XIIIA诱导的伽马链分子间交联 在端到端或半端相互作用的纤维蛋白分子之间- 摇摇晃晃的样子。原纤维中纤维蛋白的αC结构域将 被抗体结合的原纤维的电子显微镜定位 到这个区域，以及它们在AlphaC聚合物中的相互作用 在αC片段和纤维蛋白的复合体中。这个 这部分分子的功能将通过以下方式进一步研究 缺失字母C的DESA纤维蛋白单体低聚物的分析 域名。纤维蛋白肽B裂解形成的原纤维 对文酶进行电子显微镜检查。结构研究 是观察和分析纤维所必需的。 分支指向以了解分支涉及的机制。 将使用专门设计的软件进行重建和分析 从立体图像中获得的血块的定量网络 中压电子显微镜。国际关系的性质-- 将进一步研究GPIIb/IIIa与纤维蛋白原之间的作用， 使用几个络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的分子模型 在不同的纤维蛋白原结构域和GPIIb/IIIa之间。高的形状 将测定分子量激肽原，并对其结构和 功能域将被本地化，从而提供对其 行动机制。这项研究的长期目标之一是 为了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。情结 凝血/纤溶系统在许多病理疾病中失衡 制定更有效和更具体的方法来控制 在这些过程中，有必要了解分子结构 以及所涉及的蛋白质之间的相互作用。

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.