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首页> 外文学位 >Molecular scale studies of von Willebrand factor: Surface and shear dependent structure and intra -molecular binding sites.
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Molecular scale studies of von Willebrand factor: Surface and shear dependent structure and intra -molecular binding sites.

机译：von Willebrand因子的分子规模研究：表面和剪切依赖性结构以及分子内结合位点。

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摘要

von Willebrand factor (vWf) is a large, multimeric, plasma glycoprotein critical in thrombus formation and normal hemostasis. vWf is indispensable in recruiting platelets to sites of thrombosis (e.g. non-endothelial surfaces) at high fluid shear. Molecular level information about the surface/shear dependent structure, adhesion, interactions and function of critical plasma proteins, such as vWf, is necessary to further molecular understanding of hemostasis and thrombosis.;The aims of this work were: to determine the surface dependent structure of vWf under physiologic pH and ionic strength; to determine the shear dependent structure and adhesion of vWf as related to multimer size; and to determine the accessibility of intra-molecular binding sites on surface adsorbed vWf.;I designed and built a laminar flow cell system which was used with fluid atomic force microscopy (AFM), novel surfaces and purified proteins. Sources of vWf and storage conditions were examined and reliable protocols for the purification and optimal storage of human vWf were implemented. Model hydrophilic and hydrophobic surfaces were used to determine the surface dependent intra-molecular structure of vWf (improving previously published structural detail on vWf). The surface adsorbed intra-molecular structure of vWf on a hydrophilic surface was determined for the first time.;The shear dependent adhesion of vWf dimer, fibrinogen and vWf multimers was determined on hydrophobic and hydrophilic surfaces. Distinct differences in the effect of fluid shear upon the structures of these proteins were observed and correlated to differences in adhesion. These data indicate the contribution of multimerization to the adhesion of vWf. Structures of vWf layers, collagen VI layers and the structure of individual vWf and vWf layers on collagen VI surfaces, were imaged for the first time.;An immuno-gold/AFM technique to determine the accessibility of intra-molecular binding sites on surface adsorbed vWf was established. This is a direct method of correlating molecular scale changes in the structure of vWf (any protein) with the accessibility of functional domains within this structure. Using a panel of monoclonal antibodies against functional domains on vWf, we identified the differential accessibility of specific functional domains within vWf adsorbed on the hydrophobic surface and on the collagen VI surface.
机译：血管性血友病因子（vWf）是一种大型的多聚体血浆糖蛋白，对血栓形成和正常止血至关重要。 vWf是在高剪切力下将血小板募集到血栓形成部位（例如非内皮表面）必不可少的。有关表面/剪切依赖性结构，关键血浆蛋白（例如vWf）的粘附，相互作用和功能的分子水平信息对于进一步分子了解止血和血栓形成是必不可少的;该工作的目的是：确定表面依赖性结构vWf在生理pH和离子强度下的变化；确定与多聚体尺寸有关的剪切依赖性结构和vWf的附着力;我设计并建立了一个层流细胞系统，该系统与流体原子力显微镜（AFM），新颖的表面和纯化的蛋白质一起使用。检查了vWf的来源和储存条件，并实施了纯化和最佳保存人vWf的可靠方案。使用模型亲水和疏水表面来确定vWf的表面依赖性分子内结构（改进了先前发表的关于vWf的结构细节）。首次确定了vWf在亲水性表面上的表面吸附分子内结构;;确定了在疏水性和亲水性表面上vWf二聚体，纤维蛋白原和vWf多聚物的剪切依赖性粘附力。观察到流体剪切作用对这些蛋白质的结构的明显差异，并且与粘附力差异相关。这些数据表明多聚化对vWf的粘附的贡献。首次对vWf层，胶原VI层的结构以及胶原VI表面上的单个vWf和vWf层的结构进行成像。;免疫金/ AFM技术确定吸附表面上分子内结合位点的可及性vWf已建立。这是将vWf（任何蛋白质）结构中的分子尺度变化与该结构中功能域的可及性相关联的直接方法。使用一组针对vWf上功能域的单克隆抗体，我们确定了吸附在疏水表面和胶原VI表面上的vWf内特定功能域的差异可及性。

著录项

  • 作者

    Raghavachari, Madhusudan.;

  • 作者单位

    Case Western Reserve University.;

  • 授予单位 Case Western Reserve University.;
  • 学科 Engineering Biomedical.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2000
  • 页码 218 p.
  • 总页数 218
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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