VON WILLEBRAND FACTOR MULTIMER ASSEMBLY AND STRUCTURE

描述（由申请方提供）：血管性血友病因子（VWF）是一种止血所需的多聚体血液糖蛋白。在血管损伤部位，VWF结合结缔组织并介导血小板粘附。VWF的粘附功能需要最大的多聚体，并且不能组装它们导致与血管性血友病相关的出血。相反，金属蛋白酶ADAMTS13在血栓形成部位切割VWF多聚体，ADAMTS13缺乏导致血栓性血小板减少性紫癜（TTP）。因此，VWF多聚体的组装和催化之间的正常平衡具有实质性的医学重要性，并且理解VWF的功能取决于理解VWF多聚体的组装和结构。特异性目的1将表征VWF多聚体组装的前肽依赖性机制。多聚体组装依赖于成熟VWF亚基的N-末端前肽（D1D2结构域）和相邻的D'D3区域，它们在高尔基体的酸性条件下一起促进二硫键形成。使VWF的N-末端形成亚基间二硫键的结构特征将通过转染细胞中的诱变和功能研究来鉴定。将通过质谱法测定VWF的二聚体D'D3区段内的亚基间和亚基内二硫键。具体目标2将表征VWF多聚体包装在韦伯-帕拉德体内和从韦伯-帕拉德体内挤出的机制。D1 D2和D 'D3结构域之间需要非共价pH依赖性和Ca 2+依赖性相互作用，以将VWF多聚体可逆地浓缩成韦伯-帕拉德体内的管状阵列。这些相互作用将通过荧光和电子显微镜分析转染细胞中重组VWF的靶向、储存和分泌来表征。VWF前肽和D'D3结构域之间的同型和异型接触将在结构上和生物化学上表征。弗林蛋白酶对VWF前肽的切割也受pH调节，并且前肽切割在VWF储存和分泌中的作用将被表征。具体目标3将确定VWF小管内VWF结构域的三维结构和排列。在低pH和高Ca~（2+）条件下，重组D1D2和二聚D’D3片段在体外组装成类似于韦伯-帕拉德小体中的小管。来自电子显微镜图像的三维重建显示，VWF小管含有一个D'D3二聚体和两个前肽的重复单元，呈右旋螺旋，每圈4.2个单元。结构域间接触的对称性和位置表明了一种机制，即如何降低pH值沿着分泌途径协调VWF多聚体的二硫键连接组装及其管状包装。三维重建将产生的小管组装从逐渐变大的VWF结构，直到完整的VWF亚基，建立完整的VWF多聚体的分子模型。因子VIII可以与VWF一起储存在韦伯-帕拉德体内，这些研究还将证明因子VIII如何影响VWF小管的组装以及与它们结合的位置。项目叙述：VWF是血液中的一种蛋白质，在受伤部位止血是必需的，这种功能依赖于从相对较小的相同亚基组装成非常大的VWF多聚体。拟议研究的目标是了解VWF多聚体如何组装，包装在正确的细胞隔室中，并分泌到血液中。这些知识应该可以转化为更好的治疗出血和血栓形成的疾病。

DESCRIPTION (provided by applicant): Von Willebrand factor (VWF) is a multimeric blood glycoprotein that is required for hemostasis. At sites of vascular injury, VWF binds connective tissue and mediates platelet adhesion. The adhesive function of VWF requires the largest multimers, and inability to assemble them causes bleeding associated with von Willebrand disease. Conversely, ADAMTS13, a metalloprotease, cleaves VWF multimers at sites of thrombosis, and ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP). Thus, the normal balance between the assembly and catabolism of VWF multimers has substantial medical importance, and understanding the function of VWF depends on understanding the assembly and structure of VWF multimers. Specific Aim 1 will characterize the propeptide-dependent mechanism of VWF multimer assembly. Multimer assembly depends on the N-terminal propeptide (D1D2 domains) and adjacent D'D3 region of the mature VWF subunit, which together promote disulfide bond formation under the acidic conditions of the Golgi. The structural features that enable the N-terminus of VWF to form intersubunit disulfide bonds will be identified by mutagenesis and functional studies in transfected cells. Intersubunit and intrasubunit disulfide bonds within the dimeric D'D3 segment of VWF will be determined by mass spectrometry. Specific Aim 2 will characterize the mechanism of VWF multimer packing within and extrusion from Weibel-Palade bodies. Noncovalent pH-dependent and Ca2+- dependent interactions between D1D2 and D'D3 domains are required to reversibly condense VWF multimers into tubular arrays within Weibel-Palade bodies. These interactions will be characterized by analyzing the targeting, storage and secretion of recombinant VWF in transfected cells by fluorescence and electron microscopy. Homotypic and heterotypic contacts between VWF propeptide and D'D3 domains will be characterized structurally and biochemically. The cleavage of the VWF propeptide by furin also is regulated by pH, and the role of propeptide cleavage in VWF storage and secretion will be characterized. Specific Aim 3 will determine the three-dimensional structure and arrangement of VWF domains within VWF tubules. Under conditions of low pH and high Ca2+, recombinant D1D2 and dimeric D'D3 fragments assemble in vitro into tubules like those in Weibel-Palade bodies. Three-dimensional reconstructions from electron microscopy images show that VWF tubules contain a repeating unit of one D'D3 dimer and two propeptides, in a right- handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggests a mechanism for how decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Three-dimensional reconstructions will be generated for tubules assembled from progressively larger VWF constructs, up to the complete VWF subunit, to build a molecular model for intact VWF multimers. Factor VIII can be stored with VWF in Weibel-Palade bodies, and these studies will also demonstrate how factor VIII affects the assembly of VWF tubules and where it binds to them. Project Narrative: VWF is blood protein that is required to stop bleeding at sites of injury, and this function depends on the assembly of very large VWF multimers from relatively small, identical subunits. The goal of the proposed studies is to understand how VWF multimers are assembled, packaged in the correct cell compartments, and secreted into the blood. This knowledge should be translatable into better treatment for disorders of bleeding and thrombosis.