VON WILLEBRAND FACTOR MULTIMER ASSEMBLY AND STRUCTURE

描述(申请人提供)：von Willebrand因子(VWF)是一种多聚体血糖蛋白，是止血所必需的。在血管损伤部位，VWF结合结缔组织并介导血小板黏附。VWF的黏附功能需要最大的多聚体，而无法组装它们会导致与von Willebrand病相关的出血。相反，ADAMTS13，一种金属蛋白酶，在血栓形成部位裂解VWF多聚体，ADAMTS13缺乏会导致血栓性血小板减少性紫癜(TTP)。因此，VWF多聚体的组装和分解代谢之间的正常平衡具有重要的医学意义，而理解VWF的功能依赖于了解VWF多聚体的组装和结构。具体目标1将表征VWF多聚体组装的前肽依赖机制。多聚体的组装依赖于成熟VWF亚基的N-末端前肽(D1D2结构域)和邻近的D‘D3区，它们共同促进在高尔基体的酸性条件下形成二硫键。能够使VWF的N-末端形成亚单位间二硫键的结构特征将通过对转基因细胞的诱变和功能研究来鉴定。VWF二聚体D‘D3片段中的亚基间和亚基内二硫键将通过质谱学进行测定。具体目标2将描述VWF多聚体在webel-Palade体内填充和挤出的机制。D1D2和D‘D3结构域之间的非共价pH依赖和钙依赖的相互作用是可逆地将VWF多聚体凝聚成Webel-Palade小体内的管状阵列所必需的。这些相互作用将通过荧光和电子显微镜分析重组VWF在转基因细胞中的靶向、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从结构和生物化学上进行表征。呋喃对VWF前肽的裂解也受pH调节，前肽裂解在VWF储存和分泌中的作用将得到表征。具体目标3将确定VWF小管中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和二聚体D‘D3片段在体外可组装成类似于Webel-Palade小体的小管。电子显微镜图像的三维重建显示，VWF小管包含一个由一个D‘D3二聚体和两个前肽组成的重复单位，呈右旋螺旋结构，每转4.2个单位。结构域间接触的对称性和位置表明了一种机制，即沿着分泌途径降低的pH如何协调VWF多聚体的二硫键组装及其管状包装。将对由逐渐变大的VWF结构组装成的小管进行三维重建，直到完整的VWF亚单位，以建立完整的VWF多聚体的分子模型。第八因子可以与VWF一起储存在Webel-Palade小体中，这些研究也将证明第八因子如何影响VWF小管的组装以及它与它们结合的位置。项目简介：VWF是一种血液蛋白质，需要在损伤部位止血，这一功能依赖于从相对较小的、相同的亚基组装成非常大的VWF多聚体。拟议研究的目标是了解VWF多聚体是如何组装的，包装在正确的细胞室中，并分泌到血液中。这些知识应该转化为更好地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.