VON WILLEBRAND FACTOR

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的长期目标是了解血管性血友病因子（VWF）组装成二硫化物- 连接的多聚体，管状包装成分泌囊泡，并分泌到血液中。 VWF是一种巨大的多聚体糖蛋白，介导血小板粘附在 血管损伤，因此在止血中起关键作用。VWF功能取决于 组装成多聚体，可以超过20百万Da，这些大的缺陷， 多聚体引起称为血管性血友病（VWD）的出血性疾病。相反， 血液中“超大”VWF多聚体的存在与血栓形成相关 血小板减少性紫癜，一种通常致命的疾病， 微血管血栓形成因此，理解VWF多聚体组装具有实质性意义。 医学重要性 在以前的研究中，我们发现重组N-末端VWF D 'D3二硫键连接， 同源二聚体和前肽（结构域D1 D2）形成Ca 2+依赖性非共价280 kDa复合物，并进一步组装成 巨大的中空小管，螺旋圈可达30圈，长度为几百纳米， pH值与高尔基体相似。快速冷冻深蚀刻（QFDE）EM和三维 负染色图像的重建（由Egelman实验室的Ying Wang）显示， 小管由一个D3二聚体和两个前肽的重复单元组装而成 以每11nm圈4.2个单位的右旋螺旋排列。的尺寸 肾小管（外径25 nm，内径12 nm）与VWF相似 通过透射电子显微镜在内皮细胞薄片中观察到的WPB中的小管 显微镜 我们建议重建中间复合物和小管在体外由 逐渐增大的VWF片段，并增加3D的分辨率。 利用冷冻电镜进行重建。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our long-time goal is to understand von Willebrand factor (VWF) assembly into disulfide- linked multimers, tubular packing into secretory vesicles, and secretion into the blood. VWF is an enormous multimeric glycoprotein that mediates platelet adhesion at sites of vascular injury and thus plays a critical role in hemostasis. VWF function depends on assembly into multimers that can exceed 20 million Da, and defects in these large multimers cause a bleeding disorder called von Willebrand disease (VWD). Conversely, the presence of 'ultralarge' VWF multimers in the blood is associated with thrombotic thrombocytopenic purpura, an often fatal disorder characterized by extensive microvascular thrombosis. Thus understanding VWF multimer assembly has substantial medical importance n a previous study, we found that recombinant N-terminal VWF D'D3 disulfidelinked homodimer and the propeptide (domains D1D2) form a Ca2+-dependent noncovalent 280 kDa complex under conditions of neutral pH similar to the ER, and further assemble into massive hollow tubules with up to 30 helical turns and several hundred nm in length at pH values similar to the Golgi. Quick-freeze deep-etch (QFDE) EM and three-dimensional reconstruction of negatively stained images (by Ying Wang in Egelman's lab) showed that the tubules are assembled from a repeating unit of one D'D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per 11 nm turn. The dimensions of the tubule (outside diameter 25 nm, inside diameter 12 nm) are similar to those of VWF tubules in WPBs seen in thin section of endothelial cells by transmission electron microscopy. We propose to reconstruct intermediate complexes and tubules made in vitro from progressively larger fragments of VWF, and to increase the resolution of the 3D reconstructions by employing cryo EM.