VON WILLEBRAND FACTOR

血管性血友病因子

• 批准号: 8361125
• 负责人: J Evan Sadler
• 金额: $ 1.23万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361125
• 关键词: Adhesions; Blood; Blood Platelets; Blood Vessels; Caliber; Complement Factor D; Complex; Defect; Dimensions; Disease; Disulfides; Electron Microscopy; Endothelial Cells; Freezing; Funding; Glycoproteins; Goals; Golgi Apparatus; Grant; Hand; Hemorrhage; Hemostatic function; Image; In Vitro; Injury; Length; Link; Mediating; Medical; Microtomy; N-terminal; National Center for Research Resources; Play; Principal Investigator; Recombinants; Research; Research Infrastructure; Resolution; Resources; Role; Secretory Vesicles; Site; Source; Staining method; Stains; Thrombosis; Thrombotic Thrombocytopenic Purpura; Time; Transmission Electron Microscopy; Tubular formation; United States National Institutes of Health; cost; dimer; macromolecule; reconstruction; von Willebrand Disease; von Willebrand Factor

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们的长期目标是了解von Willebrand因素（VWF）组装成二硫键 - 连接的多聚体，管状包装成分泌的囊泡，并分泌到血液中。 VWF是一种巨大的多聚糖蛋白，可介导在 血管损伤，因此在止血中起关键作用。 VWF功能取决于 组装成超过2000万个DA的多聚体，并且在这些大的巨大中存在缺陷 多聚体引起一种称为von Willebrand疾病（VWD）的出血疾病。相反， 血液中的“超级兵” VWF多聚体与血栓形成有关 血小板减少性紫红色，通常是致命的疾病 微血管血栓形成。因此，了解VWF多聚机组装具有实质性 医学重要性 n先前的研究，我们发现重组N末端VWF D'D3二硫化 同型二聚体和丙肽（域D1D2）形成Ca2+依赖性非共价280 在与ER相似的中性pH条件下KDA复合物，并进一步组装 巨大的空心小管，最多30个螺旋旋转和几百nm的长度 pH值类似于高尔基。快速冻结深蚀刻（QFDE）和三维 重建否定染色的图像（通过Egelman实验室中的Ying Wang）表明 小管从一个D'D3二聚体的重复单元和两个丙肽组装 在右撇子螺旋中排列，每11 nm转弯为4.2单位。尺寸 小管（外径25 nm，直径12 nm）与VWF相似 WPB中的小管中的小管通过透射电子在内皮细胞的薄部分中看到 显微镜。 我们提议重建在体外从事的中间复合物和小管 VWF的较大片段，并增加3D的分辨率 通过使用冷冻EM进行重建。