Structure and Function of the VWF Helical Tubule Required for Hemostasis

止血所需的 VWF 螺旋管的结构和功能

• 批准号: 10705010
• 负责人: Jacob R Anderson
• 金额: $ 3.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705010
• 关键词: 3-Dimensional; Adopted; Architecture; Binding; Blood; Blood Coagulation Disorders; Blood Platelets; Blood Proteins; Blood Vessels; C-terminal; Cells; Coagulation Process; Cryo-electron tomography; Cryoelectron Microscopy; Cysteine; Data; Data Collection; Development; Dimerization; Disease; Disulfide Linkage; Disulfides; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Engineering; Environment; Factor VIII; Failure; Fellowship; Generations; Golgi Apparatus; Head; Hematological Disease; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; In Situ; In Vitro; Inherited; Injury; Length; Link; Maps; Mediating; Missense Mutation; Modeling; Molecular; Molecular Structure; Morbidity - disease rate; Mutation; Negative Staining; Peptide Hydrolases; Physicians; Play; Positioning Attribute; Process; Proteins; Research; Resolution; Role; Scientist; Series; Site; Structure; Tertiary Protein Structure; Testing; Therapeutic; Thinness; Thrombosis; Time; Training; Umbilical vein; Visualization; Weibel-Palade Bodies; Work; cleavage factor; clinically relevant; crosslink; dimer; disease-causing mutation; disorder subtype; disulfide bond; experience; insight; light scattering; model building; monomer; novel; pre-doctoral; reconstruction; tomography; von Willebrand Disease; von Willebrand Factor

PROJECT SUMMARY/ABSTRACT Von Willebrand Disease (VWD), the most common bleeding disorder worldwide, is caused by mutations in von Willebrand Factor (VWF), a large multidomain protein. In the blood, VWF circulates as a long multimer of head- to-head disulfide linked dimers of mature VWF. These long multimers are critical for VWF function as they give circulating VWF polyvalency for activating and binding platelets at sites of endothelial injury, forming a hemostatic plug to staunch bleeding. Additionally, long VWF multimers stabilize coagulation factor VIII (FVIII) in the blood. To form these long multimers, crucial for normal hemostasis, VWF forms helical tubules in the low pH of the late- Golgi and Weibel-Palade bodies (WPB). The helical tubule templates the disulfide bond formation needed to form long multimers by positioning D3 domains in close proximity. At the same time, VWF’s prodomain is cleaved, generating the mature VWF that binds FVIII in the blood. Aberrancy in these maturation steps due to VWF mutations causes several VWD subtypes. Despite the importance of the helical tubule for VWF multimerization, the high-resolution structure of the helical tubule is not known. This fellowship proposal aims to determine structures of VWF helical tubules at three stages of maturation, test Type 2A VWD mutations for causing short tubules, and interrogate the implications of prodomain cleavage for FVIII-VWF tubule association. In Aim 1, using a C-terminally truncated VWF construct, a high-resolution structure of the VWF tubule before and after head-to-head disulfide bonds form will be determined using cryo-electron microscopy (cryo-EM) and helical reconstruction. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, a three- dimensional reconstruction of the in situ VWF tubule will be determined to test if the close packing of VWF helical tubules inside the native WPB environment has consequences for the molecular structure of VWF in the tubule. Guided by this structural insight, a subset of VWD mutations will be tested for their effect on robust tubule formation and normal VWF multimer length. Aim 2 will determine the structural rearrangements in VWF upon prodomain cleavage and test if the cleaved tubule can bind FVIII. This research will elucidate the molecular mechanism of VWF head-to-head disulfide bond formation, necessary for VWF multimerization and normal hemostasis. Structural characterization of the VWF tubule will lead to a molecular understanding of VWD caused by inefficient multimerization. Identification of FVIII-VWF tubule binding will provide a novel context to understand their association and inform therapeutic efforts to modulate FVIII-VWF binding before secretion into the blood. A preliminary VWF tubule reconstruction indicates that additional data collection will allow atomic model building. This research will be carried out under the sponsorship of Dr. Timothy Springer, experienced in structural characterization of VWF, and Dr. Alan Brown, an expert in cryo-EM, creating a strong training environment for predoctoral physician-scientist training.

项目摘要/摘要 冯·威尔布兰德疾病（VWD）是全世界最常见的出血疾病，是由von突变引起的 Willebrand因子（VWF），一种大型多域蛋白。在血液中，vwf圆圈是长长的多聚体 成熟vwf的二硫化二二聚体。这些长的多聚体对于VWF功能至关重要，因为它们给出了 在内皮损伤部位激活和结合血小板的循环vWF多性价值，形成止血 插入浸水出血。另外，长VWF多聚体稳定血液中的凝结因子VIII（FVIII）。 为了形成这些长的多聚体，对于正常止血至关重要，VWF在晚期的低pH值中形成螺旋小管 Golgi和Weibel-Palade Bodies（WPB）。螺旋管模板的二硫键形成需要 通过将D3域定位在近距离位置来形成多组。同时，vwf的prodomain是 裂开，产生结合血液中FVIII的成熟VWF。由于这些成熟步骤的异常 VWF突变会导致几种VWD亚型。尽管螺旋管对VWF很重要 多层化，螺旋小管的高分辨率结构尚不清楚。该奖学金提案的目的是 在成熟的三个阶段确定VWF螺旋管的结构，测试2A型VWD突变 引起短管，并询问Prodomain裂解对FVIIII-VWF Tubele协会的含义。 在AIM 1中，使用C末端截断的VWF结构，这是VWF管的高分辨率结构 并在头到头二硫键后，将使用冷冻电子显微镜（Cryo-EM）确定 螺旋重建。使用冷冻电子层析成像（冷冻-ET）和亚图平均图，三 原位VWF管的尺寸重建将确定以测试VWF Helical的关闭堆积是否存在 天然WPB环境内部的小管对管中VWF的分子结构产生了影响。 在这种结构洞察力的指导下，将测试一部分VWD突变的对稳健管的影响 形成和正常的VWF多聚机长度。 AIM 2将确定VWF中的结构重排 Prodomain裂解并测试切割管是否可以结合FVIII。这项研究将阐明分子 VWF头对头二硫键形成的机制，VWF多层化和正常 止血。 VWF小管的结构表征将导致对VWD的分子理解 通过效率低下的多重化。 FVIIII-VWF小管结合的识别将提供一个新颖的背景来理解 他们的关联并告知治疗努力，以调节fviii-vwf在分泌为血液之前结合。一个 初步的VWF Tubele重建表明，其他数据收集将允许原子模型构建。 这项研究将在蒂莫西·斯普林格博士的赞助下进行，在结构上经验丰富 VWF的特征和Cryo-EM专家Alan Brown博士为为 专业的身体科学家培训。