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: 10380955
• 负责人: Jacob R Anderson
• 金额: $ 3.87万
• 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/10380955
• 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; Disease; Disulfide Linkage; Disulfides; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Engineering; Environment; F8 gene; 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; Polyvalence; Positioning Attribute; Process; Proteins; Research; Resolution; Role; Scientist; Series; Site; Structure; Testing; Therapeutic; Thinness; Thrombosis; Time; Training; Umbilical vein; Visualization; Weibel-Palade Bodies; Work; 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 血管性血友病(VWD)是世界上最常见的出血性疾病，由von基因突变引起 Willebrand因子(VWF)是一种大的多域蛋白。在血液中，VWF作为头部的长多聚体进行循环- 成熟VWF的二硫键连接二聚体。这些长倍增器对于VWF功能至关重要，因为它们给出了 循环VWF多价激活和结合内皮损伤部位的血小板，形成止血 用塞子止血。此外，长的VWF多聚体可以稳定血液中的凝血因子VIII(FVIII)。 为了形成这些对正常止血至关重要的长多聚体，VWF在晚期的低pH值下形成螺旋小管。 高尔基体和韦贝尔-帕莱德体(WPB)。螺旋小管模版二硫键的形成需要 通过将D3结构域放置在非常接近的位置来形成长多聚体。同时，VWF的前域是 被切割，产生成熟的VWF，在血液中结合FVIII。这些成熟步骤中的异常是由于 VWF突变导致几种VWD亚型。尽管螺旋小管对VWF很重要 多聚体，螺旋小管的高分辨率结构尚不清楚。这项奖学金提案旨在 确定VWF螺旋小管在三个成熟阶段的结构，测试2A型VWD突变 导致短小管，并询问前结构域切割对FVIII-VWF小管结合的影响。 在目标1中，使用C-末端截断的VWF结构，之前VWF小管的高分辨率结构 在头对头的二硫键形成后，将使用冷冻电子显微镜(Cryo-EM)和 螺旋重建。使用冷冻电子断层扫描(CRYO-ET)和亚断层图像平均，三个- 将确定原位VWF小管的三维重建以测试VWF的紧密堆积是否为螺旋 天然WPB环境中的小管对小管中VWF的分子结构有影响。 在这种结构洞察的指导下，VWD突变的子集将被测试它们对健壮的小管的影响 形成和正常的VWF多聚体长度。目标2将确定VWF的结构重组 前结构域的切割，并检测被切割的小管是否能结合FVIII。这项研究将阐明分子 VWF多聚体和正常所必需的头对头二硫键形成机制 止血。VWF小管的结构特征将有助于从分子水平理解VWD引起的 通过低效的多聚体。FVIII-VWF小管结合的鉴定将为理解提供一个新的背景 在分泌到血液之前，它们的相互作用和调节FVIII-VWF结合的治疗作用。一个 初步的VWF小管重建表明，额外的数据收集将允许原子模型的建立。 这项研究将在蒂莫西·斯普林格博士的赞助下进行，他在结构方面经验丰富 VWF的特征，以及冷冻-EM专家艾伦·布朗博士为 博士后内科医生-科学家培训。