Project 2: Molecular Impact of Carbohydrates on VWF Biology

项目总结：项目2 1型VWD的特点是VWF部分数量不足。尽管它占到了70%的 所有的VWD病例，1型VWD发病的分子机制仍然很差 明白了。然而，连锁研究表明，血浆VWF水平降低(特别是在患者中 在30-50IU/dL范围内轻度到中度减少)与VWF基因无关 12号染色体。来自动物和人类研究的积累数据表明， VWF上表达的复杂的糖链决定簇在调节其多个方面起着关键作用 生物学，包括合成、蛋白分解和清除。该项目的目标1将调查VWF N如何- 在一大批综合表型VWD患者中，O-糖链结构发生了变化 通过1)齐默尔曼计划，(2)加拿大1型VWD研究和(3)低VWF注册 爱尔兰队列研究。鉴于N-和O-碳水化合物结构的复杂多相性质 在人类VWF上表达，这种独特的组合临床资源将首次提供足够的 能够阐明VWF多聚糖在VWD发病机制中的作用。在目标2中，患者的子集 发现糖链异常的1型VWD和低VWF患者将被选为 进一步分析。在这个队列中，VWF的N-和O-糖链结构将使用 最新的质谱糖蛋白质组学分析。最后，采用了一些新颖的并列方式 战略，该项目的目标3将集中于(1)调查异常VWF的机制 1型VWD和低VWF患者的糖基化，以及(2)确定VWF碳水化合物 修饰在介导VWD发病中起到直接的因果作用(例如，通过减少生物合成或 调制增强型间隙)。

PROJECT SUMMARY: PROJECT 2 Type 1 VWD is characterized by a partial quantitative deficiency of VWF. Although it accounts for 70% of all cases of VWD, the molecular mechanisms underlying the pathogenesis of type 1 VWD remain poorly understood. However, linkage studies have shown that reduced plasma VWF levels (particularly in patients with mild to moderate reductions in the range 30 - 50 IU/dL) are independent of the VWF gene on chromosome 12. Accumulating data from both animal and human studies have demonstrated that the complex glycan determinants expressed on VWF play critical roles in regulating many aspects of its biology, including synthesis, proteolysis and clearance. Aim 1 of this project will investigate how VWF N- and O-glycan structures vary in a large cohort of patients with comprehensively phenotyped VWD already enrolled through 1) the Zimmerman program, (2) the Canadian type 1 VWD study and (3) the Low VWF Ireland Cohort study. Given the complex heterogeneous nature of the N- and O- carbohydrate structures expressed on human VWF, this unique combined clinical resource will for the first time provide adequate power to enable elucidation of the role of VWF glycans in VWD pathogenesis. In Aim 2, a subset of patients with type 1 VWD and Low VWF in whom glycan abnormalities have been identified will be selected for further analysis. In this cohort, the N- and O-glycan structures of VWF will be definitively sequenced using state-of-the-art mass spectroscopy glycoproteomic analysis. Finally, using a number of novel parallel strategies, Aim 3 of the project will focus on (1) investigating the mechanisms underlying abnormal VWF glycosylation in patients with type 1 VWD and Low VWF, and (2) determining whether VWF carbohydrate modifications play a direct causal role in mediating VWD pathogenesis (e.g. by reducing biosynthesis or modulating enhanced clearance).