Project 2: Molecular Impact of Carbohydrates on VWF Biology

项目概要：项目2 1型VWD的特征在于VWF的部分定量缺乏。虽然它占70%的 所有VWD病例中，1型VWD发病机制的分子机制仍不清楚 明白然而，连锁研究表明，血浆VWF水平降低（特别是在患者中）， 轻度至中度降低，范围为30 - 50 IU/dL）与VWF基因无关， 12号染色体。动物和人类研究的累积数据表明， 在VWF上表达的复合聚糖决定簇在调节VWF的许多方面起关键作用， 生物学，包括合成，蛋白水解和清除。本项目的目标1将研究VWF N- 和O-聚糖的结构在一个大的队列中的全面表型VWD的患者已经不同 通过1）齐默尔曼项目、（2）加拿大1型VWD研究和（3）低VWF入组 爱尔兰队列研究。鉴于N-和O-碳水化合物结构的复杂异质性， 表达在人VWF上，这种独特的联合临床资源将首次提供足够的 能够阐明VWF聚糖在VWD发病机制中的作用。在目标2中，一个患者子集 将选择已确定聚糖异常的1型VWD和低VWF患者， 进一步分析。在该队列中，VWF的N-和O-聚糖结构将使用 最先进的质谱糖蛋白组学分析。最后，使用一些新颖的并行 该项目的目标3将集中在（1）调查异常VWF的潜在机制 1型VWD和低VWF患者中的糖基化，以及（2）确定VWF碳水化合物 修饰在介导VWD发病机制中起直接的因果作用（例如通过减少生物合成或 调节增强的清除率）。

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).