Biosynthetic and Functional Consequences of von Willebrand Disease Mutations

描述(申请人提供)：von Willebrand病(VWD)是世界上最常见的出血性疾病，影响大约1%的人类人口。在2型VWD中，大多数突变导致VWF中单一氨基酸的替换。由于VWF合成和结构的复杂性，这些突变的功能后果很难预测。在本申请中，将以两个具体目标来解决类型2 VWD的问题。在具体目标1中，提出了多种方法来检测2B型功能增益突变对VWF A1-A2-A3区域二硫键结构的影响。这些研究解决了这样的假设，即改变Cys1272-Cys1458二硫键区域的Cys残基数量或增加负电荷的突变会改变该区域的二硫键结构。2BVWF突变体的二硫键结构将通过质谱学(MS)进行检测，并与功能变化相关联。在具体目标2中，来自2型VWD家系的大量特征良好的患者的血浆将使用VWF质量和数量的数学指标进行评估，该指标考虑了多聚体中包含的突变单体的百分比、不同大小的多聚体的相对含量以及VWF的数量。这一指数将与VWF功能参数和出血表型相关，通过有效的出血评分问卷进行量化。这些研究将与对6名2B型VWD患者(突变R1308C)的纵向研究相补充，以将该指数与同时测定的VWF功能参数进行比较。预计这些研究将描绘出VWF质量指数的个体内和个体间的差异。总之，拟议的研究将阐明2型VWD的复杂病理生理学，并有可能为VWD患者的出血风险提供准确和定量的评估。

DESCRIPTION (provided by applicant): Von Willebrand disease (VWD) is the most prevalent bleeding disorder in the world, affecting approximately 1% of the human population. It is divided into three general categories, types 1, 2 and 3. In type 2 VWD, most mutations result in replacement of a single amino acid in VWF. The functional consequences of these mutations are difficult to predict because of the complex nature of VWF synthesis and structure. The problem of type 2 VWD will be approached in this application with two specific aims. In Specific Aim 1, a variety of approaches are proposed to examine the effects of type 2B gain-of-function mutations on the disulfide bond structure of the VWF A1-A2-A3 region. These studies address the hypothesis that mutations that change the number of Cys residues or increase the negative charge in the region of the Cys1272-Cys1458 disulfide bond alter the disulfide-bond structure of the region. The disulfide-bond structure of 2B VWF mutants will be examined by mass spectrometry (MS) and correlated with functional alterations. In Specific Aim 2, plasma from a large number of well-characterized patients from a type 2 VWD kindred will be evaluated with a mathematical index of VWF quality and quantity that takes into account the percentage of mutant monomers incorporated into multimers, the relative content of multimers of different sizes, and the VWF quantity. This index will be correlated with VWF functional parameters and with the bleeding phenotype as quantified with a validated bleeding score questionnaire. These studies will be complemented with a longitudinal study of six patients with type 2B VWD (mutation R1308C) to compare the index with contemporaneously determined parameters of VWF function. It is expected that these studies will delineate both intra- and inter-individual variation in the VWF quality index. In aggregate, the proposed studies will shed light on the complex pathophysiology of type 2 VWD and have the potential to provide an accurate and quantitative assessment of bleeding risk in VWD patients.