Biosynthetic and Functional Consequences of von Willebrand Disease Mutations

描述（由申请人提供）：血管性血友病（VWD）是世界上最普遍的出血性疾病，影响约1%的人口。它被分为三大类，类型1、2和3。在2型VWD中，大多数突变导致VWF中单个氨基酸的替换。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。2型VWD的问题将在本应用中有两个具体目标。在Specific Aim 1中，提出了多种方法来研究2B型功能获得突变对VWF A1-A2-A3区域二硫键结构的影响。这些研究的假设是，突变改变了Cys残基的数量或增加了Cys1272-Cys1458二硫键区域的负电荷，改变了该区域的二硫键结构。2B VWF突变体的二硫化物键结构将通过质谱（MS）进行检测，并与功能改变相关。在Specific Aim 2中，将对大量具有良好特征的2型VWD亲属患者的血浆进行VWF质量和数量的数学指标评估，该指标考虑了多聚体中突变单体的百分比、不同大小的多聚体的相对含量以及VWF数量。该指标将与VWF功能参数和出血表型相关，并通过有效的出血评分问卷进行量化。这些研究将辅以一项对6例2B型VWD（突变R1308C）患者的纵向研究，将该指数与同时确定的VWF功能参数进行比较。预计这些研究将描绘出VWF质量指数的个体内部和个体之间的变化。总的来说，拟议的研究将揭示2型VWD的复杂病理生理学，并有可能提供准确和定量的VWD患者出血风险评估。

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.