MOLECULAR MECHANISMS OF VWF ALTERATION IN VITRO/VIVO

血管性血友病（VWD）是最常见的遗传性出血性疾病，其特征在于： 血管性血友病因子（VWF）水平降低或功能缺陷。新的机制，最近 在VWD患者中发现的缺陷包括VWF的细胞内滞留， 和分泌，并增加血浆中VWF的清除率。调节的分子/结构要求 正常的VWF功能，清除和体内降解还不完全清楚，我们预测， 大规模的患者调查将揭示描述VWF结构域结构-功能的独特突变 关系。因此，项目2的目标是确定导致临床和实验室 在项目1、3和4中鉴定的VWD患者的表型和基因型。这一目标将通过 实现以下两个具体目标。目标1将检查候选1型和3型VWD的影响 突变对VWF的合成、细胞内加工、分泌和清除的影响。细胞系表达 将检查改变形式的VWF的翻译后修饰和分泌的异常， VWF。我们将使用鼠模型检查选定的1型VWD变体从血浆中的清除。在 目的2，我们将确定功能性VWF变体的新机制。关于这一点仍有许多未知之处。 2A型VWD的潜在机制。我们将检查2A型变异，以确定相对丰度 以下机制：细胞内滞留和降解，缺陷的多聚化，或增加 通过血浆蛋白酶的蛋白水解。2A型和选定的1型VWF突变对VWF结合的影响 还将测定血小板、FVIII和胶原蛋白。在所有研究中，将体外数据集与 患者数据以确定基因型/表型相关性。由于启动子和剪接点突变， 在项目4中鉴定或在项目3中修饰基因，这些改变将在小鼠模型中探索， 进一步明确其发病机制。通过这些研究，我们希望找出独特的 表型/基因型，进一步定义VWF结构/功能关系。的 从这个项目中获得的知识将增加我们对VWD的理解，并将导致开发 更精确的诊断技术以及帮助开发更有效的治疗策略。

von Willebrand disease (VWD) is the most common inherited bleeding disorder and is characterized by decreased levels or defective function of von Willebrand factor (VWF). Novel mechanisms that have recently been identified in individuals with VWD include the intracellular retention of VWF, defective regulated storage and secretion, and increased VWF clearance from plasma. The molecular/structural requirements that regulate normal VWF function, clearance, and degradation in vivo are not completely understood, and we predict that a large-scale survey of patients will reveal unique mutations that delineate VWF domain structure-function relationships. Therefore, the goal of Project 2 is to define the mechanisms causing the clinical and laboratory phenotypes and genotypes in VWD patients identified in Projects 1, 3 and 4. This goal will be accomplished by completing the following two specific aims. Aim 1 will examine the effect of candidate type 1 and type 3 VWD mutations on the synthesis, intracellular processing, secretion, and clearance of VWF. Cell lines expressing altered forms of VWF will be examined for abnormalities in post-translational modification and secretion of VWF. We will examine the clearance of selected type 1 VWD variants from plasma using a murine model. In Aim 2, we will determine novel mechanisms of functional VWF variants. Much remains unknown regarding the mechanisms underlying type 2A VWD. We will examine type 2A variants to determine the relative abundance of the following mechanisms: intracellular retention and degradation, defective multimerization, or increased proteolysis by plasma proteases. The effects of type 2 A and selected type 1 VWF mutations on binding of platelets, FVIII, and collagen will also be determined. In all studies, the in vitro data set will be compared to patient data to determine genotype/phenotype correlation. As promoter and splice junction mutations are identified in Project 4 or modifying genes in Project 3, these alterations will be explored in murine models the further define their pathogenetic mechanisms. Through these studies we expect to identify unique phenotypes/genotypes in individual VWD patients that further define VWF structure/function relationships. The knowledge gained from this project will increase our understanding of VWD, and will lead to the development of more precise diagnostic techniques as well as aid the development of more effective treatment strategies.