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，其特点是 血管性血友病因子(VWF)水平降低或功能缺陷。最近有了新的机制 在VWD患者中发现的VWF包括VWF细胞内滞留，调节存储缺陷 和分泌，并增加VWF从血浆中的清除。调控的分子/结构要求 体内正常的VWF功能、清除和降解尚不完全清楚，我们预测 对患者的大规模调查将揭示描绘VWF结构-功能的独特突变 两性关系。因此，项目2的目标是定义导致临床和实验室 项目1、3和4中确定的VWD患者的表型和基因类型。这一目标将通过 完成以下两个具体目标。目标1将检查候选类型1和类型3的VWD的影响 VWF的合成、细胞内加工、分泌和清除上的突变。细胞系表达 改变形式的VWF将被检查翻译后修饰和分泌的异常 VWF。我们将使用小鼠模型检查选定的1型VWD变异体从血浆中的清除情况。在……里面 目的2，我们将确定功能性VWF变体的新机制。很多事情仍然是未知的。 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、有缺陷的多聚体或增加 血浆蛋白水解酶的蛋白分解作用。A型和选定的1型VWF突变对血管紧张素转换酶结合的影响 还将测定血小板、FVIII和胶原蛋白。在所有研究中，体外数据集将与 患者数据，以确定基因型别/表型相关性。AS启动子和剪接接头突变是 在项目4中确定或在项目3中修改基因，这些变化将在小鼠模型中进行探索 进一步明确其致病机制。通过这些研究，我们希望确定独特的 进一步定义VWF结构/功能关系的个体VWD患者的表型/基因型别。这个 从这个项目中获得的知识将增加我们对VWD的了解，并将导致VWD的发展 提供更精确的诊断技术，并帮助制定更有效的治疗策略。

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.