MOLECULAR MECHANISMS OF VWF ALTERATION IN VITRO/VIVO

体外/体内 VWF 改变的分子机制

• 批准号: 7885355
• 负责人: SANDRA HABERICHTER
• 金额: $ 18.25万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2012-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885355
• 关键词: Adhesions; Adhesives; Affect; Affinity; Alleles; Binding; Biological Assay; Biology; Blood Platelets; Blood Vessels; C-terminal; Canis familiaris; Carbohydrates; Carrier Proteins; Cell Line; Cells; Classification; Clinical; Coagulation Process; Code; Collagen; Confocal Microscopy; DDAVP; Data; Data Set; Defect; Development; Diagnostic Procedure; Disease; Dominant-Negative Mutation; Electrophoresis; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Factor VIII; Gene-Modified; Genes; Genotype; Glycoproteins; Goals; Golgi Apparatus; Grant; Half-Life; Hemorrhage; Hemostatic function; Hour; In Vitro; Individual; Inherited; Injury; Knowledge; Laboratories; Lead; Mediating; Modeling; Modification; Molecular; Molecular Weight; Mus; Mutation; N-terminal; Pathway interactions; Patients; Penetrance; Peptide Hydrolases; Peptide Signal Sequences; Phenotype; Plasma; Plasmids; Post-Translational Protein Processing; Predisposition; Process; Proteins; Proteolysis; Recombinants; Relative (related person); Reporting; Ristocetin; Role; Route; Secretory Vesicles; Sepharose; Site; Splice-Site Mutation; Structure-Activity Relationship; Surveys; Testing; Time; Tissues; Variant; botrocetin; dimer; disulfide bond; effective therapy; in vivo; intravenous injection; novel; platelet typing; promoter; protein folding; secretion process; sulfation; trafficking; treatment strategy; von Willebrand Disease; von Willebrand Factor

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.

冯·维勒布兰德病 (VWD) 是最常见的遗传性出血性疾病，其特征是 血管性血友病因子 (VWF) 水平降低或功能缺陷。最近出现的新机制 在患有 VWD 的个体中已发现的缺陷包括 VWF 的细胞内滞留、调节储存缺陷 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、清除和体内降解尚不完全清楚，我们预测 对患者的大规模调查将揭示描述 VWF 结构域结构功能的独特突变 关系。因此，项目2的目标是明确引起临床和实验室异常的机制。 项目 1、3 和 4 中确定的 VWD 患者的表型和基因型。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 1 型和 3 型 VWD 的效果 VWF 合成、细胞内加工、分泌和清除的突变。表达细胞系 改变形式的 VWF 将被检查翻译后修饰和分泌的异常情况 VWF。我们将使用小鼠模型检查血浆中选定的 1 型 VWD 变体的清除情况。在 目标 2，我们将确定功能性 VWF 变异的新机制。关于该问题还有很多未知之处 2A 型 VWD 的潜在机制。我们将检查 2A 型变体以确定相对丰度 以下机制：细胞内滞留和降解、多聚化缺陷或增加 通过血浆蛋白酶进行蛋白水解。 2 A 型和选定的 1 型 VWF 突变对结合的影响 血小板、FVIII 和胶原蛋白也将被测定。在所有研究中，体外数据集将与 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是 项目 4 中确定的基因或修改项目 3 中的基因，这些改变将在小鼠模型中进行探索 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 个体 VWD 患者的表型/基因型进一步定义了 VWF 结构/功能关系。这 从这个项目中获得的知识将增加我们对 VWD 的理解，并将导致发展 更精确的诊断技术以及帮助制定更有效的治疗策略。