CATABOLISM OF COAGULATION FACTOR VIII

凝血因子 VIII 的分解代谢

• 批准号: 6702276
• 负责人: EVGUENI L. SAENKO
• 金额: $ 0.48万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702276
• 关键词: aminoacid; binding sites; bioengineering /biomedical engineering; biotransformation; blood disorder; blood disorder chemotherapy; clearance rate; coagulation factor VIII; disease /disorder model; drug metabolism; gene expression; genetic manipulation; hemophilia As; heparin; intermolecular interaction; laboratory mouse; low density lipoprotein receptor; mutant; protein protein interaction; protein structure function; proteoglycan; receptor binding; recombinant proteins; tissue /cell culture; von Willebrand factor

DESCRIPTION: (Investigator's abstract) Factor VIII (fVIII) is an important plasma component required for haemostasis, since genetic defects in this molecule cause a life-threatening coagulation disorder known as Hemophilia A. This genetic disease is treated by repeated infusions of expensive fVIII products. A more effective therapy can be provided if the molecular basis of fVIII clearance is understood and a novel recombinant fVIII protein with a prolonged lifetime in circulation is developed. We have previously found that the low density lipoprotein receptor-related protein (LRP), the main endocytic liver receptor, and cell surface heparan sulfate proteoglycans (HSPGs) cooperate in the clearance of fVIII, since simultaneous blocking of these two receptor systems dramatically prolonged the lifetime of fVIII in mice. While in purified system both LRP and HSPGs were shown to interact with fVIII via the sites located within the A2 domain, the precise molecular events responsible for fVIII catabolism are currently not well characterized. We propose to identify the amino acid residues critical for fVIII interaction with LRP and HSPGs by mutational analysis of the regions previously identified as LRP and HSPGs binding sites of fVIII. The mutations will be introduced into B-domain depleted recombinant fVIII, which is functionally identical to plasma-derived fVIII and is presently used for Hemophilia A therapy. We will express these fVIII mutants in mammalian cells and test them for binding to LRP and heparin, used as model of HSPGs, in purified systems. The catabolism of the mutants will be examined in vitro using LRP-expressing cells and in vivo in a murine model of Hemophilia A. These experiments will identify fVIII mutants with reduced binding to LRP and HSPGs and will clarify the role of these two receptor systems in fVIIII clearance. The proposed studies should develop an insight into the mechanism of fVIII regulation in circulation and will provide a basis for generation of a novel type of recombinant fVIII products, having a prolonged lifetime in circulation. Development of such fVIII derivatives, which may be prospective for less expensive Hemophilia A therapy, is the long-term goal of our studies.

描述：（研究者摘要）因子VIII（fVIII）是一种重要的 止血所需的血浆成分，因为这种基因缺陷， 这种分子会导致一种危及生命的凝血障碍，即血友病A。 这种遗传性疾病的治疗方法是反复注射昂贵的凝血因子VIII 产品.如果能够从分子水平上研究肿瘤的分子基础， 了解了fVIII清除，并发现了一种新的具有 延长了循环寿命。我们先前发现， 低密度脂蛋白受体相关蛋白（LRP），主要的内吞 肝受体和细胞表面硫酸乙酰肝素蛋白聚糖（HSPGs） 合作清除fVIII，因为同时阻断这两个 受体系统显著延长了小鼠中FVIII的寿命。而在 纯化的系统LRP和HSPG都显示出通过 位于A2结构域内的位点， 对于FVIII catastrophic目前没有很好地表征。我们建议 鉴定对FVIII与LRP相互作用至关重要的氨基酸残基， 通过对先前鉴定为LRP的区域的突变分析， fVIII的HSPG结合位点。突变将被引入B结构域 耗尽的重组fVIII，其功能上与血浆来源的 FVIII，目前用于血友病A治疗。我们将表达这些 在哺乳动物细胞中的FVIII突变体，并测试它们与LRP和肝素的结合， 在纯化系统中用作HSPG的模型。变种人的大屠杀 使用LRP表达细胞在体外和在鼠模型中在体内进行检查 血友病A这些实验将鉴定具有降低的 结合LRP和HSPGs，并将阐明这两种受体的作用 fVIII-R系统。拟议中的研究应有助于深入了解 进入流通中的FVIII调节机制，并将提供基础 用于产生新型重组FVIII产物，其具有 延长循环寿命。开发这种FVIII衍生物， 可能是前瞻性的便宜血友病A治疗，是长期的 我们学习的目标。