CATABOLISM OF COAGULATION FACTOR VIII

凝血因子 VIII 的分解代谢

• 批准号: 6629143
• 负责人: EVGUENI L. SAENKO
• 金额: $ 30.84万
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629143
• 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：（研究者的摘要）因子VIII（FVIII）是重要的 止血所需的血浆成分，因为这是遗传缺陷 分子引起威胁生命的凝血障碍，称为血友病。 这种遗传疾病通过昂贵的FVIII的反复输注来治疗 产品。如果分子基础 FVIII清除被理解为一种新型的重组FVIII蛋白， 开发了循环延长的寿命。我们以前发现 低密度脂蛋白受体相关蛋白（LRP），主要内吞蛋白质（LRP） 肝受体和细胞表面硫酸盐蛋白聚糖（HSPGS） 在清除FVIII的情况下合作，因为这两者同时阻止 受体系统急剧延长了小鼠FVIII的寿命。同时在 纯化系统LRP和HSPG均显示通过通过该系统与FVIII相互作用 位于A2域内的位点，确切的分子事件负责 对于FVIII目前，分解代谢目前尚未得到很好的特征。我们建议 确定对FVIII与LRP相互作用至关重要的氨基酸残基 通过对先前确定为LRP和 HSPG的FVIII结合位点。突变将引入B域 耗尽的重组FVIII，在功能上与等离子衍生相同 FVIII，目前用于血友病A疗法。我们将表达这些 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， 用作HSPG的模型，在纯化系统中。突变体的分解代谢将 使用表达LRP的细胞和体内在体外检查鼠模型 血友病A。这些实验将鉴定出降低的FVIII突变体 与LRP和HSPG结合，将阐明这两个受体的作用 FVIIII清除系统。拟议的研究应开发出洞察力 进入流通中FVIII调节的机制，并将提供基础 用于生成一种新型的重组FVIII产品，具有 循环长期寿命。开发这种FVIII衍生物，这是 可能是预期较便宜的血友病治疗，是长期的 我们学习的目标。