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：(研究人员摘要)第八因子(FVIII)是一种重要的 止血所需的血浆成分，因为这是遗传缺陷 分子会导致一种称为血友病A的危及生命的凝血障碍。 这种遗传病是通过反复输注昂贵的FVIII来治疗的 产品。如果分子基础不同，则可以提供更有效的治疗方法 了解FVIII清除和一种新的重组FVIII蛋白 开发了长寿命的循环使用装置。我们之前已经发现， 低密度脂蛋白受体相关蛋白(LRP)是主要的内吞蛋白 肝受体和细胞表面硫酸肝素蛋白多糖(HSPGs) 合作清除FVIII，因为同时阻止了这两个 受体系统显著延长了FVIII在小鼠体内的寿命。当在 纯化系统LRP和HSPG均可与FVIII通过 位于A2结构域内的位置，负责的精确分子事件 对于FVIII的分解代谢目前还没有很好的描述。我们建议 确定FVIII与LRP和LRP相互作用的关键氨基酸残基 通过突变分析先前确定的LRP和HSPG区域 FVIII的HSPG结合位点。突变将被引入B结构域 耗尽的重组FVIII，其功能与血浆来源的相同 FVIII，目前用于血友病A治疗。我们将会表达这些 FVIII突变体在哺乳动物细胞中，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。突变体的分解代谢会 用表达LRP的细胞进行体外检测和在小鼠模型中进行体内检测 血友病A。这些实验将确定FVIII突变体具有降低的 与LRP和HSPG的结合，将阐明这两个受体的作用 系统处于fVIIII许可状态。拟议的研究应该发展一种洞察力 为探讨FVIII在循环中的调控机制提供依据 用于产生一种新型的重组FVIII产物，具有 在循环中使用寿命延长。开发这种FVIII衍生品，这是 可能是未来较便宜的血友病A疗法，是长期的 我们研究的目标。