Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII.,Molecular Therapy - Methods & Clinical Development

当前位置： X-MOL 学术 › Mol. Ther. Methods Clin. Dev. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII.
Molecular Therapy - Methods & Clinical Development ( IF 4.6 ) Pub Date : 2020-01-15 , DOI: 10.1016/j.omtm.2019.12.019
Jenni Firrman _{1,

2} , Qizhao Wang ₃ , Wenman Wu ₃ , Biao Dong ₃ , Wenjing Cao ₃ , Andrea Rossi Moore ₃ , Sean Roberts ₃ , Barbara A Konkle _{4,

5} , Carol Miao ₅ , LinShu Liu ₂ , Dong Li ₆ , Weidong Xiao _{1,

2,

3}

Affiliation

It is well known that canine factor VIII (cFVIII) has a higher specific activity than does human FVIII (hFVIII), and it has been previously demonstrated that cFVIII light chain is able to enhance hFVIII activity. The goal of this study was to first determine which amino acids in cFVIII light chain were responsible for enhancing hFVIII activity, and second to use these amino acids to develop a hFVIII variant with enhanced functional activity. We systemically screened segments of cFVIII light chain by testing an array of human-canine light chain hybrids and found that canine amino acids 1857-2147 were key to this enhancement. Each canine amino acid within this span was screened individually using a negative selection method, which led to the identification of 12 aa (JF12) in the FVIII light chain that could enhance activity. Substitution of the corresponding 12 aa into hFVIII (hFVIIIJF12BDD) elevated the specific activity profile in vitro. Furthermore, hFVIIIJF12BDD expressed an in vivo-displayed increased coagulation activity compared to wild-type, while maintaining normal secretion efficiency. In conclusion, we identified the amino acids in cFVIII that are the key determinants for higher specific activity and may be the basis for future development of therapeutic treatments for hemophilia A.

中文翻译：

犬因子 VIII 中关键凝血活性决定元素的鉴定。

众所周知，犬因子 VIII (cFVIII) 具有比人 FVIII (hFVIII) 更高的比活性，并且先前已经证明 cFVIII 轻链能够增强 hFVIII 活性。本研究的目标是首先确定 cFVIII 轻链中的哪些氨基酸负责增强 hFVIII 活性，其次使用这些氨基酸开发具有增强功能活性的 hFVIII 变体。我们通过测试一系列人-犬轻链杂交体系统地筛选了 cFVIII 轻链片段，发现犬氨基酸 1857-2147 是这种增强的关键。使用负选择方法单独筛选此跨度内的每个犬氨基酸，从而鉴定出 FVIII 轻链中可增强活性的 12 aa (JF12)。将相应的 12 个氨基酸替换为 hFVIII (hFVIIIJF12BDD) 提高了体外比活性谱。此外，与野生型相比，hFVIIIJF12BDD 表达的体内凝血活性增加，同时保持正常的分泌效率。总之，我们确定了 cFVIII 中的氨基酸，它们是更高比活性的关键决定因素，并且可能是未来 A 型血友病治疗方法开发的基础。

更新日期：2020-01-15

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文