Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII.
Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII.
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犬因子 VIII 中关键凝血活性决定元件的鉴定。
DOI：
10.1016/j.omtm.2019.12.019
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Xiao,Weidong
中科院分区：
文献类型：
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作者：
Firrman,Jenni;Wang,Qizhao;Wu,Wenman;Dong,Biao;Cao,Wenjing;Moore,AndreaRossi;Roberts,Sean;Konkle,BarbaraA;Miao,Carol;Liu,LinShu;Li,Dong;Xiao,Weidong
众所周知，犬凝血因子VIII(CFVIII)比人凝血因子VIII(HFVIII)具有更高的比活性，并且已经证明cFVIII轻链能够增强hFVIII的活性。本研究的目的是首先确定cFVIII轻链上的哪些氨基酸负责增强hFVIII的活性，然后利用这些氨基酸开发具有增强功能活性的hFVIII变异体。我们通过测试一系列人-狗轻链杂交来系统地筛选cFVIII轻链片段，发现狗的氨基酸1857-2147是这种增强的关键。使用负选择方法对这一跨度内的每一种犬氨基酸进行单独筛选，从而鉴定出FVIII轻链中可以增强活性的12个氨基酸(JF12)。将相应的12个氨基酸替换到hFVIII(HFVIIIJF12BDD)中，可提高体外的比活性。此外，在体内表达的hFVIIIJF12BDD与野生型相比显示出更强的凝血活性，同时保持了正常的分泌效率。总之，我们确定了cFVIII中的氨基酸是具有更高比活性的关键决定因素，并可能为未来血友病A的治疗开发奠定基础。
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 profilein vitro. Furthermore, hFVIIIJF12BDD expressed anin 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.