凝血因子ⅧTrp1707Ser突变患者相关抑制物的结合作用机制研究  

The binding mechanisms of FⅧ Trp1707Ser mutation-associated inhibitor

作　　者：吴希[1] 陆晔玲[2] 丁秋兰[2] 戴菁[2] 奚晓东[1] 王鸿利[1] 王学锋[2] 

机构地区：[1]上海交通大学医学院附属瑞金医院、上海血液学研究所医学基因组学国家重点实验室,200025 [2]上海交通大学医学院附属瑞金医院、上海血液学研究所检验科,200025

出　　处：《中华血液学杂志》2013年第8期691-695,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（81170480）;国家自然科学基金青年科学基金（81000206）

摘　　要：目的对1例凝血因子Ⅷ（FⅧ）Trpl707Ser突变患者相关抑制物的结合作用机制进行探讨。方法对患者进行APTT、PT、纤维蛋白原和FⅧ活性（FⅧ：c）等血友病A相关检测；Bethesda法进行FⅧ抑制物检测；采用长链PcR（LD．PCR）和两组序列特异的PCR分别进行FⅧ基因内含子22倒位和内含子1倒位检测；采用核酸直接测序法对患者FⅧ基因各外显子及其侧翼序列进行检测；分别将含抑制物血浆与FⅧ重链及其片段（AI、A2）、轻链及其片段（A3、C1和c2）进行反应，加入等体积正常人混合血浆纠正后测定剩余FⅧ：C。以FⅧ片段作为抗原，生物素标记的经纯化的抑制物作为抗体与之反应，Westemblot法进一步验证抑制物与FⅧ各片段的结合反应。结果患者FⅧ：C为1．1％，临床诊断为血友病A，Bethesda法检测其体内FⅧ抑制物滴度达18．4BU，基因分析发现患者FⅧ基因14号外显子存在错义突变c．97223C〉G（p．Trp1707Ser）。纠正试验法检测显示含抑制物血浆分别与重链和轻链反应后，剩余FⅧ：C均有升高，且与重链反应时升高更明显；与重链的A2和轻链的c2片段反应后，也出现了剩余FⅧ：C升高。以还原变性的B区缺失重组FⅧ、A2和c2作为抗原，抑制物作为抗体时Western blot均能检测到相应条带，且重链相应的条带更深。结论FⅧTrp1707Ser突变相关抑制物结合FⅧ的位点位于重链的A2亚基和轻链的c2亚基，且与重链的结合能力更强。Objective To investigate the binding mechanisms ofFVllI Trpl707Ser mutation-associ- ated inhibitor. Methods The APPT, PT, TT, Fg and FV^I ： C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR （LD-PCR） and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FⅧ coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FⅧ, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining FⅧ： C （%） by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments. Results The haemophilia A patient had mild deficiency of FⅧ ：C （1.1%） and had high FⅧ inhibitor titer of 18.4 BU. A mutation c.97223C〉G in exon 14 ofF8 gene resulted to p.Trp 1707Ser was identified by DNA sequencing. Corrected test showed that the remaining FⅧ ： C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FⅧ ： C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the At, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FⅧ, A2 and C2 were used as antigens. Conclusion The binding sites of FⅧTrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.

关 键 词：血友病A 因子Ⅷ 抑制物 突变 

分 类 号：R554.1[医药卫生—血液循环系统疾病]