His99Arg突变患者血浆凝血因子Ⅷ活性的稳定性研究

Factor Ⅷ A1 domain substitution His99Arg resulting in super-instability of factor Ⅷ in a mild hemophilia A patient

目的探讨His99Arg突变致患者血浆凝血因子Ⅷ(FⅧ)活性检测结果变化大及一期法和二期法FⅧ活性检测结果不相符的分子发病机制。方法以凝固法检测先证者及家系成员凝血酶原时间(PT)、活化部分凝血活酶时间(APTT),FⅧ活性(FⅧ∶C)、FⅨ∶C等。基于APTT途径的一期法和基于发色底物法途径的二期法检测FⅧ∶C,测定患者血浆分别在37、25、4、-20及-80℃时的FⅧ∶C,测定56℃时患者血浆FⅧ∶C稳定性(一期法)及FⅧ蛋白的热稳定性(EILSA法);以长链PCR及序列特异PCR检测F8基因内含子22及内含子1倒位,对F8基因所有外显子及其侧翼序列测序;以PyMOL软件分析FⅧ突变蛋白的三维结构;构建His99Arg突变表达载体,转染HEK293T细胞后博莱霉素筛选稳定表达细胞株,测定培养上清FⅧ∶C稳定性。结果先证者血浆FⅧ∶C:一期法检测为14.7%,二期法检测为1.2%。和正常人相比,患者血浆在室温放置<2 h时FⅧ∶C迅速下降,>2 h后FⅧ∶C基本稳定在1%左右;在4℃孵育时FⅧ∶C下降的趋势和室温放置时基本相似;FⅧ∶C在37℃随孵育时间延长而明显下降,孵育10 min后FⅧ∶C下降了93.9%;不管在-20还是在-80℃,患者血浆放置1个月后,其FⅧ∶C基本完全丧失;56℃时患者血浆FⅧ∶C的半衰期仅为正常人的1/4(2 min/8 min);在56℃时患者FⅧ稳定性下降明显,重链和轻链解离速度是正常人的3倍(2 min/6 min)。F8基因分析发现患者存在g.30716A>G突变而导致His99Arg氨基酸改变;三维结构模型分析显示His99Arg突变后,Arg99残基与His1957及Ser1959残基间的距离由3.49和3.42分别变为4.19和2.39。体外表达研究显示培养上清液中的FⅧ具有与患者血浆FⅧ相似的不稳定表现。结论 His99Arg突变引起FⅧ空间结构发生变化和FⅧ重链和轻链解离速度加快。His99Arg突变后FⅧ∶C的稳定性明显下降,造成常规条件下FⅧ∶C检测结果变化大及一期法和二期法FⅧ∶C检测结果的不相符。

Objective To investigate the molecular mechanism of super-instability of F~ and discrepancy of one- stage and two-stage FⅧ： C assay in a mild hemophilia A patient with His99Arg mutation. Methods Coagulation function was assayed for establishing phenotype diagnosis. FⅧ： C was detected by one stage method and chromogenic method. Pa- tient＇s plasma was incubated at routine storage temperature （37 ℃ ,25 ℃,4℃, -20℃, -80℃ ） and aliquots were taken to assess the FⅧ： C at the indicated times. One-stage method and ELISA were applied to measure activity and amount of re- maining FⅧ when the plasma was incubated at 56℃ at different time. LD-PCR and sequence specific PCR were adopted for deteeting intron 22 and intron 1 inversion of F8 gene. All the exons of F8 gene and its flanking sequences were amplified and sequenced directly. Three-dimensionai structure analysis was performed by PyMOL program to analyze the impact of His99Arg mutation on the spatial structure of FⅧ. The B-domaiuless FV/H mutant expression plasmid His99Arg was con- strueted and transfected into HEK293T,the stability of the stable expression products were assayed. Results The FⅧ： C of the proband was 14.7% detected by one-stage method but just 1.2% measured by chromogenic method. The activity of mutant FⅧ exhibited significantly increased rates of loss of activity when the plasma incubated at routine storage tempera- ture. Furthermore,the activity of FⅧK His99Arg was less stable following heat denaturation analysis,this reduced stability appeared to result from an about 300% increase in the dissociation rate judged by ELISA. Gene analysis revealed a A30716G substitution in exon 3 resulting in a His99Arg missense mutation. Three-dimensional structural analysis showed that His99 residue closed to His1957 （3.49） arid Ser1959 （3. 42 ） of A3 subunit, when His99 mutated into Arg99, the distance changed into 4.19 and 2.39 ,respectively. The FⅧ activity of stable expression products showed similar decrease trend in accordance with that of patient＇s FⅧ. Conclusion His99Arg nmtation caused changes in the spatial structure of FⅧ and increased the dissociation rate of the hear） chain and light chain. His99Arg mutation resulted in instability of FⅧ： C arid discrepancy of one-stage and two-stage FⅧ： C assay.