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STUDIES FOR TREATMENT OF HEMOPHILIA A WITH INHBITOR USING CANINIE SYSTEM

使用 CANINIE 系统使用抑制剂治疗 A 型血友病的研究

基本信息

批准号：
06670819
负责人：
SHIMA Midori
金额：
$ 1.41万
依托单位：
NARA MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06670819/
关键词：
hemophilia A inhibitor factor VIII 抗第VIII因子同種抗体

项目摘要

Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor JVIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated woth a recombinant factor VIII preparation. The correlation between the neutralizing activity and reactivity of the IgG4 antibody with factor VIII was high with correlation coefficients of 0.912.We constructed a fusion protein, glutathione-C2, which binds to immobillized von Willebrand factor (vWF), suggesting that the factor VIII C2 domain contains a binding site for vWF.Immunoblotting using a panel of recombinant light chain fragments demonstrated that the binding regions of antibodies in cases 1 to 5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognised an epitope within residues 2248-2312, completely inhibited FVIII/vWF binding. Antibodies from case 3 recognising 2170-2312 and case 5 recognising 2170-2327 also inhibited FVIII/vWF binding. Case 4 antibodies recognising 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognising the 44-kDa heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical. Synthetic peptides consisting of 15 residues inhibited factor VIII binding to vWF,phospholipid and anti-C2 inhibitor.Caninie factor VIIII could act as cofactor in human system too. We tested reactivity of caninie factor VIII with several inhibitor alloantibodies. Inhibitor alloantiboies which recognized the C2 domain in the factor VIII light chain cross reacted well with canine factor VIII.On the other hand, inhibitors alloantibodies which recognized the A2 domain in the heavy chain did not react with caninie factor VIII.We are going to administer the neutralyzing synthetic peptides to caninie model.

用酶联免疫吸附试验(EL ISA)检测重组第VIII因子制剂治疗的甲型血友病患者血清中的抗-JVIII免疫球蛋白G(Ig G)、Ig G4和Ig M。抗体的中和活性和反应性与因子的相关性很高，相关系数为0.912。我们构建了谷胱甘肽-C2融合蛋白，该融合蛋白与固定化的von Willebrand因子(VWF)结合，表明C2区含有vWF的结合部位。来自病例1和病例2的抗体识别残基2248-2312内的表位，完全抑制FVIII/vWF结合。病例3识别2170-2312的抗体和病例5识别2170-2327的抗体也抑制FVIII/vWF结合。识别2218-2307的病例4抗体几乎检测不到抑制作用，识别44 kDa重链的病例6和7抗体不抑制。我们的结果表明，所有具有延伸到2312残基的表位的抗C2同种异体抗体都抑制vWF结合，并且抑制物表位与残基2308-2312的重叠是至关重要的。由15个残基组成的合成肽可抑制凝血因子VIII与vWF、磷脂和抗C2抑制剂的结合。犬凝血因子VIIII在人体内也可作为辅因子发挥作用。我们检测了犬因子与几种抑制物同种抗体的反应性。识别因子VIII轻链C2结构域的抑制物同种异体抗体与犬凝血因子VIII交叉反应良好，而识别重链A2结构域的抑制物同种抗体不与犬凝血因子VIII反应。