STUDIES FOR TREATMENT OF HEMOPHILIA A WITH INHBITOR USING CANINIE SYSTEM

采用酶联免疫吸附试验（ELISA）测定重组因子VIII制剂治疗的A型血友病患者血清中抗因子JVIII免疫球蛋白G （IgG）、IgG4和IgM的含量。IgG4抗体的中和活性和反应性与因子VIII相关性较高，相关系数为0.912。我们构建了一个融合蛋白，谷胱甘肽-C2，它与固定化血管性血液病因子（vWF）结合，表明因子VIII C2结构域含有vWF的结合位点。使用重组轻链片段进行免疫印迹分析表明，病例1至5中抗体的结合区位于轻链的C2结构域。病例1和病例2的抗体在2248-2312残基内识别一个表位，完全抑制了FVIII/vWF的结合。病例3中识别2170-2312的抗体和病例5中识别2170-2327的抗体也抑制了FVIII/vWF的结合。病例4识别2218-2307的抗体几乎没有检测到抑制作用，病例6和7识别44-kDa重链的抗体没有抑制作用。我们的研究结果表明，所有具有延伸到残基2312的表位的抗c2同种抗体都能抑制vWF的结合，并且抑制剂表位与残基2308-2312的重叠是至关重要的。由15个残基组成的合成肽抑制因子VIII与vWF、磷脂和抗c2抑制剂的结合。犬类因子viii在人体系统中也可作为辅助因子。我们测试了犬因子VIII与几种抑制剂同种抗体的反应性。识别因子VIII轻链中C2结构域的异体抗体抑制剂与犬因子VIII交叉反应良好。另一方面，识别重链A2结构域的抑制异体抗体不与犬因子VIII发生反应。我们将给犬模型注射中和性合成肽。

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.