大疱性类天疱疮并获得性血友病A患者血浆IgG亚型及FⅧ结合表位鉴定

Identification of IgG subclass and FⅧ binding epitope of an acquired FⅧ inhibitor in a bullous pemphigoid patient

目的确定1例大疱性类天疱疮并获得性血友病A患者的临床和实验室诊断；通过体内外干预实验观察获得性血友病A患者体内的FⅧ抑制物对正常人血浆及兔FⅧ凝血活性（FⅧ：C）的影响；确定患者FⅧ抑制物的IgG亚型和结构表位，以揭示该病发病的分子机制。方法用Ⅰ期法测定患者血浆FⅧ：C；以Bethesda单位（BU）表示FⅧ：C抑制物滴度；用蛋白A一琼脂糖柱纯化患者及正常人血浆IgG；用活化部分凝血活酶时间（APTT）分析其对FⅧ：C的抑制作用；观察FⅧ抑制物对兔血浆FⅧ：C活性的抑制作用；分别用抗IgG1、IgG2、IgG3、IgG4抗体作Western blot，结合灰度扫描确定患者血浆IgG亚型相对表达量；散射比浊法测定患者及正常人体内各IgG亚型浓度；用FⅧ／FⅧ抑制物固相结合试验及Western blot鉴定患者IgG抗体（FⅧ抑制物）作用于FⅧ的具体“表位”。结果①患者APTT明显延长，正常血浆不能纠正；FⅧ：C〈1．5％，FⅧ抑制物滴度为147．8BU；②纯化的患者IgG以剂量依赖方式抑制正常人混合血浆FⅧ：C；动物实验显示患者血浆能以时间依赖方式延长兔血浆APTT；@Western blot结果显示FⅧ抑制物成分主要为IgG4，其次为IgG1，FⅧ抑制物作用于FⅧ：C的结构相对分子质量为44×10^3。散射比浊法测定结果显示患者体内IgG4、IgG4含量明显高于正常人。结论研究结果证明大疱性类天疱疮并获得性血友病A患者体内FⅧ抑制物对FⅧ：C有抑制作用，FⅧ抑制物的IgG亚型主要为IgG4，其次为IgG1；患者FⅧ抑制物作用于FⅧ的表位为相对分子质量为44×10^3的肽段，揭示了该获得性血友病A发生、发展的分子机制。

Objective To identify the clinical and aboratory diagnosis of a bullous pemphigoid patient with acquired hemophilia A （AH-A）. To identify FⅧ binding epitope and IgG subclass of the FⅧ inhibitor, and explore the molecular mechanism for AH-A pathogenesis. Methods Plasma FⅧ activity（FⅧ： C） was determined by one-stage assay, the titre of FⅧ inhibitor by Bethesda Unit（BU）. IgG purification of patient plasma or normal pooled plasma was finished by protein A-agrose column chromatography. Activated partial thromboplastin time（AP3T） was assayed for uncovering FⅧ inhibitor effect on FⅧ in vivo. Combined Western blot analysis by anti-IgG1 ,IgG2,IgG3 and IgG4 antibodies was used to determine the relative concentration of patient＇ s IgG subclass. IgG subclass concentrations were quantified by nephelometric method. Sol- id-phase binding assay of FⅧ and FⅧ inhibitor, combined with Western blot was used to recognize the binding epitope at which the FⅧ inhibitor bound to FⅧ. Results （1）Plasma APTT value of patient was prolonged evidently and could not be corrected by normal pooled plasma. Patient＇ s FⅧ： C was 〈 1.5%. The titre of FⅧ inhibitor in patient plasma was 147.8 BU.（2）The purified patient IgG was able to inhibit FⅧ： C of normal pooled plasma significantly with a dose dependent mannar, and the patient plasma could prolong rabbit plasma APTI＇ markedly with a time dependent manner. （3）The FⅧ inhibitor was predominantly then of IgG4 subtype with a minority IgG1 , and the concentration of IgG4 and IgG1 in the patient was higher than that in normal. The FⅧ inhibitor reacted with FⅧ 44× 10^3 fragment epitope. Conclusions The inhibiting effect of FⅧ inhibitors on FⅧ： C in the bullous pemphigoid patient with AH-A is determined and the IgG subclass of the FⅧ inhibitor is identified. A binding epitope for the FⅧ inhibitor is a FⅧ 44 -103 fragment. The resuits provides evidence for understanding the pathogenesis of AH-A.