Healey J F, Lubin I M, Nakai H, Saenko E L, Hoyer L W, Scandella D, Lollar P
Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 1995 Jun 16;270(24):14505-9. doi: 10.1074/jbc.270.24.14505.
The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.
人凝血因子VIII（fVIII）的A2结构域（残基373 - 740）包含抑制性同种抗体和自身抗体的主要表位。我们利用抑制性抗体对人和猪fVIII的不同反应性，通过使用一系列有活性的重组人/猪fVIII杂交分子，绘制了A2表位的主要决定簇。在人A2结构域的410 - 508、445 - 508或484 - 508片段含有猪序列替代的杂交分子，不受鼠单克隆抗体A2抑制性抗体mAb 413的抑制，而在387 - 403、387 - 444和387 - 468含有替代的杂交分子则受mAb 413抑制。这表明由Arg484和Ile508界定的片段包含A2表位的主要决定簇。mAb 413不抑制另外两个在残基484 - 488和489 - 508含有猪替代的杂交分子，表明在Arg484 - Ile508片段内Ser488 - Arg489键两侧的氨基酸侧链对A2表位有贡献。484 - 508、484 - 488和489 - 508猪替代杂交分子对来自4例患者血浆的A2抑制剂的抑制作用降低，提示抑制剂群体中A2表位的结构变化很小。
