Nogami Keiji, Shima Midori, Giddings John C, Takeyama Masahiro, Tanaka Ichiro, Yoshioka Akira
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Int J Hematol. 2007 May;85(4):317-22. doi: 10.1532/IJH97.06192.
Some factor VIII (FVIII) inhibitor alloantibodies block FVIII binding to von Willebrand factor (VWF) and phospholipid (PL) and recognize a C2 domain epitope that overlaps both binding sites. We previously showed that FVIII peptide 2315-2330 neutralized FVIII inhibitors and that Cys2326 and Glu2327 contributed to the maximum neutralizing effect. In the present study, we investigated the relationship between the essential binding sites for VWF, PL, and anti-C2 inhibitors by means of competitive-inhibition assays with overlapping synthetic peptides that span the C terminus of the C2 domain (residues 2288-2332). We identified 2 peptides (residues 2303-2317 and 2315-2330) that specifically blocked FVIII binding to VWF or PL by approximately 80% (50%-inhibitory concentration [IC50], 9.0 microM) and 95% (IC50, 0.12 microM), respectively. To examine in detail the residues responsible for PL binding, we prepared mutants of peptide 2315-2330 in which we sequentially substituted each residue with Gly. Two residues, Ile2317 and Met2321, were shown to be essential for PL binding. Their substitution with Gly reduced the inhibitory effect by >90%. The data suggest that the binding sites for VWF, PL, and anti-C2 inhibitors in the C2 domain are in very close proximity but are not identical.
一些因子VIII（FVIII）抑制性同种抗体可阻断FVIII与血管性血友病因子（VWF）及磷脂（PL）的结合，并识别一个与两个结合位点均重叠的C2结构域表位。我们之前曾表明，FVIII肽2315 - 2330可中和FVIII抑制物，且半胱氨酸2326和谷氨酸2327对最大中和效应有作用。在本研究中，我们通过使用跨越C2结构域C末端（残基2288 - 2332）的重叠合成肽进行竞争性抑制试验，研究了VWF、PL及抗C2抑制物的关键结合位点之间的关系。我们鉴定出2种肽（残基2303 - 2317和2315 - 2330），它们分别可特异性地阻断FVIII与VWF或PL的结合，阻断率约为80%（50%抑制浓度[IC50]，9.0微摩尔/升）和95%（IC50，0.12微摩尔/升）。为详细研究负责PL结合的残基，我们制备了肽2315 - 2330的突变体，其中我们将每个残基依次替换为甘氨酸。结果显示，两个残基异亮氨酸2317和甲硫氨酸2321对PL结合至关重要。将它们替换为甘氨酸可使抑制效应降低>90%。数据表明，C2结构域中VWF、PL及抗C2抑制物的结合位点非常接近，但并不相同。
