Relationship between the binding sites for von Willebrand factor, phospholipid, and human factor VIII c2 inhibitor Alloantibodies within the factor VIII c2 domain
Relationship between the binding sites for von Willebrand factor, phospholipid, and human factor VIII c2 inhibitor Alloantibodies within the factor VIII c2 domain
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DOI：
10.1532/ijh97.06192
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发表时间：
2007-05-01
影响因子：
2.1
通讯作者：
Yoshioka, Akira
中科院分区：
文献类型：
--
作者：
Nogami, Keiji;Shirria, Midori;Yoshioka, Akira
Some factorVIII (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 Cys(2326) and Glu(2327) 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 mu M) and 95% (IC50, 0.12 mu M), 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, Ile(2317) and Met(2321), 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.
