title: [The binding mechanisms of F VIII Trp1707Ser mutation-associated inhibitor].
abstract: OBJECTIVE
  To investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.
  
  
  METHODS
  The APPT, PT, TT, Fg and FVIII:C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR (LD-PCR) and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FVIII coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FVIII, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining F VIII:C (% ) by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments.
  
  
  RESULTS
  The haemophilia A patient had mild deficiency of FVIII:C (1.1%) and had high FVIII inhibitor titer of 18.4 BU. A mutation c.97223C>G in exon 14 of F8 gene resulted to p.Trp1707Ser was identified by DNA sequencing. Corrected test showed that the remaining F VIII:C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FVIII:C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the A1, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FVIII, A2 and C2 were used as antigens.
  
  
  CONCLUSIONS
  The binding sites of FVIIITrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.
authors: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Department of Clinical Laboratory, Ruijin Hospital of Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
authors: Xi
authors: X
authors: Wu
authors: Ye-ling
authors: YL
authors: Lu
authors: Qiu-lan
authors: QL
authors: Ding
authors: Jing
authors: J
authors: Dai
authors: Xiao-dong
authors: XD
authors: Xi
authors: Hong-li
authors: HL
authors: Wang
authors: Xue-feng
authors: XF
authors: Wang
mesh_terms: D001665:Binding Sites / Q000235:genetics; D005091:Exons; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000235:genetics*; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D055815:Young Adult
chemical_list: D005169:Factor VIII