Summary. Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3-C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy-terminal region of the C2 domain (residues 2288±2332). We found one peptide (2315±2330, L9) which neutralized the anti-FVIII activity of four out of ®ve different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173±2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218±2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315±2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg 2320 and Cys 2326or Glu 2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.
SummaryWe found a patient with mild hemophilia A who had no detectable factor VIII antigen (FVIII:Ag), as shown by two-site ELISA using inhibitor alloantibodies (TK). We then analyzed A2, A2/B, and C2 antigen of the patient's DDAVP-induced FVIII using several anti-FVIII monoclonal antibodies. Factor VIII activity (FVIII : C) was increased from 12 to 42 Uldl by the administration of DDAVP. The DDAVPinduced increases in the A2 and A2/B antigens were 40 and 36 Uldl, respectively. However, the increase in the C2 antigen was only 7.5 Uldl. SSCP analysis and subsequent sequencing demonstrated an Arg to Cys transition at codon 2159. The anti-FVII1:C titer of monoclonal antibody, NMC-VIII15 which recognized the C2 domain, against normal plasma was 450 Bethesda Ulmg of IgG. However, the titer against DDAVP-treated patient's plasma was only 15 Bethesda Ulmg. We also tested DDAVP-induced increase in the FVIII : Ag in another mild hemophilia A patient with the same mutation at Arg2159. Increase in his C2 antigen levels was only 19% of those in the A2 and A2/B antigen. We designate this abnormal FVIII as FVIII Ise. Our results show that a missense mutation at Arg2159 to Cys modifies the antigenicity of the C2 domain.
We have established an ELISA for detecting thrombin cleavage of the FVIII light chain at Arg1689. The method used a coating alloantibody which recognized amino acid residues 2248-2312 in the C2 domain, together with a second monoclonal antibody, NMC-VIII/10, which recognized residues 1675-1684 in the amino-terminal region of the light chain. FVIII antigen (FVIII:Ag) was measured after treatment of plasma with various concentrations of thrombin. The FVIII:Ag of normal plasma was reduced in a dose-dependent manner by the thrombin, falling to 28% in the presence of 100 U/ml enzyme. The concentration of thrombin that achieved 50% reduction (IC50) was approximately 1.0 U/ml. The plasma of four haemophilia A positive (A+) and two haemophilia A reduced (AR) patients were analysed. The IC50 of all patients was more than 1.0 U/ml, indicating that thrombin cleavage of the FVIII light chain was defective. One haemophilia A+ plasma did not respond to thrombin in this ELISA system. The patient (TI) was a haemophiliac with FVIII coagulant activity of 0.04 U/ml and FVIII:Ag of 1.78 U/ml. In addition, immunoblotting of the purified FVIII from TI showed that thrombin cleavage of the 80 kilodalton (kD) light chain was impaired. The patient's DNA was amplified using the polymerase chain reaction with a set of synthetic oligonucleotide primers spanning amino acid residues 1646-1714. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine 1689 to cysteine. This abnormal FVIII was designated as FVIII Hiroshima. Our ELISA system is a simple and useful method of evaluating the proteolytic cleavage by thrombin at Arg1689.
SummaryWe report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRMR) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti- C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient’s peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.
Haemophilia A is the most common X‐linked blood coagulation disorder; it is caused by deficiency of factor VIII activity (FVIII:C). Half of the affected patients do not have detectable levels of FVIII protein in their plasma, whereas about 5% have normal levels of the FVIII antigen (FVIII:Ag) (> 50 u/dl), and are called cross‐reacting material (CRM) positive (CRM+ or A+). About 45% of patients have reduced levels of the FVIII:Ag (1–50 u/dl), classified as CRM reduced (CRMR or AR). We screened the FVIII gene of 13 Japanese patients (five CRM+ and eight CRMR) by single‐strand conformation polymorphism, and identified 11 different mutations in 13 patients by analysing all 26 exons (Trp255Cys, Tyr473Cys, Gly479Arg, Arg531His, Thr667Arg, Arg1689Cys, Arg1941Gln, Arg2150His, Arg2159Cys, Thr2245Ala and Gly2285Val). Seven mutations were identified in the A domains (four in the A2 domain). All the mutations are point mutations resulting in missense codons. Four mutations (Trp255Cys, Thr667Arg, Thr2245Ala and Gly2285Val) have not been described previously.
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