Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti-C2 antibody inhibiting allogeneic but not autologous factor VIII activity.
BACKGROUND
In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence.


OBJECTIVE
To investigate the mechanism(s) of inhibitor development in MHA.


RESULTS
A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity (
C ~10 IU dL(-1) ), despite the development of an inhibitor (5.6 BU mL(-1) ). Purified HA78-IgG significantly depressed
C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated ~3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an ~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with ~10(2) -fold lower affinity compared with ESH8.


CONCLUSIONS
These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
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http://orcid.org/0000-0002-4400-499X
K
Yada
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
K
K
Nogami
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
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M
Takeyama
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
K
K
Ogiwara
Department of Biochemistry and Biophysics, University of Rochester School, Rochester, NY, USA.
H
H
Wakabayashi
Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
M
M
Shima
D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood*; D001777:Blood Coagulation / Q000235:genetics*; D001780:Blood Coagulation Tests; D003175:Complement C2 / Q000276:immunology*; D003429:Cross Reactions; D004252:DNA Mutational Analysis; D018604:Epitope Mapping; D000939:Epitopes; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D020125:Mutation, Missense; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012720:Severity of Illness Index; D013329:Structure-Activity Relationship
D001323:Autoantibodies; D003175:Complement C2; D000939:Epitopes; C078147:F8 protein, human; D005169:Factor VIII
blood coagulation factor inhibitors; epitopes; factor VIII; hemophilia A; mutation missense