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The Molecular Basis for Cross-Reacting Material-Positive Hemophilia
A Due to Missense Mutations Within the A2-Domain of Factor VIII
Kagehiro Amano,
Rita Sarkar,
Susan Pemberton,
Geoffrey Kemball-Cook,
Haig H. Kazazian Jr, and
Randal J. Kaufman
From The Howard Hughes Medical Institute and the Department of
Biological Chemistry and University of Michigan Medical Center, Ann
Arbor, MI; the Department of Genetics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Haemostasis Research Group,
Medical Research Council Clinical Sciences Centre, London, UK; and the
Department of Clinical Pathology, Tokyo Medical College, Tokyo, Japan.
Factor VIII (FVIII) is the protein defective in the bleeding
disorder hemophilia A. Approximately 5% of hemophilia A patients have
normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of
the genetic defects within the A2-domain for FVIII function we
constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T,
V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the
conditioned medium from transiently transfected COS-1 cells correlated
with published data for the patients plasma-derived FVIII, confirming
the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis analysis of immunoprecipitated FVIII protein
radiolabeled in COS-1 cells showed that all CRM-positive mutant
proteins were synthesized and secreted into the medium at rates similar
to wild-type FVIII. The majority of the DeltaF652/3 mutant was
defective in secretion and was degraded within the cell. All mutant
FVIII proteins were susceptible to thrombin cleavage, and the A2-domain
fragment from the I566T mutant had a reduced mobility because of use of
an introduced potential N-linked glycosylation site that was confirmed
by N-glycanase digestion. To evaluate interaction of FVIII
with factor IXa, we performed an inhibition assay using a synthetic
peptide corresponding to FVIII residues 558 to 565, previously shown to
be a factor IXa interaction site. The concentration of peptide required
for 50% inhibition of FVIII activity (IC50) was reduced for the I566T
(800 µmol/L) and the S558F (960 µmol/L) mutants compared with
wild-type FVIII (>2,000 µmol/L). N-glycanase digestion increased
I566T mutant FVIII activity and increased its IC50 for the peptide
(1,400 µmol/L). In comparison to S558F, a more conservative mutant
(S558A) had a sixfold increased specific activity that also correlated
with an increased IC50 for the peptide. These results provided support
that the defects in the I566T and S558F FVIII molecules are caused by
steric hindrance for interaction with factor IXa.
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 538-548
© 1998 by The American Society of Hematology.
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