Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor.
Factor VIII (FVIII) inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVIII binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1-5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248-2312, completely inhibited FVIII/vWF binding in an ELISA (IC50: 5.0 and 9.0 micrograms/ml, respectively). Antibodies from case 3 recognizing 2170-2312 and case 5 recognizing 2170-2327 also inhibited FVIII/vWF binding (IC50: 110 and 400 micrograms/ml, respectively). Case 4 antibodies recognizing 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti-C2 domain inhibitor alloantibodies.
Shima
M
M
Department of Paediatrics, Nara Medical University, Kashihara City, Japan.
Nakai
H
H
Scandella
D
D
Tanaka
I
I
Sawamoto
Y
Y
Kamisue
S
S
Morichika
S
S
Murakami
T
T
Yoshioka
A
A
D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D005169:Factor VIII / Q000378:metabolism*; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain; D006467:Hemophilia A / Q000378:metabolism*; D006801:Humans; D007518:Isoantibodies / Q000378:metabolism; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000939:Epitopes; D007518:Isoantibodies; D014841:von Willebrand Factor; D005169:Factor VIII