| Research Abstract |
Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor JVIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated woth a recombinant factor VIII preparation. The correlation between the neutralizing activity and reactivity of the IgG4 antibody with factor VIII was high with correlation coefficients of 0.912.
We constructed a fusion protein, glutathione-C2, which binds to immobillized von Willebrand factor (vWF), suggesting that the factor VIII C2 domain contains a binding site for vWF.
Immunoblotting using a panel of recombinant light chain fragments demonstrated that the binding regions of antibodies in cases 1 to 5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognised an epitope within residues 2248-2312, completely inhibited FVIII/vWF binding. Antibodies from case 3 recognising 2170-2312 and case 5 recognising 2170-2327 also inhibited FVIII/vWF binding. Case 4 antibodies recognising 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognising the 44-kDa 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. Synthetic peptides consisting of 15 residues inhibited factor VIII binding to vWF,phospholipid and anti-C2 inhibitor.
Caninie factor VIIII could act as cofactor in human system too. We tested reactivity of caninie factor VIII with several inhibitor alloantibodies. Inhibitor alloantiboies which recognized the C2 domain in the factor VIII light chain cross reacted well with canine factor VIII.On the other hand, inhibitors alloantibodies which recognized the A2 domain in the heavy chain did not react with caninie factor VIII.
We are going to administer the neutralyzing synthetic peptides to caninie model.
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