Phospholipid vesicles interfere with binding of antibody fragments to the light chain of factor VIII

Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223,W2313 andV2314 at the tips of β-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factorV form a phospholipid binding site, but residues in the A3 and CI domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factorVIII inhibitors. Phospholipid vesicles inhibited the binding of factorVIII to scFvsWRI and WR16 (epitope: E2181-M2199) with half saturation values of 23 and 47 μM respectively.The single point mutant F2200A factor VIII light chain bound to WRI and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factorVIII to C2-specific scFvs WR13 and EL14 (epitope: K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factorVIII to theA3-CI-specific scFvs KM36 (epitope: Q1778-D1840) and KM38 (epitope: S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 μM, respectively, suggesting that the A3 and/or CI domains may contribute to membrane binding of the cofactor.