title: Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII.
abstract: Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of beta-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V forma phospholipid binding site, but residues in the A3 and C1 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 factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvs WR1 and WR16 (epitope : E2181-M2199) with half saturation values of 23 and 47 muM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 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 factor VIII 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 factor VIII to the A3-C1-specific scFvs KM36 (epitope : Q1778-D1840) and KM38 (epitope : S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 microM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.
authors.affiliation: Division of Hematology, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Lewis
authors.forename: Niels
authors.initials: N
authors.lastname: Bovenschen
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Ortel
mesh_terms: D000918:Antibody Specificity; D001665:Binding Sites; D019774:Blood Coagulation Factor Inhibitors / Q000737:chemistry; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008958:Models, Molecular; D019151:Peptide Library; D010743:Phospholipids / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013997:Time Factors
chemical_list: D019774:Blood Coagulation Factor Inhibitors; D017077:Culture Media, Conditioned; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D010743:Phospholipids; D005169:Factor VIII; D015951:Factor Xa