title: Binding of factor VIII inhibitors to discrete regions of the factor VIII C2 domain disrupt phospholipid binding.
abstract: We characterized seven factor VIII inhibitors with epitopes in the C2 domain of factor VIII using a series of factor V C2 domain chimeras that substituted exon-sized fragments of the C2 domain of factor VIII for the corresponding regions of factor V. All inhibited co-factor activity of factor VIII and six inhibited binding of factor VIII to phosphatidylserine. Inhibitors Hz, JN and GK32 bound epitopes within amino acids S2173-K2281; inhibitors GK24 and TO bound epitopes within amino acids V2223-Y2332; and inhibitors UNC11 and UNC12 bound epitopes throughout the C2 domain (amino acids S2173-Y2332). Inhibitors Hz, JN and UNC12 inhibited the co-factor activity of chimera 5A, which substituted amino acids S2173-Q2222 of factor VIII for the corresponding region of factor V, in a prothrombinase assay. This inhibition could be partially reversed by pre-incubation of chimera 5A with phospholipid vesicles, suggesting that these antibodies interfered with phospholipid binding. Inhibitors UNC11 and UNC12, on the other hand, did not inhibit the binding of chimera 1 A to phosphatidylserine, suggesting that binding to the segment spanning amino acids V2282-Y2332 does not necessarily block phospholipid binding. These results agree with the model of the phospholipid-binding site determined by crystal structure of the C2 domain of factor VIII.
authors.affiliation: Department of Medicine, Division of Hematology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Lewis
authors.forename: Karen D
authors.initials: KD
authors.lastname: Moore
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Ortel
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D019774:Blood Coagulation Factor Inhibitors / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005165:Factor V / Q000037:antagonists & inhibitors / Q000378:metabolism; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D019774:Blood Coagulation Factor Inhibitors; D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D005165:Factor V; D005169:Factor VIII