title: The N-terminal epidermal growth factor-like domain of coagulation factor IX. Probing its functions in the activation of factor IX and factor X with a monoclonal antibody.
abstract: The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k(cat) for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu(78)-Arg(94) salt bridge is not important for maintaining the structure of FIX.
authors.affiliation: Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden.
authors.forename: Kristina E M
authors.initials: KE
authors.lastname: Persson
authors.forename: Bruno O
authors.initials: BO
authors.lastname: Villoutreix
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Thämlitz
authors.forename: Karin E
authors.initials: KE
authors.lastname: Knobe
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D003198:Computer Simulation; D017931:DNA Primers; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D005164:Factor IX / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005170:Factor X / Q000276:immunology*; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D015335:Molecular Probes; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005164:Factor IX; D005170:Factor X