title: Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity.
abstract: Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33(scFv), and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 --> Ala/Ala (fVIII(YFP 2092/93)) and 2 single mutants Lys2092 --> Ala and Phe2093 --> Ala. KM33(scFv) inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII(YFP 2092/93) had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33(scFv) was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33(scFv) inhibited binding to platelets and fVIII(YFP 2092/93) had reduced binding to A23187-stimulated platelets. fVIII(YFP 2092) exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII(YFP 2093) showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII(YFP 2092/93) had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands.
authors.forename: Henriët
authors.initials: H
authors.lastname: Meems
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
authors.forename: David B
authors.initials: DB
authors.lastname: Cullinan
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gilbert
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008567:Membranes, Artificial; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D003067:Coenzymes; D000939:Epitopes; D008567:Membranes, Artificial; D010649:Phenylalanine; D005169:Factor VIII; D008239:Lysine