title: A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function.
abstract: Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the V(max) for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the V(max) of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.
authors.affiliation: Department of Medicine, Veterans Administration Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Junhong
authors.initials: J
authors.lastname: Lü
authors.forename: Steven W
authors.initials: SW
authors.lastname: Pipe
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Miao
authors.forename: Marc
authors.initials: M
authors.lastname: Jacquemin
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gilbert
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013499:Surface Properties / Q000187:drug effects; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D010743:Phospholipids; D014841:von Willebrand Factor; D005169:Factor VIII; D005170:Factor X; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant