title: Membrane-binding properties of the Factor VIII C2 domain.
abstract: Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa-Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.
authors.affiliation: Department of Medicine, Veterans Affairs Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02132, USA.
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Novakovic
authors.forename: David B
authors.initials: DB
authors.lastname: Cullinan
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
authors.forename: James D
authors.initials: JD
authors.lastname: Baleja
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gilbert
mesh_terms: D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008894:Milk Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride
chemical_list: D000954:Antigens, Surface; C098942:MFGE8 protein, human; D008894:Milk Proteins; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D012965:Sodium Chloride; C078147:F8 protein, human; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant