title: The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII.
abstract: Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.
authors.affiliation: Center for Vascular and Inflammatory Diseases, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA. NAnanyeva@som.umaryland.edu
authors.forename: Natalya M
authors.initials: NM
authors.lastname: Ananyeva
authors.forename: Yevgen M
authors.initials: YM
authors.lastname: Makogonenko
authors.forename: Diana V
authors.initials: DV
authors.lastname: Kouiavskaia
authors.forename: Jose
authors.initials: J
authors.lastname: Ruiz
authors.forename: Vincent
authors.initials: V
authors.lastname: Limburg
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Khrenov
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
authors.forename: Dudley K
authors.initials: DK
authors.lastname: Strickland
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
mesh_terms: D001665:Binding Sites / Q000502:physiology; D001777:Blood Coagulation / Q000502:physiology; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000502:physiology; D015944:Factor VIIIa / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008079:Lipoproteins, VLDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology; D011973:Receptors, LDL / Q000502:physiology*; D020349:Surface Plasmon Resonance
chemical_list: D008079:Lipoproteins, VLDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009363:Neoplasm Proteins; D011973:Receptors, LDL; C036053:VLDL receptor; D015944:Factor VIIIa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant