title: Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484-509.
abstract: Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this 'consensus' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.
authors.affiliation: Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, MD 21201, 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: Andrey G
authors.initials: AG
authors.lastname: Sarafanov
authors.forename: Igor V
authors.initials: IV
authors.lastname: Pechik
authors.forename: Natalya
authors.initials: N
authors.lastname: Gorlatova
authors.forename: Klaus P
authors.initials: KP
authors.lastname: Radtke
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000502:physiology; D001665:Binding Sites; D016384:Consensus Sequence; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism*; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000596:Amino Acids; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; C036053:VLDL receptor; D014841:von Willebrand Factor; C078147:F8 protein, human; D005169:Factor VIII; D013917:Thrombin