title: Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.
abstract: Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. asarafanov@som.umaryland.edu
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Sarafanov
authors.forename: Evgeny M
authors.initials: EM
authors.lastname: Makogonenko
authors.forename: Igor V
authors.initials: IV
authors.lastname: Pechik
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Radtke
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Khrenov
authors.forename: Natalya M
authors.initials: NM
authors.lastname: Ananyeva
authors.forename: Dudley K
authors.initials: DK
authors.lastname: Strickland
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D026563:LDL-Receptor Related Protein-Associated Protein / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011971:Receptors, Immunologic / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000939:Epitopes; D026563:LDL-Receptor Related Protein-Associated Protein; D011971:Receptors, Immunologic; D005169:Factor VIII; D000409:Alanine