title: A3 domain region 1803-1818 contributes to the stability of activated factor VIII and includes a binding site for activated factor IX.
abstract: A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.
authors.forename: Esther
authors.initials: E
authors.lastname: Bloem
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.
authors.forename: Henriet
authors.initials: H
authors.lastname: Meems
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and.
authors.forename: Maartje
authors.initials: M
authors.lastname: van den Biggelaar
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and; Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 1066 CX Amsterdam, The Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and; Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 1066 CX Amsterdam, The Netherlands. Electronic address: s.meijer@sanquin.nl.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015944:Factor VIIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D015944:Factor VIIIa; C078147:F8 protein, human; D005169:Factor VIII; D005164:Factor IX
keywords: Enzyme Kinetics; Factor VIII; Protein Stability; Protein Structure; Surface Plasmon Resonance (SPR)