title: Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit. Results from an increased apparent affinity for the A2 subunit.
abstract: Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.
authors.affiliation: Morgan Monaghan, PO Box 712, Department of Biochemistry and Biophysics, 601 Elmwood Ave., Rochester, NY 14642, USA, Tel.: +1 585 275 6576, Fax: +1 585 275 6007, E-mail: Morgan_monaghan@urmc.rochester.edu.
authors.forename: M
authors.initials: M
authors.lastname: Monaghan
authors.affiliation: Hironao Wakabayashi, PO Box 712, Department of Biochemistry and Biophysics, 601 Elmwood Ave., Rochester, NY 14642, USA, Tel.: +1 585 275 6576, Fax: +1 585 275 6007, E-mail: Hironao_Wakabayashi@URMC.Rochester.edu.
authors.forename: H
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: A
authors.initials: A
authors.lastname: Griffiths
authors.forename: J
authors.initials: J
authors.lastname: Wintermute
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics
chemical_list: D009363:Neoplasm Proteins; D021122:Protein Subunits; C078147:F8 protein, human; D005169:Factor VIII; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant
keywords: Factor VIII; factor VIIIa; inter-subunit binding affinity; protein stability