title: Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability.
abstract: Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ∼3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ∼4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ∼4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Amy E
authors.initials: AE
authors.lastname: Griffiths
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry*; D015944:Factor VIIIa / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D021122:Protein Subunits; D015944:Factor VIIIa