title: Replacing the factor VIII C1 domain with a second C2 domain reduces factor VIII stability and affinity for factor IXa.
abstract: Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIIIC2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (~11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIIIC2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (~2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIIIC2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIIIC2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (~10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIIIC2C2 variant (~4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIIIC2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.
authors.affiliation: From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D015949:Factor IXa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII; D015949:Factor IXa
keywords: Blood Coagulation Factors; Factor VIII; Phospholipid Vesicle; Protein Stability; Protein Structure
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