Details

Autor(en) / Beteiligte

• Wakabayashi, Hironao
• Fay, Philip J.

Titel

Modification of inter-domain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity

Ist Teil von

• Biochemistry (Easton), 2013-05, Vol.52 (22), p.3921-3929

Erscheinungsjahr

2013

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Factor (F)VIII consists of a heavy chain [A1(a1)A2(a2)B domains] and light chain [(a3)A3C1C2 domains]. Several reports have shown significant changes in FVIII stability and/or activity following selected mutations at the interfaces of the A1-A2, A1-A3, A2-A3, or A1-C2 domains. In this study the remaining inter-FVIII subunit interfaces (A3-C1 and C1-C2) were examined for their contributions to FVIII/FVIIIa stability and activity. We prepared FVIII mutants with a nascent disulfide bridges between A3 and C1 domains (Gly1750Cys/Arg2116Cys and Ala1866Cys/Ser2119Cys) or C1 and C2 domains (Ser2029Cys/Pro2292Cys). We also prepared mutants replacing Arg2116 with hydrophobic residues (Ala and Val) since this C1 domain residue appears to face a pocket of positive electrostatic potential in the A3 domain. Stability was assessed following the rates of loss of FVIII activity at 55°C and the spontaneous loss of FVIIIa activity from A2 subunit dissociation. FVIII Gly1750Cys/Arg2116Cys showed a marked increase in thermal stability (~3.7 fold) as compared with WT FVIII while the stability of FVIII Ala1866Cys/Ser2119Cys was reduced (~4.7 fold). Although the Ser2029Cys/Pro2292Cys variant showed a modest loss in FVIII stability, the specific activity and thrombin generation potential of this variant were increased (up to 1.2-fold) compared with WT. Furthermore, this variant demonstrated an ~2-fold reduced K m for FX. Mutation of Arg2116 to hydrophobic residues resulted in variable decreases in stability and thrombin generation parameters suggesting a role of this Arg residue contributing to FVIII structure. Taken together, selective modification of the contiguous domain interfaces in FVIII light chain may improve FVIII stability and/or cofactor function.