Modification of Interdomain Interfaces within the A3C1C2 Subunit of Factor VIII Affects Its Stability and Activity

Abstract

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 Km 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.

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

“…This value falls within the range of possible C␣ distances at disulfide bond forming Cys residues (4 -7 Å) (29 (31). Interestingly, in the above report, we also prepared seven additional FVIII variants with double Cys mutations that did not express and/or showed no disulfide bond formation, although C␣ distance values for five of the seven variants ranged from 4 to 7 Å.…”

Prediction of hemophilia A severity using a small-input machine-learning framework

“…2 ). As it happens with other proteins 15 , 16 , this indicates that the substitution of conserved FVIII amino acids buried at the core of FVIII interferes with its conformation, function, and ability to bind other proteins 17 – 19 .…”

Stabilizing interactions between D666‐S1787 and T657‐Y1792 at the A2‐A3 interface support factor VIIIa stability in the blood clotting pathway

“…Previously, the FVIII structures and the homology model derived from ceruloplasmin have been guides for targeted mutagenesis to identify residues important for FVIIIa stability and A2 retention, as measured by biochemical analysis . A panel of polar residues at the A2 interface were mutated to examine the contribution that the residues made to FVIII/FVIIIa stability , providing evidence that several residues at both the A2‐A1 and A2‐A3 interfaces were important for FVIII and FVIIIa stability.…”