Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Hironao Wakabayashi,Amy E Griffiths,Philip J Fay

doi:10.1074/jbc.m111.241109

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.

Highlights

We recently reported that an Factor VIII (FVIII) variant lacking the C2 domain retained the capacity to bind phospholipid membranes, albeit with a marked reduction in affinity [5], supporting a direct role for the C1 domain in this interaction
We investigate interactions at the interface between FVIII A1 and C2 domains following preparation of two novel variants, R121C/L2302C FVIII, possessing a nascent disulfide bond spanning the domains, and A108I FVIII, which has a larger hydrophobic side chain to better fill the inter-domain space
These variants yielded low specific activity values, possibly resulting from unfavorable changes in conformation, and their characterization was not pursued further. Both variants studied exhibited enhanced inter-A1-C2 domain affinity resulting in increases in the observed stability of the FVIII variants, especially related to thermal denaturation

Summary

EXPERIMENTAL PROCEDURES

Materials—Recombinant FVIII (KogenateTM) and the monoclonal antibodies 58.12 and 2D2 were generous gifts from Dr Lisa Regan of Bayer Corp. (Berkeley, CA). Reactions were immediately quenched by hirudin (10 units/ml) to inactivate thrombin, aliquots were removed at the indicated times, and activity was determined using the FXa generation assay following the addition of FIXa (40 nM) and FX (300 nM). Samples were diluted 1:20 with buffer B containing 20 ␮M PS/PC/PE vesicles, and reconstituted FVIIIa activity was measured directly by FXa generation assay in the absence of the thrombin activation step. Data Analysis—For activity decay analysis of FVIII/FVIIIa, activity values as a function of time were fitted to a single exponential decay curve by non-linear least squares regression using the equation,. Where A is residual FVIIIa activity (nM/min/nM FVIII), A0 is the initial activity, k is the apparent rate constant, and t is the time (minutes) of reaction of FVIII (for FVIII thermal decay experiments) or of FVIIIa after thrombin was quenched (for FVIIIa decay measurements). A Student’s t test was performed for statistical analysis

RESULTS

FVIIIa decay rate

DISCUSSION