Amy E. Griffiths

Factor VIII lacking the C2 domain retains cofactor activity in vitro

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated ΔC2) to characterize the contributions of the C1 domain to function. Binding affinity of the ΔC2 variant to phospholipid vesicles as measured by energy transfer was reduced ∼14-fold. However, the activity of ΔC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions (∼4-fold) were observed in the functional affinity of ΔC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability (∼3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as ∼60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function.

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations

Summary.  Background: Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 112, 2761, 2008). Objectives: In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Methods: Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 °C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L−1) FVIII concentration. Results and conclusions: Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.

Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction.

Summary.  Background: Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods: Reactive epitopes to an immobilized IgG fraction prepared from a high‐titer, FVIII inhibitor plasma were determined after immuno‐precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography‐mass spectrometry (LC‐MS). Results: Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC‐MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues. Conclusions: IP coupled with LC‐MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein–protein and protein–membrane interactions.

Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability.

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.

Factor VIIIa A2 Subunit Shows a High Affinity Interaction with Factor IXa: Contribution of A2 Subunit Residues 707-714 to the Interaction with Factor IXa

Background: FVIIIa binds FIXa through interactions involving A2 and A3C1C2 subunits. Results: A2 subunit shows a high affinity interaction for FIXa with contribution of residues 707-714 to this interaction. Conclusion: Sequences in A2 subunit make significant contributions to binding FIXa. Significance: This study shows an important contribution of A2 subunit in forming FXase and identifies residues participating in this interaction. Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 ± 1.6 nm) that was similar to the A3C1C2 subunit (Kd = 3.6 ± 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. Vmax values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent Kd relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys707, Asp712, and Lys713 demonstrated the greatest increases in apparent Kd (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.

Enhancing factor VIII and VIIIa stability by combining mutations at the A2 domain interface and A1-C2 domain interface.

Factor (F)VIII, a plasma protein that is decreased or defective in individuals with hemophilia A, consists of a heavy chain (HC) comprised of A1A2B domains and a light chain (LC) comprised of A3C1C2 domains (see Ref [1] for review). FVIII is activated by thrombin or FXa to yield FVIIIa, a heterotrimer comprised of subunits designated A1, A2, and A3C1C2, that functions as a cofactor for the serine protease FIXa in the membrane-dependent conversion of zymogen FX to the serine protease, FXa (see Ref. [1] for review). FVIIIa is labile resulting from weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer [2,3] and its dissociation leads to dampening of factor Xase activity. Several FVIII point mutations have been shown to facilitate the rate of dissociation of A2 relative to wild type (WT) and these residues localize to the interface of the A2 domain with either A1 or A3 domains (see Ref [4] for review). Previously, we identified a number of the residues at the A1–A2 and A2–A3 domain interfaces that differentially contribute to the stabilization of FVIII and/or FVIIIa [5]. Subsequent studies identified several mutants at these regions possessing increased stability, and in particular enhanced retention of the A2 subunit in FVIIIa [6]. Based on that study we identified an Asp519Val/Glu665Val variant as the best combination of mutations to maximize FVIIIa stability [7]. Recently, we demonstrated that modifying the A1–C2 interface interaction also enhanced the stability of FVIII [8]. Arg121 at the A1 domain and Leu2302 at the C2 domain (7.73 A separating Cα atoms; PDB#3CDZ) [9]) were chosen for the site of a nascent disulfide bridge formed by mutating these residues to Cys to yield the variant, Arg121Cys/Leu2302Cys. That study also showed that mutation of Ala108 at A1 domain to Ile improved stability by presumably increasing hydrophobic interactions. These FVIII variants possessed 3–4 fold increased FVIII thermal stability [8].

Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit

Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.

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

Essentials Factor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2‐A3 interface. We present evidence for stabilizing interactions between D666‐S1787 and T657‐Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2‐A3 linkage has a FVIIIa decay rate that is 1% of wild‐type.