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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0361.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biochemistry and Biophysics and
the Department of Medicine, University of Rochester Medical Center, NY.
The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms
a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIIIWT) and B-domainless FVIII
containing the hemophilia A-associated mutations Ser558Phe, Val559Ala,
Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant
Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the
mutant and wild-type proteins had similar functional affinities for
FIXa (dissociation constant [Kd]
values ~5 nM-20 nM and ~100 nM-250 nM in the presence and absence
of phospholipid, respectively). The catalytic activities of the factor
Xase complex composed of the hemophilia A-associated FVIII species
were markedly reduced both in the presence and absence of phospholipid.
FVIIIWT and FVIIIArg562Ala showed catalytic
rate constant (kcat) values of approximately 60 minute The generation of a fibrin clot in response to
vascular injury is mediated by the regulated and sequential activation
of a series of serine proteases and their cofactors.1 The
glycoprotein factor VIII (FVIII) in its activated form, FVIIIa, acts as
a cofactor to the serine protease FIXa, in the conversion of the
zymogen FX to the active enzyme (FXa). Both FVIII and FIX are essential for normal coagulation; deficiencies of either are associated with the
bleeding diatheses hemophilia A and hemophilia B, respectively.
FVIII is synthesized as a 2332-residue single-chain glycoprotein,
composed of 3 distinct domain types in the arrangement (NH2) A1-A2-B-A3-C1-C2 (COOH).2,3 As a result of intracellular proteolysis at the B-A3 junction plus additional sites within the B
domain, FVIII circulates as a heterodimer of a variable length heavy
chain (A1-A2-B domains) and light chain (A3-C1-C2).4-6 FVIII is activated by thrombin cleavage at residues 372 (A1-A2 junction) and 740 (A2-B junction) in the heavy chain, and residue 1689 in the light chain.7 The resulting FVIIIa is a trimer composed of A1, A2, and A3-C1-C2 subunits.8,9
On activation FVIIIa forms a stoichiometric complex with FIXa. This
complex is formed on a phospholipid surface and requires the presence
of Ca++.10 The effect of phospholipid is to
limit the interactions to 2 dimensions, thereby reducing the
Kd for the FVIIIa-FIXa interaction and the
Km for substrate factor X.11 The
effect of FVIIIa is to increase the catalytic rate constant
(kcat) of FX conversion to FXa by several orders
of magnitude.12 The mechanisms by which FVIIIa acts as a
cofactor for FIXa remain unclear. In contrast to the isolated A1 and
A3-C1-C2 subunits, the isolated A2 subunit can stimulate FIXa, though
this ability is fractional as compared with the intact
FVIIIa.13 The Ser558-Gln565 region within the A2 subunit
has been shown to be critical for VIIIa-IXa
interaction.14,15 This region contains the activated
protein C (APC) cleavage site at Arg562.16 Interaction of
FVIIIa with FIXa selectively protects this site from APC cleavage,
suggesting that there is a direct interaction between FVIIIa and FIXa
in this region.17 This has been confirmed by peptide
studies. Synthetic peptides spanning the 558-565 residues of FVIIIa
noncompetitively inhibit tenase activity.14 Fluorescence
anisotropy studies of FIXa labeled with the fluorophore at the active
site crevice demonstrate that the 558-565 peptide blocked the increase
in anisotropy contributed by the A2 subunit of FVIIIa on interaction of
the labeled FIXa, indicating the importance of this region in the
A2-FIXa interaction.15
Mutations within the 558-565 region resulting in hemophilia A are
described in the hemophilia A mutation database.18 The mutations Ser558Phe, Val559Ala, Asp560Ala, and Gln565Arg are described as cross reactive material-positive (CRM+), that is, having normal levels of FVIII antigen but defective FVIII activity associated with
mild hemophilia A, indicating functional defects of secreted protein.
In order to determine the mechanism by which the 558-565 region
contributes to the cofactor activity of FVIIIa, we have stably expressed and partially purified FVIII forms containing the
mutated residues within this region. In addition, we have expressed and purified FVIII containing an Arg562Ala mutation. Using a functional assay, we demonstrate that the effect of the mutations associated with
hemophilia A is not to alter the affinity of FVIIIa for FIXa but rather
to directly affect the catalytic rate constant of the complex, and thus
the effect of the mutations is to reduce the cofactor potential of
FVIII. This conclusion is supported by fluorescence anisotropy data
showing defective interaction of Fl-FFR-IXa with mutant FVIIIa in the
presence of FX. In contrast, the Arg562Ala mutation has no effect on
either Kd or kcat,
indicating that the conservation of this residue is not critical for
cofactor activity.
Reagents
The B-domainless FVIII (B-FVIII) expression construct RENeoFVIII was a
gift kindly provided by Dr Pete Lollar. Baby hamster kidney
(BHK) cells were obtained from American Type Culture Collection (Manassas, VA). Fetal bovine serum (FBS) was obtained from Gemini Bioproducts (Woodland, CA). All other reagents used for the BHK cell
culture were obtained from Gibco BRL (Gaithesburg, MD).
Plasmid mutagenesis
DNA transfection and FVIII expression The FVIII expression constructs were stably transfected in BHK cells by liposome-mediated transfection. Plasmid DNA (1 µg) was coated with lipofectamine-plus reagent and added to the cells in serum-free Dulbecco modified Eagle medium-F12 (DMEM-F12). After incubation at 37°C, 5% CO2 for 3 hours, the cells were incubated in 10% FBS in DMEM-F12. Selection was initiated 24 hours after transfection by addition of geneticin (500 µg/mL) to the serum-supplemented DMEM-F12. Geneticin-resistant colonies were picked and expanded by plating, and FVIII expression levels determined by enzyme-linked immunosorbent assay (ELISA). The highest expressing colonies were grown and subclones prepared by limiting dilution. These were assayed for FVIII expression levels and high-expressing subclones were transferred and expanded, and aliquots frozen until required.Approximately 27 × 106 cells were transferred to a sterile roller bottle, incubated in serum-supplemented DMEM-F12 overnight, and rotated at approximately 4 rpm. The following day the medium was replaced with 100 mL serum-free AIM-V and the bottles rotated at approximately 4 rpm at 30°C and 5%CO2 (Gibco BRL). The conditioned medium was collected after 24 hours and replaced. Conditioned medium was collected daily for a total of 4 days. Protein purification and concentration After daily collection the conditioned medium was spun at approximately 2500g at 4°C for 10 minutes to pellet cell debris. The supernatant was then decanted, and the protein was precipitated with ammonium sulfate (40% wt/vol) overnight at 4°C. The supernatant/salt mix was centrifuged at 10 000g, 4°C for 10 minutes and the supernatant decanted. The ammonium sulfate precipitate was resuspended in 0.1 M NaCl-buffer A (20 mM MES [2-(N-morpholino) ethanesulfonic acid], pH 6.0, 5 mM CaCl2, 0.01% Tween-20) at one-tenth the original supernatant volume. The collections were pooled and dialyzed overnight in 0.2 M NaCl-buffer A (2 L) with 2 changes of buffer. FVIII was partially purified from the dialyzed protein solution after application to an SP sepharose ion-exchange column. After adsorption onto the column the protein was washed with 10 column volumes of dialysis buffer and eluted with 0.8 M NaCl-buffer A. Protein concentration of each fraction was determined by the Coomassie dye binding method of Bradford,21 and FVIII content determined by one-stage clotting assay. The partially purified FVIII was further concentrated and purified after overnight dialysis in 0.1 M NaCl-buffer B (20 mM Hepes, pH 7.5, 5 mM CaCl2, 0.01% Tween-20) and applied onto a Q-sepharose column. After application, the column was washed with 10 column volumes of 0.1 M NaCl-buffer B, and then 10 column volumes of 0.2 M NaCl-buffer B. FVIII was eluted with 0.8 M NaCl-buffer B. Protein and FVIII content were determined as described above, and the partially purified FVIII dialyzed overnight with 0.1 M NaCl-buffer B. FVIII activity and antigen were determined by one-stage clotting assay and ELISA, respectively.FVIII activity and antigen measurement One-stage FVIII assays were performed in chemically depleted FVIII-deficient pooled human plasma. Alternatively, FVIII activity was determined following the rate of conversion of FX to FXa using a purified system in the presence or absence of phospholipid. FVIII wild type and mutants were initially activated in 0.1 M NaCl-buffer B, 100 µg/mL bovine serum albumin, by the addition of thrombin (10 nM) in the presence of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine (PSPCPE) vesicles (10 µg/mL), and FIXa (5 nM). Thrombin activity was inhibited after 1 minute by the addition of hirudin (2.5 U/mL). The conversion of substrate FX to FXa was initiated by the addition near Vmax (maximum kinetic velocity) levels of FX (~300 nM). Aliquots were removed after an appropriate time to assess initial rates of FXa production and added to tubes containing ethylenediaminetetraacetic acid (EDTA) (80 mM final concentration). Rates of FXa generation were measured by the addition of the chromogenic substrate S-2765 (0.46 mM final concentration) and the reactions read at 405 nm using a Vmax microtiter plate reader (Molecular Devices, Royal Oak, MI). Factor Xa generation reactions in the absence of phospholipid were performed in 50 mM NaCl-buffer B, 100 µg/mL bovine serum albumin, as described above. Reactions were initiated by the addition of 1 µM FX.FVIII antigen was measured by sandwich ELISA. Each well was coated overnight with 1 µg of ESH-8 monoclonal antibody (20 µg/mL, 0.15 M Na2CO3, 0.035 M NaHCO3; pH 9.6). The wells were washed with phosphate buffered saline (PBS)-0.01% Tween 20, and coated in block solution (5% nonfat dry milk dissolved in PBS-0.01% Tween 20). On removal and further washing of the plate, 50 µL of samples or standard was added and incubated at room temperature. Standards were comprised of purified FVIII of known concentration varying from 1 µg/mL to 15.6 ng/mL. After washing, 1 µg of biotinylated R8B12 (20 µg/mL) was added to each well. On further incubation and washing, horseradish peroxidase-labeled strepavidin (Calbiochem, San Diego, CA) was added to each well. After incubation and washing, the reaction was visualized by the addition of substrate containing 1 mg/mL O-phenylenediamine dihydrochloride (Sigma). The reaction was stopped by addition of 2 M H2SO4, and the absorbance measured at 490 nm using a Vmax microtiter plate reader. Electrophoresis and western blotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli,22 using a Bio-Rad minigel system (Hercules, CA). Electrophoresis was performed at 200 v for 40 minutes and protein bands were visualized using Coomassie blue staining. Proteins were transferred onto polyvinylidenefluoride (PVDF) membrane using a Bio-Rad minitransblot apparatus at 0.2 A overnight in a buffer containing 10 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), pH 11, and 10% (vol/vol) methanol. Western blotting was performed using the primary antibodies indicated followed by goat anti-mouse horseradish peroxidase-conjugated secondary antibody. The secondary antibody signal was produced using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL) with luminol as substrate, and the blot exposed to film for times varying between 1 minute and 10 minutes.Fluorescence spectroscopy Fluorescence anisotropy measurements were made using an Amico-Bowman series 2 spectrometer equipped with automatic polarizers arranged in an L-format (Spectronic Instruments, Rochester, NY). Reactions were performed at room temperature in 0.1 M NaCl-buffer B containing 100 µg/mL bovine serum albumin. Reactions contained 50 nM Fl-FFR-IXa, 200 nM FVIIIa, 50 µg/mL PSPCPE vesicles, in the absence or presence of 500 nM FX. FVIII was activated by the addition of thrombin (20 nM) for one minute, and the thrombin then inhibited by the addition of hirudin (5 U/mL). Samples were excited at 495 nm, and the emission intensity was monitored at 520 nm (4-nm band pass) for 2 seconds at each polarizer position. Each reaction was read a total of 20 times in each position, and 3 reactions performed for FVIIIWT and each mutant. Anisotropy values were calculated automatically after subtraction of blank readings and data then averaged for each measurement.Data analysis Data were fitted using the single-site ligand binding model where amount bound = (capacity × free)/(kd + free), using the Marquart algorithm and computed using UltraFit software (v3.04; BioSoft, Ferguson, MO). As the concentration of FVIIIa was more than the FIXa for all FVIIIa levels, the value for free factor VIIIa used the total FVIIIa concentration, therefore the Kd determined is an apparent Kd. Probability (P) values were determined using the Student t test.
Expression and purification of recombinant factor VIII Mutations of the 558-565 loop of FVIIIa were constructed in the expression vector RENeoFVIII and expressed in BHK cells. Between 100 µg and 300 µg of expressed protein was purified from approximately 1 L conditioned medium. The material obtained from the chromatographic steps employed was more than 30% pure as judged by gel electrophoresis and Coomassie staining (results not shown). The major contaminating protein was albumin.The FVIII obtained from each mutant and wild type was fully activated
by thrombin. A time-course analysis of FVIIIa activity generation
demonstrated peak activity at 1 minute to 2 minutes, with a 10- to 20- fold increase in activity of FVIIIa as compared with the preactivated
levels (data not shown). Western blotting analysis of the intact and
thrombin-cleaved FVIII forms using an anti-A2 domainal monoclonal
antibody is shown in Figure 1. The intact
protein demonstrated prominent high-molecular-weight bands (> 130 000) consistent with expression of B-FVIII as a single chain. A
less-abundant band of approximately 90 000 represented the contiguous
A1-A2 domains of the FVIII heavy chain, consistent with its absence
following blotting with an anti-A3 domain-specific antibody (results
not shown). Low (variable) levels of an approximately 45 000 band
representing the A2 domain was observed, suggesting some degradation
and/or activation of the B-FVIII. All B-FVIII forms exhibited similar
peptide patterns following cleavage by thrombin, consistent with the
potentiation of activity. High-molecular-weight bands (> 90 000) were
converted to bands of approximately 45 000 and 50 000, representing
the A2 subunit forms. The origin of the higher Mr A2 band is unclear,
but may represent additional glycosylation and/or partial extension of
residues at its C-terminal end.
Comparison of factor activity and antigen The profiles of the expressed proteins and of the database entries are summarized in Table 1. Of the 5 mutants, 4 were associated with mild to severe hemophilia A. B-domainless wild-type FVIII demonstrated an FVIII activity-antigen ratio of approximately 1.57, higher than the expected value of unity. One possibility that this value is greater than unity may reflect low levels of activation in the FVIII preparation. Alternatively, this value may represent an underestimate in the concentration of FVIII protein. FVIIIArg562Ala also demonstrated a ratio greater than unity (1.86), further indicating that the mutation at residue 562 does not have a deleterious effect on FVIII function. In contrast, all the mutations associated with hemophilia A had reduced FVIII Ac/Ag ratios ranging from 2% to 10% of activity as compared with antigen, consistent with the available database entries.
Cofactor activity of wild-type and mutant FVIIIa forms FVIIIa-mediated generation of FXa was performed in a purified system with varying concentrations of each of the expressed FVIII mutants and wild type, and fixed concentrations of FIXa. Experiments were performed both in the presence and absence of PSPCPE as described with near Vmax levels of FX. In all experiments FVIII was converted to FVIIIa by the addition of thrombin for 1 minute, followed by inhibition of thrombin by addition of hirudin.In the presence of PSPCPE, FVIIIWT and
FVIIIArg562Ala demonstrated similar abilities to act as
cofactors for FIXa-catalyzed generation of FXa. Similar levels of FXa
were generated with increasing FVIIIa levels and saturation of FIXa was
observed at similar FVIIIa concentrations (Figure
2A). This was reflected in the derived functional affinity and catalytic constants. FVIIIWT and
FVIIIArg562Ala yielded similar kcat
values of 64.2 minute
Surface-independent experiments were performed in the absence of PSPCPE
in order to evaluate the direct protein-protein interaction of FVIIIa
with FIXa. Experiments were performed at low salt concentration (50 mM
NaCl) in order to minimize the dissociation of the FVIIIa A2 subunit
from the A1/A3-C1-C2 dimer.23 The ability of the FVIIIaWT-FIXa complex to convert FX to FXa was considerably
reduced in the absence of PSPCPE. The Kd and
kcat values for FVIIIWT of 133.5 nM
minute
Similar to results obtained in the presence of surface, the mutation at residue 562 did not markedly affect the relative ability of FVIIIArg562Ala to act as a cofactor in the generation of FXa in the absence of phospholipid. Similar FXa levels were generated upon increasing FVIIIaArg562Ala concentration and the kcat derived approached that obtained for wild-type FVIIIa. FXa generated in the presence of saturating FVIIIaSer558Phe, FVIIIaVal559Ala, FVIIIaAsp560Ala, or FVIIIGln565Arg was fractional as compared with FVIIIWT and FVIIIArg562Ala. The determined kcat values for FVIIISer558Phe, FVIIIGln565Arg, and FVIIIVal559Ala were reduced to approximately 3% of that for FVIIIWT, whereas that for FVIIIAsp560Ala was approximately 13% of that of FVIIIWT. Taken together, these results demonstrate that FVIII molecules possessing mutations within the 558-565 loop region form FVIIIa-FIXa complexes with marked defects in transducing this binding event to yield enhanced enzymatic activity of FIXa. However, with the exception of FVIIIaGln565Arg, all the mutants form complexes with wild-type-like affinities for FIXa. FVIIIaGln565Arg demonstrated moderately reduced ability to form the FVIIIa-FIXa complex in the presence of phospholipid, but this reduction is marginal in comparison with its inability when in complex with FIXa to promote increased catalytic activity. Effect of mutations in the 558-565 region of FVIII on fluorescence anisotropy of Fl-FFR-FIXa The above results indicate that mutations in the 558-565 loop region modulate the cofactor effect of FVIIIa. In order to gain additional insights into the mechanism(s) for altered function of the mutant proteins, local topographical effects of the FVIIIa-dependent modulation of the FIXa active site crevice were assessed by fluorescence anisotropy. Reactions were performed in the presence of PSPCE, both in the absence and presence of FX. As the fluorophore is attached via a tripeptide to the active site Ser, any changes in anisotropy are a direct reflection of changes in the local conformation of the active site cleft of FIXa, as detected by altered rotational freedom of the fluorophore.Results in Table 3 demonstrate the change
in anisotropy on addition of FVIIIa in the absence or presence of
further addition of FX. In the presence of FVIIIaWT, there
was an increase of 0.033 in Fl-FFR-IXa anisotropy as compared with
Fl-FFR-IXa alone. Variable increases ranging from 0.021 to 0.064 were
detected upon addition of the mutated FVIIIa forms. The significance of
these differences for the cofactor-enzyme interaction is unclear, but
suggests that amino acid substitutions in this region differentially
modulate the mobility of the active site-specific probe upon binding
of cofactor to enzyme.
Upon addition of FX to the complex of Fl-FFR-IXa and FVIIIaWT, there was an increase in anisotropy of 0.072 as compared with FX added to Fl-FFR-FIXa alone. In contrast, only the reaction containing FVIIIaArg562Ala yielded a similar increase in anisotropy observed on addition of FX. No increase to a similar level of FX-dependent anisotropy was detected in any of the hemophilia A-associated mutant FVIIIa-FIXa interactions, although this value was approached with FVIIISer558Phe (0.061). These results suggest that there is modulation of the FIXa active site region by the mutant FVIIIa forms. However, with the exception of FVIIIaArg562Ala, which possesses essentially wild-type activity, this modulation is ineffective in presenting the optimum conformation for FIXa-FX interaction in the catalytic region of FIXa.
The 558-565 region of FVIII is a conserved region. It is identical across canine, murine, and porcine FVIII-A2 domains,24-26 and shares sequence conservation across homologous A domains.27 The 558-565 loop of activated FVIII has been shown by various studies to be an FIXa interactive site. Initial studies demonstrated that FIXa interaction selectively protected FVIIIa from cleavage by APC at Arg562.17 Further studies demonstrated that interaction with FIXa could be blocked by peptides spanning the 558-565 region, as measured by FXa generation assays and by the loss of the A2-dependent increase in fluorescence anisotropy.14,15 In addition to being an FIXa interactive site and containing an APC cleavage site, the 558-565 site interacts with heparan sulfate proteoglycans as demonstrated recently by peptide analysis.28 Thus the FVIIIa Ser558-Gln565 loop represents a key interactive region of FVIIIIa. Its importance is highlighted by the association of missense mutations with hemophilia A resulting in defective FVIII molecules.29 By stable expression and subsequent partial purification of 558-565 mutant proteins we have been able to perform kinetic studies on the effects of mutations in this region on the interaction of FVIIIa with FIXa and subsequent generation of FXa. The mutant proteins FVIIISer558Phe, FVIIIVal559Ala, FVIIIAsp560Ala, and FVIIIGln565Arg, all demonstrated defective FVIII function in one-stage clotting assays to 2% to 10% of activity as compared with FVIII antigen. This reduction in activity was not due to defective cleavage of FVIII to FVIIIa by thrombin, as activity and gel analysis showed full activation of FVIII. The expressed mutant proteins reproduced the patient plasma-based assays, confirming that the mutations directly cause hemophilia A as a result of the reduced ability of secreted FVIII to act as a cofactor for FIXa. Assays performed with varying concentrations of FVIIIa in the presence of phospholipid and fixed concentrations of FIXa and FX showed reduced FXa generation. This effect was directly due to impaired cofactor activity of FVIIIa as the kcat for the FXase complexes were reduced for hemophilia A-associated mutants by 5% to 12% as compared with wild type. In contrast, the affinity of cofactor forms for FIXa was similar for mutants FVIIISer558Phe, FVIIIVal558Ala, and FVIIIAsp560Ala (Kd values from 6.2 nM-12.4 nM) as compared with FVIIIWT (5.4 nM), whereas FVIIIGln565Arg showed a modest increase in Kd (20.9 nM). Previous analysis by Amano et al30 performed on transiently expressed FVIII containing the Ser558Phe mutation led to the conclusion that the likely effect of the mutation was to affect FVIIIa-FIXa interaction. This was based on an indirect determination by competition of a peptide corresponding to the 558-565 sequence with mutant FVIIIa for FIXa interaction. Therefore, while a proportionally greater reduction in mutant cofactor activity was observed as compared with wild type, no direct determination of either interprotein affinity or catalytic rate constants was performed. Our results demonstrate that the interaction of FVIIIa with FIXa and ability to form a complex were not substantially affected by any of the mutations in the presence of phospholipid, whereas the catalytic activity of the tenase complex was greatly impaired by each mutation associated with CRM+ hemophilia A. Factor VIIIa binds to phospholipid with high affinity, with reported
Kd values ranging between 10 Titration of FVIIIaWT and FVIIIaArg562Ala in
FXa generation experiments in the absence of phospholipid with fixed FX
and FIXa concentrations gave similar Kd and
kcat values to those previously reported of 90 nM minute Fluorescence anisotropy measurements of interactions of Fl-FFR-IXa with wild-type and mutant FVIIIa in the absence and presence of FX lends further support to the data obtained by the functional assays. In the absence of FX, inclusion of all FVIII forms yielded increased anisotropy values compared with FIXa alone, suggesting formation of cofactor-enzyme complexes. The variability in these values suggests that changes in composition of the 558-565 loop directly influence constraints imposed on the active site label. Of particular interest are results obtained in the presence of FX. Earlier studies by Lollar et al42 showed that presence of substrate FX had little influence on the anisotropy of Fl-FFR-FIXa alone, but a marked increase was observed in the complete system consisting of surface-bound enzyme, cofactor, and substrate. Under these conditions, we observed maximal increases in anisotropy with FVIIIWT and FVIIIArg562Ala, consistent with maximal rates of FXa generation, suggesting that constraints on fluorophore rotation may represent an indicator of catalytic activity. Interestingly, under these conditions a significant increase in anisotropy was obtained for the FVIIISer558Phe mutation. We speculate that the presence of the bulky Phe residue may in itself contribute to rotational constraints in the fluorophore in the complex of enzyme, cofactor, and substrate. In addition to FVIII residues 558-565 comprising an interactive region with FIXa, a FIXa-interactive region has been identified in the A3 domain of the light chain.43 Inhibition studies by monoclonal antibody demonstrated the location of a high-affinity FIXa interactive site within residues Gln1778-Asp1840, and subsequent peptide inhibition studies have localized the minimum sequence required for this interaction to Glu1811-Lys1818.40 The Kd for the FVIIIa light chain-FIXa interaction is similar at 14 nM to the intact FVIIIa. Thus, interaction of FIXa likely occurs with 2 regions of FVIIIa, via the high-affinity binding site with FVIIIa light chain, and the much lower affinity site within the A2 domain. The finding that mutations within the 558-565 loop region do not greatly affect FIXa binding supports the hypothesis that in the presence of phospholipid the binding of FVIIIa and FIXa is a critical first step followed by high-affinity binding involving the FVIIIa A3 domain to FIXa.44 The low-affinity binding of FVIIIa A2 to the serine protease domain, and the effect of mutations in the 558-565 loop on the kcat of the complex support the proposal that this region is key in modulating FIXa activity. The residues 330-339 of the serine protease of FIXa represent an FVIIIa-interactive site,45,46 and more recent studies have demonstrated that this region interacts with the 558-565 loop of FVIIIa.41 The presence of hemophilia B mutations in this region also demonstrates the importance of these residues.45 As a result of studies of wild-type and mutant FIXa interaction with the isolated A2 domain of FVIIIa, an interface model of the surface of the A2 domain and FIXa protease domain has been proposed.41 In this model, FVIIIa residues Asp560, Gln561, and Arg562 are shown as interacting with Arg338 and Asp332 of FIXa, respectively. The moderately increased Kd for FVIIIAsp560Ala in the absence of phospholipid suggests that Asp560 has a role in stabilizing the interaction in this region, but that in the presence of phospholipid this interaction is not a major contribution to the binding energy of FVIIIa in its interaction with FIXa. The comparable Kd for FVIIIArg562Ala and FVIIIWT both in the presence and absence of phospholipid suggests that any conservation of FVIIIaArg562 interaction with Asp332 of FIXa is not critical in maintaining either the interaction with FIXa or modulating cofactor activity of FVIIIa. Substitution of Arg562 has previously been shown not to cause a defect in cofactor activity.47 The lack of effect of the Arg562 substitution on FVIII cofactor activity is analogous to the FVArg506Gln, referred to as factor V Leiden. The Leiden mutation is the most common inherited defect associated with venous thrombosis as a result of resistance to APC cleavage, but the substitution does not affect FVa cofactor activity.48 Similarly, the substitution of Arg562 affects cleavage by APC.47 Hemophilia B-associated mutations within the helical 330 region of FIXa have been expressed and interaction with subsaturating levels of FVIIIa studied by 2 separate groups.45,46 In one study,45 8 separate residue substitutions were expressed and purified. In 7 of the 8, the result was decreased cofactor-mediated, FIXa activity, attributed primarily to the result of an increased Kd of FVIIIa-FIXa interaction of between 10- and 100-fold as compared with wild-type FIXa. In another report,46 2 residues within the 330 helix, FIX-Arg333 and FIX-Leu337, were substituted with Gln and Phe, respectively, which differed from the Leu and Ile substitutions made in the prior study. In addition to the residue substitutions, these investigators also substituted the residues FIXa 333-339 for the corresponding FX sequence. Results from these experiments indicated that the point mutations caused a decrease in FIXa-catalyzed FXa generation as compared with FIXa wild type. However, this effect resulted from a decrease in kcat for the tenase complex to between 3% and 4% of wild type, with a relatively small increase in Kd detected. The differing mechanisms indicated above may reflect the different mutations studied, and their effect on this highly structured region. In contrast to the FIXa 330-339 region that contains a helical secondary structure, the FVIII 558-565 region is likely an exposed loop region containing a number of highly exposed side chains.49 Substitutions within this region are not predicted to affect secondary structure. Our data suggest that the primary feature of this region is that it is critical in modulating cofactor activity through direct interaction near the active site of FIXa, rather than making substantial contribution to the binding energy with FIXa per se. The mutations associated with hemophilia A lead to a defective ability to act as cofactor for FIXa, and thus a dramatically reduced ability to generate FXa.
The authors thank Dr Pete Lollar and John Healey for kind provision of the RENeoB-FVIII construct and many helpful discussions. The authors acknowledge the input of Dr Noelene Quinsey and Jennifer Chandler in the early stages of this project.
Submitted December 28, 2001; accepted February 1, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0361.
Supported by grants HL 30616 and HL 38199 from the National Institutes of Health and a Judith Graham Pool Postdoctoral Award to P.V.J. from the National Hemophilia Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Philip J. Fay, Department of Biochemistry and Biophysics, PO Box 610, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642; e-mail: philip_fay{at}urmc.rochester.edu.
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© 2002 by The American Society of Hematology.
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  I. Jagannathan, H. T. Ichikawa, T. Kruger, and P. J. Fay Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa J. Biol. Chem., November 20, 2009; 284(47): 32248 - 32255. [Abstract] [Full Text] [PDF]
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J. L. Newell and P. J. Fay Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., April 24, 2009; 284(17): 11080 - 11089. [Abstract] [Full Text] [PDF]
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M. Steen, S. Tran, L. Autin, B. O. Villoutreix, A.-L. Tholander, and B. Dahlback Mapping of the Factor Xa Binding Site on Factor Va by Site-directed Mutagenesis J. Biol. Chem., July 25, 2008; 283(30): 20805 - 20812. [Abstract] [Full Text] [PDF]
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J. L. Newell and P. J. Fay Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., August 31, 2007; 282(35): 25367 - 25375. [Abstract] [Full Text] [PDF]
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A. J. Gale, S. Yegneswaran, X. Xu, J.-L. Pellequer, and J. H. Griffin Characterization of a Factor Xa Binding Site on Factor Va near the Arg-506 Activated Protein C Cleavage Site J. Biol. Chem., July 27, 2007; 282(30): 21848 - 21855. [Abstract] [Full Text] [PDF]
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F. Varfaj, H. Wakabayashi, and P. J. Fay Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C J. Biol. Chem., July 13, 2007; 282(28): 20264 - 20272. [Abstract] [Full Text] [PDF]
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K. Nogami, Q. Zhou, H. Wakabayashi, and P. J. Fay Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site Blood, June 1, 2005; 105(11): 4362 - 4368. [Abstract] [Full Text] [PDF]
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K. Nogami, Q. Zhou, T. Myles, L. L. K. Leung, H. Wakabayashi, and P. J. Fay Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain J. Biol. Chem., May 6, 2005; 280(18): 18476 - 18487. [Abstract] [Full Text] [PDF]
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R. J. Kerschbaumer, K. Riedrich, M. Kral, K. Varadi, F. Dorner, J. Rosing, and F. Scheiflinger An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex J. Biol. Chem., September 24, 2004; 279(39): 40445 - 40450. [Abstract] [Full Text] [PDF]
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K. Nogami, K. A. Lapan, Q. Zhou, H. Wakabayashi, and P. J. Fay Identification of a Factor Xa-interactive Site within Residues 337-372 of the Factor VIII Heavy Chain J. Biol. Chem., April 16, 2004; 279(16): 15763 - 15771. [Abstract] [Full Text] [PDF]
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H. Wakabayashi, J. Freas, Q. Zhou, and P. J. Fay Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity J. Biol. Chem., March 26, 2004; 279(13): 12677 - 12684. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1 in the presence of phospholipid, whereas
the hemophilia A-associated mutants showed
kcat values ranging from 3.3 minute
-thrombin, factor IXa
, factor X, factor Xa
(Enzyme Research Laboratories, South Bend, IN); Fl-FFR-factor IXa (Molecular Innovations, Southfield, MI); and hirudin,
phosphotidylethanolamine (PE), phosphotidylcholine (PC), and
phospholtidylserine (PS) (Sigma, St Louis, MO) were purchased from the
indicated vendors. Phospholipid vesicles composed of 40% PE, 20% PS,
and 20% PC were prepared using octyl glucoside as previously
described.
) and FVIIIArg562Ala
(
). (B) Data generated for FVIIISer558Phe (
),
FVIIIVal559Ala (
), FVIIIAsp560Ala (*),
FVIIIGln565Arg (
). The data were plotted and fitted
using a single-site ligand binding model as described in "Materials
and methods."
