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INTRODUCTION |
The proteolytically activated form of factor VIII, factor VIIIa,
serves as a cofactor for the serine protease factor IXa in the
conversion of factor X to factor Xa. This complex of enzyme and
cofactor, assembled on an anionic phospholipid surface, is referred to
as the intrinsic factor Xase. The role of factor VIIIa is to increase
the catalytic rate constant (kcat) by several
orders of magnitude. The phospholipid surface is primarily involved in reducing molecular interactions to a two-dimensional space, thereby markedly decreasing the Km for factor X. The
association of factor VIIIa and factor IXa and the mechanism(s) by
which factor VIIIa stimulates reaction rate are not fully understood.
The importance of this interaction is indicated by defects or
deficiency in factor VIII that result in hemophilia A.
Factor VIII circulates as a heavy chain (A1-A2-B domains) and a light
chain (A3-C1-C2 domains) associated in a divalent metal ion-dependent heterodimer. Proteolysis by thrombin yields
factor VIIIa, a trimer of A1, A2, and A3-C1-C2
subunits1 (1, 2). The A1 and
A3-C1-C2 subunits retain the divalent metal ion-dependent
linkage and can be isolated as a stable dimer. Conversely, the A2
subunit is associated with the A1/A3-C1-C2 dimer in a primarily
electrostatic interaction and readily dissociates from the dimer at
physiological pH and ionic strength. However under appropriate reaction
conditions, factor VIIIa can be reconstituted from isolated A1/A3-C1-C2
dimer and A2 subunit (2-4). The affinity of A2 subunit for the
A1/A3-C1-C2 has been measured following functional assay (4, 5) as well
as physical assay employing surface plasmon resonance (6). In human
factor VIIIa, the affinity of A2 subunit for the A1/A3-C1-C2 dimer
(Kd ~260 nM at physiological pH) is
increased 10-fold under slightly acidic conditions (Kd ~30 nM at pH = 6.0) (5).
Little is known about residues in the A2 subunit and A1/A3-C1-C2 dimer
that are involved in the intersubunit interaction. Several lines of
evidence suggest that the C-terminal acidic region of the A1 subunit
(residues 337-372) participates in the retention of A2 subunit
following cleavage at the A1-A2 junction. These observations include
failure of A2 subunit to bind the A1/A3-C1-C2 dimer in which the A1
subunit has been truncated at residue 336 (6, 7).
At least two subunits of the factor VIIIa heterotrimer have been
implicated as possessing factor IXa interactive sites. The factor VIII
light chain-derived A3-C1-C2 subunit likely possesses a high affinity
site for factor IXa. The free light chain of factor VIII shows similar
affinity for factor IXa (Kd ~14 nM, Ref. 8) as is observed for factor VIIIa (Kd ~2-20 nM, Refs. 9 and 10). This suggests little if any of the
binding energy for the interaction is contributed by the factor VIII
heavy chain-derived subunits, A1 and A2. This interactive site was
localized to the A3 domain following studies using inhibition by a
monoclonal antibody whose epitope is represented by residues 1778-1840
(8), and was further localized to within residues 1811-1818 based upon studies employing synthetic peptides (11).
Based upon the identification of an activated protein C cleavage site
at Arg562 in the A2 subunit (7), and the capacity for
factor IXa to selectively protect from cleavage at this site (12), a
factor IXa interactive site in the A2 subunit was postulated. Synthetic peptides spanning residues 558-565 noncompetitively inhibited factor
Xase activity (13). The peptide also blocked the A2
subunit-dependent increase in fluorescence anisotropy of
factor IXa labeled in its active site with fluorescein (14). Taken
together, these results suggest that residues contained in A2 sequence
558-565 are critical to the interaction between cofactor and enzyme.
Recently, we showed that the isolated A2 subunit associates with the
protease in the absence of other factor VIIIa subunits (15). The result
of this interaction is an approximate 100-fold enhancement in the rate
of substrate factor X conversion by a mechanism affecting
kcat rather than Km. This
functional effect is unique to A2 and was not observed with the other
isolated factor VIIIa subunits. However, the magnitude of this effect
is ~1% of that observed for factor VIIIa, indicating contributions of other factor VIIIa subunits to the catalytic rate exist. In this
study we show that A1 subunit alters the interaction of A2 subunit with
factor IXa to synergistically enhance the A2 effect by ~10-fold, thus
yielding a kcat ~10% of that observed for the intact cofactor.
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MATERIALS AND METHODS |
Reagents--
Recombinant factor VIII preparations (KogenateTM)
were a gift from James Brown of Bayer Corp. Purified recombinant factor
VIII also was a generous gift from Debbie Pittman of the Genetics
Institute. The murine monoclonal antibody R8B12, which reacts with the
C-terminal region of the factor VIII A2 domain (7), was prepared as
described previously (2). Monoclonal antibody specific for the
N-terminal region of A1 subunit was also a kind gift from James Brown.
Anti-FVIII337-372 IgG was obtained from a rabbit immunized
with the synthetic peptide consisting of factor VIII residues 337-372
as described previously (16). The reagents
-thrombin, factor
IXa
, factor X, and factor Xa (Enzyme Research Labs);
Fl-FFR-factor IXa2 (Molecular
Innovations), hirudin, and phospholipids (Sigma); and the chromogenic
substrate S-2765
(N-
-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginyl-p-nitroanilide-dihydrochloride; Kabi-Pharmacia) were purchased from the indicated vendors.
Proteins--
The KogenateTM concentrate was fractionated to
separate factor VIII from albumin following S-Sepharose chromatography
(16). Factor VIII was converted to factor VIIIa using thrombin as
described (2). Purification of the A2 subunit and A1/A3-C1-C2 dimer by Mono-S chromatography was as described (17). The A1 and A3-C1-C2 were
prepared from the A1/A3-C1-C2 dimer following dissociation of the dimer
by EDTA and chromatography on Mono-Q (17). In some instances, proteins
were concentrated using a MicroCon concentrator (Millipore, 10 kDa
cut-off). Factor VIII activity was measured by a one-stage clotting
assay using plasma that had been chemically depleted of factor VIII
activity as described previously (18). Protein concentrations were
determined by the Coomassie Blue dye binding method of Bradford
(19).
Factor Xa Generation Assays--
The rate of conversion of
factor X to factor Xa was monitored in a purified system (20). Factor
VIIIa subunits were reacted with factor IXa in 20 mM Hepes,
pH 7.2, 100 mM NaCl, 5 mM CaCl2, and 0.01% Tween (Buffer A) in the presence of 200 µg/ml bovine serum
albumin and 10 µM PS/PC/PE vesicles. The phospholipid
vesicles containing 20% PS, 40% PC, and 40% PE were prepared using
octyl glucoside as described previously (21). Time course reactions were initiated with the addition of factor X (see figure legends for
reactant concentrations). Aliquots were removed at appropriate times to
assess initial rates of product formation and were added to tubes
containing EDTA (80 mM final concentration) to stop the reaction. Rates of factor Xa generation were determined by addition of
the chromogenic substrate, S-2765 (0.46 mM final
concentration). Reactions were read at 405 nm using a
Vmax microtiter plate reader (Molecular Devices).
Data Analysis--
The influence of A1 on the affinity of A2
subunit for factor IXa was determined from the rate of factor Xa
generation as a function of A2 concentration. Data were fitted to a
single site ligand model where, amount bound = capacity × free/Kd + free, using the Marquart algorithm and
UltraFit software (BioSoft). Because the concentration of A2 subunit
was more than double the concentration of factor IXa for all A2 levels,
the value for free A2 used the total A2 concentration. For this reason,
the Kd determined is an apparent
Kd. Using these conditions, the capacity term
reflects the maximal rate enhancement at saturating A2 subunit. Data
from initial rate kinetics were fitted to the Michaelis-Menten equation
(UltraFit) to determine Km and
kcat values.
Fluorescence Anisotropy--
Fluorescence anisotropy
measurements were made using a SPEX Fluorolog 212 spectrometer operated
in the L format. The excitation wavelength was 495 nm (5-nm band pass),
and the emission wavelength was 520 nm (14.4-nm band pass). Reactions
(0.2 ml) were carried out at room temperature in Buffer A containing 30 nM Fl-FFR-factor IXa, 50 µM PS/PC/PE
vesicles, and the indicated concentrations of factor VIIIa (or factor
VIIIa subunits) and factor X in a quartz micro cell. Anisotropy
measurements were made by manually rotating the polarizers and
monitoring fluorescence for 5 s at each position. Fluorescence
intensity determinations (3-5) were made at each position, and the
average value was obtained. Blank readings for the buffer
containing phospholipid were subtracted from all determinations.
Electrophoresis--
SDS-polyacrylamide gel electrophoresis was
performed using the method of Laemmli (22) with a Bio-Rad minigel
electrophoresis system. Electrophoresis was carried out at 200 V for
1 h. Bands were visualized following staining with silver nitrate.
Alternatively, the proteins were transferred to polyvinylidene
diflouride membranes (Bio-Rad, 0.2 µm) using a Bio-Rad mini-transblot
apparatus at 500 mA (constant current) for 30 min in buffer containing
10 mM CAPS, pH 11, and 10% (v/v) methanol. Western
blotting was performed using the indicated antibodies followed by a
goat anti-mouse horseradish peroxidase-conjugated secondary antibody.
The secondary antibody signal was detected using the ECL system
(Amersham Pharmacia Biotech) with luminol as the substrate, and the
blots were exposed to film for various times.
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RESULTS |
Synergistic Effect of A1 and A2 Subunits on the Stimulation of
Factor X Conversion--
In a previous study (15) we showed that
isolated A2 subunit increased the rate of factor IXa-catalyzed
activation of factor X by ~100-fold. Because this
A2-dependent rate increase was fractional compared with
that observed for intact factor VIIIa, we examined the effect of A2 in
combination with other factor VIIIa subunits on cofactor activity.
Titration of A1 subunit in the presence of fixed levels of A2 subunit
yielded marked increases in the rate of factor Xa generation relative
to A2 subunit alone (Fig. 1). In this
experiment, isolated A1 and A2 subunits were recombined in the reaction
mixture for approximately 10 min prior to addition of factor IXa, and
reactions were initiated with addition of factor X. In the absence of
added A2 subunit, the A1 subunit showed no effect on the rate of factor
Xa generation, consistent with our earlier observation (15). In the
presence of a concentration of A2 subunit (50 nM) that was
markedly less than the Kd for the A2-factor IXa
interaction (~300 nM, Ref. 15), we observed little
stimulation in the absence of A1. However, saturating levels of A1
subunit resulted in an approximate 20-fold stimulation in the rate of
substrate conversion. Using a higher level of A2 subunit (400 nM) resulted in a significant stimulation of reaction rate that was further increased ~5-fold in the presence of saturating A1
subunit. Because A1 subunit alone shows no direct factor IXa stimulatory activity, and because the maximal levels of stimulation occur at A1 subunit concentrations in excess of the concentration of
the A2 subunit, these results suggest that the stimulation observed in
the presence of A1 subunit is mediated through its interaction with the
A2 subunit.

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Fig. 1.
A1 subunit stimulates the A2
subunit-dependent enhancement of factor X activation.
Factor Xa generation reactions contained 1 nM factor IXa,
300 nM factor X, 10 µM PS/PC/PE vesicles and
were performed as described under "Materials and Methods" in the
absence (triangles) and presence of 50 nM
(circles) or 400 nM (squares) A2
subunit plus the indicated concentrations of A1 subunit.
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A1 Subunit but Not A3-C1-C2 Subunit Stimulates
A2-dependent Enhancement of Factor X Conversion--
To
determine whether this synergy was specific for the interaction of A2
with A1 or if A3-C1-C2 subunit also enhanced the A2 effect, the
following experiment was performed. A2 subunit was titrated into the
factor Xa generation reaction either alone or in the presence of 1 µM A1 subunit or 1 µM A3-C1-C2 subunit (Fig. 2). In the presence of A1 subunit,
an approximate 7-fold increase in the A2 subunit-dependent
rate of factor X activation was observed. At 1 µM A1
subunit, the level of stimulation of the A2-dependent
effect was similar over the range of A2 concentrations employed in this
analysis, suggesting that fold stimulation of factor Xa generation was
directly dependent upon A2 subunit concentration. This result also
points to the role of A1 in modulating A2 rather than another
component(s) of the reaction. Because the A1 subunit likely provides
the primary contact for A2 subunit in factor VIIIa (5, 16), we
speculate that the affinity for the A1-A2 interaction is approximately
that observed for the interaction of A2 with A1/A3-C1-C2 dimer (~260
nM, Refs. 4 and 5). Thus, the concentration of A1 employed
in this experiment was consistent with near saturation of A2 subunit at
all concentrations. However, titration of A2 subunit in the presence of
1 µM A3-C1-C2 subunit resulted in no incremental increase
in the rate of substrate conversion compared with A2 alone. This result
indicated that the light chain-derived factor VIIIa subunit was
ineffective in modulating the A2-dependent stimulation of
factor IXa-catalyzed activation of factor X.

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Fig. 2.
Effects of A1 and A3-C1-C2 subunits on the A2
subunit-dependent enhancement of factor IXa-catalyzed
activation of factor X. Factor Xa generation assays were performed
as described in the legend to Fig. 1 using the indicated concentrations
of A2 subunit alone (circles) or in the presence of either 1 µM A1 subunit (squares) or 1 µM
A3-C1-C2 subunit (triangles). Data points were fitted to a
single site ligand binding equation as described under "Materials and
Methods."
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Fitting the rate versus concentration data for the three
experimental conditions to a single site ligand binding model was performed to evaluate the functional affinity of A2 subunit for factor
IXa and the effects of the other factor VIIIa subunits on this
parameter. In the absence of other additions, the A2 interaction with
factor IXa yielded a functional (apparent) Kd of 377 ± 112 nM, similar to the value of 314 ± 89 nM obtained in our earlier study (15). A2 subunit in the
presence of 1 µM A1 or A3-C1-C2 revealed similar affinity
values (Kd = 410 ± 130 and 447 ± 194 nM, respectively). Thus the presence of a saturating level
of A1 subunit did not significantly alter the affinity of A2 subunit
for factor IXa. Interestingly, A3-C1-C2 subunit, which itself possesses
relatively high affinity for factor IXa, did not alter the (functional)
affinity of A2 for the enzyme. This result is consistent with the
failure to observe direct interaction between these two factor VIIIa
subunits. Using the capacity term of the ligand binding equation as an
indicator of maximal rate enhancement, we obtained an ~7-fold
increase in the presence of A1 (19.4 ± 2.9 nM factor
Xa/min/nM factor IXa) compared with its absence (3.2 ± 0.4 nM factor Xa/min/nM factor IXa). Taken
together, these data suggest that the A1 subunit of factor VIIIa
modulates the A2 subunit, altering its interaction, but not its
affinity, with factor IXa and resulting in an approximate order of
magnitude increase in the rate of substrate conversion.
Effects of A1 Subunit on Kinetic Parameters for A2
Subunit-dependent Factor Xa Generation--
In the
previous study, we showed an increase in the
kcat for factor Xa conversion from 0.013 min
1 to 0.98 min
1 in the absence and
presence of 600 nM A2, respectively, (15). A further
increase in this parameter of approximately 10-fold was observed with
A2 plus A1 subunits (Fig. 3). The value
for kcat obtained from the fitted curve
(13.7 ± 1.6 min
1) is approximately 5-10% that
obtained with intact factor VIIIa (200 min
1, Ref. 15).
Inclusion of A1 subunit had no significant effect on the
Km for factor X (107 ± 28 nM)
compared with this value in the absence of A1 subunit (101 ± 28 nM, Ref. 15), consistent with the primary effect of A1 in
modulating the activity of the A2 subunit.

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Fig. 3.
Effects of A1 and A2 subunits on the kinetics
of factor Xa generation. Reactions were run as described under
"Materials and Methods" using 600 nM each A1 and A2
subunits, 1 nM factor IXa, and the indicated concentrations
of factor X. Initial rates of factor Xa generation were plotted as a
function of substrate concentration and fitted to a Michaelis-Menton
equation using UltraFit software. Extracted values for
Km and kcat were 107.4 ± 27.9 nM and 13.7 ± 1.6 min 1,
respectively.
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Inhibition of the A1-dependent Enhancement by
Anti-factor VIII337-372 Antibody--
The association of
A2 subunit with A1 appears to be mediated through the A1 C-terminal
acidic region. In an earlier study, we showed that a polyclonal
antibody prepared to this sequence (residues 337-372) inhibited the
reassociation of A2 subunit with the A1/A3-C1-C2 dimer (16). An
experiment was performed to determine whether this reagent affected the
A1-dependent enhancement of the A2 effect. In the absence
of antibody, we observed an ~3-fold rate increase for the reaction
mixture supplemented with A1 subunit (Fig.
4). Inclusion of the IgG resulted in a
dose-dependent elimination of this A1-dependent
enhancement of factor Xa generation in the presence of A2 subunit. In
the absence of A1 subunit, the IgG showed no effect on A2 stimulation
of factor Xa generation. This result indicated that disruption of the
interaction between A1 and A2 subunits eliminated the synergy observed
in the presence of the two subunits and supported the notion that the
effect of A1 subunit in this assay is to directly modulate A2
subunit.

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Fig. 4.
Effect of anti-FVIII337-372
IgG on factor Xa generation in the presence of A1 and A2
subunits. Indicated levels of the IgG were reacted with A2 subunit
(120 nM) in the absence (circles) and presence
(squares) of A1 subunit (70 nM) for 2 h at
room temperature. Factor Xa generations were performed following
addition of factor IXa (1 nM) and factor X (300 nM).
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Reconstitution of Factor VIIIa from Isolated
Subunits--
Although A2 in the presence of A1 subunit results in a
significant increase in kcat for factor Xa
generation, this value reflects ~10% of the increase observed with
factor VIIIa. To ensure that the reagents employed retained the
potential to yield native cofactor-like activity, factor VIIIa was
reconstituted from the three isolated subunits in a two-step reaction
and evaluated in a factor Xa generation assay. In the first
reconstitution step, the A1/A3-C1-C2 dimer was prepared following
overnight reaction of the individual subunits in either a Ca(II) or
Ca(II)/Cu(II) buffer. The products of these reactions were then mixed
with A2 subunit prior to addition of factor IXa, and the reactions were
initiated following addition of the substrate factor X. As shown in
Fig. 5, authentic factor VIIIa-like
activity (>100 nM factor Xa/min/nM factor IX)
required reassociation of the three individual subunits. Although
Ca(II) alone was sufficient to promote formation of significant factor VIIIa activity, the presence of Cu(II) during the association of A1 and
A3-C1-C2 subunits enhanced the specific activity of the cofactor by
~2-fold, consistent with our earlier findings (23). The factor VIIIa
reconstituted under these conditions showed a similar
kcat (~160 nM factor
Xa/min/nM factor IXa) as observed earlier for native factor
VIIIa (~200 nM factor Xa/min/nM factor IXa,
Ref. 15). This result indicated that (i) the subunits employed in this
study were functionally capable of generating the native cofactor, and
(ii) A2 and A1 subunits are insufficient to yield the maximal
kcat effect, identifying an essential role for
the A3-C1-C2 in contributing to this kinetic parameter.

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Fig. 5.
Reconstitution of factor VIIIa from isolated
A1, A3-C1-C2, and A2 subunits in the absence and presence of
Cu(II). The A1/A3-C1-C2 dimer was prepared following
reconstitution of A1 and A3-C1-C2 (500 nM each) overnight
at 4 °C in Buffer A supplemented with 10 mM
CaCl2 in the absence (squares) or presence
(circles) of 10 µM CuCl2. Factor
VIIIa was reconstituted in a reaction containing 50 nM of
either dimer preparation plus indicated levels of A2 subunit. Factor Xa
generation assays were performed as described under "Materials and
Methods."
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Fluorescence Analysis of the Factor VIIIa Subunit-Factor IXa
Interaction--
This active site modulating activity of A1 in
combination with A2 was further investigated for effects on the
fluorescence anisotropy of Fl-FFR-factor IXa (Table
I). In the previous report we observed
that the presence of A2 subunit resulted in a modest increase in the
fluorescence anisotropy of Fl-FFR-factor IXa (
r = 0.015), suggesting that A2 subunit effects the orientation of the
factor IXa active site (15). Furthermore, the increase in anisotropy of
Fl-FFR-factor IXa plus A2 in the presence of saturating factor X
(
r = 0.086) was significantly greater than the
factor X-dependent effect observed for factor IXa alone
(
r = 0.044), suggesting that this factor VIIIa
subunit made a significant contribution to the orientation of the
active site of the enzyme relative to the substrate. We now show that
inclusion of both A1 and A2 subunits yielded further increases in
anisotropy values obtained in the absence (
r = 0.041) and presence (
r = 0.073) of factor X. These results suggested that the increased rotational constraints imposed in
the presence of both factor VIIIa subunits reflects the functional synergy observed in factor Xa generation rates. Furthermore, these values approach those observed with the native factor VIIIa (Ref. 14;
Table I), suggesting a gradient to the factor VIIIa
subunit-dependent increases in anisotropy that parallels
the subunit-dependent effects on catalytic function.
Isolated A1 Subunit Is Not a Substrate for Cleavage by Factor
IXa--
The above data demonstrate the A1 subunit contributes to
cofactor activity when combined with A2. However, unlike A2 subunit, the A1 subunit is also a substrate for factor IXa cleavage at Arg336 (24, 25). The A1 subunit is cleaved by factor IXa at
an appreciable rate (~0.2-0.5 min
1, Ref. 24) in the
A1/A3-C1-C2 dimer and in a phospholipid-dependent reaction.
This rate is increased ~3-fold for the factor VIIIa substrate,
suggesting a contribution of A2 subunit to this reaction (24). The
following experiment was performed to determine whether the isolated A1
subunit remains a substrate for factor IXa and, if so, whether cleavage
rate is influenced by the presence of A2 subunit. A1 subunit (300 nM) in the absence or presence of A2 subunit (300 nM) was reacted with factor IXa (20 nM) in the presence of PS/PC/PE vesicles. Samples were removed during the time
course and assayed for both factor Xa generating activity and A1
subunit composition, the latter by blotting with an antibody that
detects both intact and cleaved A1 subunit. The results of this
experiment, shown in Fig. 6, indicate
that the A1 contribution to A2-dependent activity remained
relatively stable during the course of the reaction, retaining >80%
of the initial activity level after a 2-h reaction with factor IXa.
Consistent with this retention of activity, no detectable proteolysis
of A1 was observed either in the absence or presence of A2 subunit.
Alternatively, near total cleavage of A1 subunit in the A1/A3-C1-C2
dimer was observed following a 10-min reaction under near identical
reaction conditions. These results show a dissociation of the cofactor from substrate activities of A1 relative to factor IXa and indicate the
importance of A3-C1-C2 subunit in defining A1 as substrate. Thus,
whereas A3-C1-C2 subunit is needed for maximal cofactor activity, this
subunit appears essential for factor IXa to recognize A1 as
substrate.

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Fig. 6.
A, stability of the
A1-dependent stimulation of A2 cofactor activity. The A1
and A2 subunits (300 nM each) were reacted with 20 nM factor IXa in buffer A containing 200 µg/ml bovine
serum albumin and 100 µM PS/PC/PE vesicles. At the
indicated times, aliquots were removed and residual factor Xa
generating activity was determined following addition of factor X to
300 nM. B, A1 subunit as a substrate for factor
IXa. Western blotting using the 58.12 anti-A1 N terminus monoclonal
antibody was performed as described under "Materials and Methods."
Blot a shows samples obtained from the reaction described in
panel A above. Blot b is a similar reaction run
in the absence of A2 subunit. Blot c is a reaction
containing 100 nM A1/A3-C1-C2 dimer in place of the
isolated factor VIIIa subunits and under the conditions described
above. Arrowhead identifies the A1 subunit cleaved at
Arg336. Lanes 1-6 indicate time points at 0, 10, 40, 60, 90, and 120 min, respectively.
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DISCUSSION |
In the present study we demonstrate a significant contribution of
A1 subunit to the A2-dependent stimulation of the catalytic activity of factor IXa toward factor X. The role of A1 appears to
modulate the A2 subunit which in turn alters the interaction of enzyme
with substrate. This effect is suggested by the ~10-fold increase in
kcat for the A2-dependent effect in
factor Xa generation, whereas little if any change in A2 affinity for
factor IXa is observed in the presence of A1 subunit. That A1 modulates
A2 rather than factor IXa and/or factor X is suggested by several
observations including (i) the lack of any contribution to catalytic
rate in the presence of A1 subunit alone, (ii) a requirement for a
stoichiometric excess of A1 relative to A2 subunit, and (iii) the
inhibition of the A1-dependent contribution to activity by
an antibody that disrupts the interaction between the two factor VIIIa
subunits. These results implicate the A1 subunit as contributing to the cofactor function of factor VIIIa through an indirect mechanism relative to enzyme in the intrinsic factor Xase.
Relatively little information is available on the interactions between
A2 and other factor VIIIa subunits. The affinity of A2 for A1/A3-C1-C2
is both pH (5, 26) and ionic strength (2) sensitive, with slightly
acidic conditions increasing affinity by ~10-fold (5). The
dissociation rate constant (~0.35 min
1) is about
3-fold faster for the human protein than the porcine
material (4). A2 subunit exhibits little if any affinity for the
A3-C1-C2 subunit based upon failure of the latter subunit to inhibit
reassociation of A2 with A1/A3-C1-C2 in a functional assay. This result
is supported by the present study showing lack of effect of A3-C1-C2 on
the A2-dependent stimulation of factor IXa activity even
though the A3-C1-C2 possesses high affinity for the enzyme (8).
Conversely, A1 subunit was demonstrated to effectively inhibit the
association of A2 subunit with the A1/A3-C1-C2 dimer in that near
equivalent concentrations of A1 and dimer yielded 50% inhibition of
factor VIIIa reconstitution (5). Thus A2 likely interacts primarily
with A1 subunit in the factor VIIIa heterotrimer.
The C-terminal region of A1 subunit appears critical for the
association of A2 subunit. In an early experiment, it was observed that
activated protein C-catalyzed cleavage of the A1/A3-C1-C2 dimer at
Arg336 yielded a truncated version of the dimer
(A1336/A3-C1-C2), which was ineffective in competing with
the native dimer for A2 association (7). Similarly, the truncated dimer failed to bind A2 subunit as judged by surface plasmon resonance (6).
Subsequent studies showed direct interaction of the A2 subunit with a
fluorescently labeled peptide corresponding to A1 C-terminal residues
337-372 (16) and localized a covalent linkage formed by the
zero-length cross-linker, ethyl-dimethylaminopropylcarbodiimide, between the C-terminal region of A1 and the N-terminal half of A2 (27).
Finally, antibody prepared to a synthetic peptide corresponding to the
acidic residues 337-372 blocked the reconstitution of factor VIIIa
activity from A2 plus dimer, presumably by interfering with the
inter-subunit interaction (16). This immunologic reagent also blocked
the contribution of A1 subunit to the A2-dependent stimulation of factor IXa activity, consistent with the role of the A1
C-terminal region in representing an A2 interactive site.
The above data support the notion that A1 stimulation is mediated
through its interaction with A2. However, the mechanism by which A1
modulates the "cofactor" activity of A2 subunit toward factor IXa
remains to be determined. One possibility is that A1 alters A2
conformation, and this alteration potentiates the cofactor role without
altering affinity for the enzyme. Support for such a conformation
change is suggested by changes in the affinity of the apolar dye,
bisanilinonapthalsulfonic acid, for a surface-exposed hydrophobic
pocket in the free A2 subunit compared with the A2 domain/subunit in
factor VIII/factor VIIIa (28). In addition, thrombin cleavage of the
factor VIII heavy chain results in the formation of a new salt linkage
between A1 and A2 subunits (27) that could yield a conformation change
in the latter.
An alternative mechanism of A1 subunit directly modulating substrate
factor X appears unlikely, but cannot be discounted. The anisotropy of
Fl-FFR-factor IXa was increased in the presence of the A1/A3-C1-C2
dimer, and a further increase was observed in the presence of factor X
(29). However, these effects may result in part from contribution of
the A3-C1-C2 to factor IXa binding. Recently we showed that A1 subunit
possesses an interactive site for factor X that is also contained
within the C-terminal acidic region (17). The affinity for this
interaction (Kd ~1-3 µM, (17)) is
markedly lower than the concentration of factor X used in the factor Xa
generation reactions performed in the present study. Furthermore, the
degree of stimulation of the A2 effect by A1 subunit was independent of
factor X concentration. Finally, the presence of A1 subunit did not
affect the Km for factor X. Taken together with the
inability of isolated A1 subunit alone to enhance the rate of factor X
conversion, these observations argue against this factor VIIIa subunit
substantially affecting the enzyme-substrate interaction by an
A2-independent mechanism.
The cofactor activity resulting from the presence of both A1 and A2
subunits yields an ~1000-fold increase in the
kcat for factor IXa-catalyzed conversion of
factor X compared with factor IXa alone. This magnitude increase is
~10% of that observed in the presence of intact factor VIIIa. The
reason for the disparity does not appear to reflect inactive/defective
subunits resulting from the isolation procedures because native-like
factor VIIIa activity can be reconstituted from the isolated A1,
A3-C1-C2, and A2 subunits. Thus the difference in
kcat values obtained for A1 plus A2
versus intact factor VIIIa may be attributed to
contributions by the A3-C1-C2 subunit. This result is consistent with
observations showing that thrombin cleavage of the light chain actually
contributes to the specific activity of factor VIIIa (30, 31).
The A3-C1-C2 subunit also appears to be responsible for the factor
VIIIa-dependent reduction in Km for
factor X. Km values for factor X are similar for
reactions run with factor IXa alone (121 nM; Ref. 15),
factor IXa plus A2 (101 nM; Ref. 15) and factor IXa plus A2
and A1 subunits (107 nM; this study). However, the
Km for factor X interactions run with intact factor
VIIIa (33 nM; Ref. 15) is reduced by severalfold. This
effect is likely dependent upon the phospholipid surface binding
properties localized within the C2 domain of this subunit (32).
Finally, A3-C1-C2 appears to be critical for physiological interactions
of factor VIIIa with factor IXa. First, this subunit contains a high
affinity site for factor IXa (Kd~14
nM; Ref. 8) that approaches the affinity of factor VIIIa
for the enzyme (Kd ~2 nM; Ref. 9). As
noted earlier, the affinity of A2 subunit for factor IXa is markedly
weaker (Kd ~300 nM; Ref. 15), and as
determined in the present report, this value is unchanged by the
presence of the A1 subunit. Second, the capacity for factor VIIIa to be
recognized as substrate for factor IXa (that is, cleaved at
Arg336 in the A1 subunit) appears dependent upon A3-C1-C2
and influenced by A2. Earlier studies showed that factor IXa cleaved
intact factor VIIIa ~3-fold faster than A1/A3-C1-C2, implying a role
for A2 in the rate increase (24). This result is not surprising given the recent observation of A2 enhancing factor IXa-catalyzed cleavage of
substrate factor X. However, in the absence of A3-C1-C2, we observe no
detectable cleavage of A1 subunit independent of the presence of A2
subunit and at concentrations that approach near maximal levels for the
effects of these isolated subunits on factor Xa generating activity.
This observation suggests A3-C1-C2 is likely to be required for proper
orientation of A1 subunit for cleavage by factor IXa in the
surface-dependent reaction. This orientation of A1 imposed
by A3-C1-C2 may also be required for maximal cofactor activity. Thus
the restriction in factor IXa proteolytic activity toward A1 subunit
may also account for the sub-maximal rates of factor X conversion in
the presence of A1 and A2 factor VIIIa subunits compared with the
intact cofactor.