(Received for publication, July 13, 1995; and in revised form, October 17, 1995)
From the
The intrinsic factor Xase complex (FXase) is comprised of a
serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a
phospholipid surface. Activity of FXase decays with time and reflects
the lability of FVIIIa. Two mechanisms potentially contribute to this
decay: (i) a weak affinity interaction between the FVIIIa A2 subunit
and A1/A3-C1-C2 dimer and (ii) FVIIIa inactivation resulting from
FIXa-catalyzed proteolysis of the A1 subunit. At low reactant
concentrations (0.5 nM FVIIIa; 5 nM FIXa), FXase
decay is governed by the inter-FVIIIa subunit affinity and residual
activity approaches a value consistent with this equilibrium, as judged
by reactions containing exogenous A2 subunit. Analysis using a mutant
form of FVIII (FVIII) possessing an altered FIXa
cleavage site, showed similar rates of FXase decay (0.12
min
) and confirmed the lack of contribution of
proteolysis under these conditions. When the concentration of FIXa was
increased 10-fold, the initial rate of decay of FXase containing native
FVIIIa increased (0.82 min
) and paralleled the rate
of proteolysis of A1 subunit. However, the rate of decay of FXase
containing the FVIIIa
was reduced (0.048
min
) consistent with the elevated concentration of
FIXa stabilizing the labile subunit structure of the cofactor.
Reconstitution of FVIII with FIXa-cleaved light chain showed that
cleavage at the alternate FIXa site (A3 domain) was not inhibitory to
FXase. The presence of substrate FX resulted in a 10-fold reduction in
the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest
a model whereby decay of FXase results from both FVIIIa subunit
dissociation and FIXa-catalyzed cleavage, dependent upon the relative
concentration of reactants, with greater contribution of the former at
low values and, in the absence of substrate, greater contribution of
the latter at high values.
FVIII ()and FIX are essential plasma glycoproteins
that when absent or defective, result in hemophilia A and B,
respectively. The proteolytically activated forms of these proteins;
FIXa, a serine protease, and FVIIIa, a protein cofactor, form a
Ca
- and surface-dependent complex referred to as the
intrinsic FXase complex, that efficiently converts zymogen FX to FXa
(see (1) for review). The role of FVIIIa in this complex is to
increase the k
for this reaction by several
orders of magnitude(2) .
FVIIIa is a heterotrimer composed
of the A1, A2, and A3-C1-C2 subunits. ()The A1 and A3-C1-C2
subunits retain the Me
ion linkage responsible for
the association of the heavy and light chains in heterodimeric factor
VIII and can be isolated as a stable dimer(3, 4) . The
A2 subunit is weakly associated with the dimer (K
= 260 nM; Refs. 5 and 6) primarily through
electrostatic interaction (7) and at physiologic pH readily
dissociates resulting in the loss of FVIIIa
activity(5, 6, 7, 8) . Under the
appropriate reaction conditions, FVIIIa activity can be reconstituted
from the isolated A2 subunit and the A1/A3-C1-C2 dimer (6, 7, 8, 9, 10) .
Association of FVIIIa with FIXa in the presence of phospholipid and
Ca stabilizes cofactor activity(11) .
Furthermore, reconstitution of heterotrimeric FVIIIa from the isolated
A2 subunit and A1/A3-C1-C2 dimer is enhanced severalfold in the
presence of FIXa and phospholipid(9) . While prolonged
interaction of FVIII(a) with FIXa results in a loss of FVIII(a)
activity due to proteolytic cleavage within the A1
domain/subunit(12, 13) , stable enhancement of FVIIIa
reconstitution can be achieved in the presence of active site-inhibited
FIXa(9) . Thus FIXa exhibits the capacity to modulate FVIIIa
activity, thereby affecting the catalytic efficiency of FXase.
FXase
activity is labile, whereas FVIIIa-independent conversion of FX by FIXa
is relatively stable. In this report we examine the FVIIIa-dependent
lability of FXase, which has been referred to as a
``self-damping''(14) . Experiments assess the
relative contributions of FVIIIa subunit dissociation and
FIXa-catalyzed proteolysis of FVIIIa to the decay of FXase activity.
This study was aided by use of a mutant FVIII molecule,
FVIII, resistant to FIXa-catalyzed cleavage at the A1
site. Results of this analysis yield a model for the intrinsic
instability of this enzyme complex.
which represents the reversible dissociation of FVIIIa to A2 and
A1/A3-C1-C2 subunits. In this and following steps, FVIIIa (or
A1/A3-C1-C2) is bound to the PL surface. The rate constants, k and k
, are 0.32
min
and 1.3
10
M
min
,
respectively(6) , and the K
is 260
nM(5) . The steady state concentration of FVIIIa was
solved by quadratic equation as determined
from,
where [A2] = [A1/A3-C1-C2] in the
absence of exogenous A2 and [FVIIIa] is the
initial concentration of FVIIIa following activation and equals the
initial concentration of FVIII, [FVIII]
.
The interaction of FIXa with FVIIIa is given by,
where the K = 23 nM(22) is equivalent to the interaction of FIXa with
A1/A3-C1-C2 dimer (11 nM; (23) ) and is assumed to be
equivalent for the interaction with the mutant FVIIIa
form. The concentration of FIXa-FVIIIa-PL is calculated using a
(single site) ligand binding
curve,
where [FIXa-FVIIIa-PL] = B
= [FVIIIa-PL]
and
[FIXa]
[FVIIIa]. The reversible dissociation
of FVIIIa subunits in the presence of FIXa is given
by,
At low FVIIIa concentrations, the inter-FVIIIa subunit
interaction is stabilized by the presence of FIXa and
phospholipid(11, 12) . Furthermore, preliminary data
suggest the association rate constants in the absence (k) and presence (k
) of FIXa
are similar. (
)Thus we assume that k
> k
. Because reactions use low (sub
nM) concentrations of FVIIIa, the contribution of the
association rate constants (k
and k
) for the dimer/A2 subunit interaction is
negligible (e.g. at 1 nM FVIIIa, k
= 0.0013 min
) so only
the dissociation rate constants need to be considered in assessing
rates of FXase decay. Therefore, under these
conditions,
where k` is the experimentally observed
dissociation rate constant for FVIIIa at a given concentration of FIXa.
The denominator of the equation represents 1 + a stabilization
factor. This factor, which we define as the ratio of dissociation
rate constants in the absence (k
) and presence (k
) of FIXa multiplied by the mole fraction of
FVIIIa in complex with FIXa, retards the dissociation rate constant for
FVIIIa (k
) and thus would reduce the overall rate
of FXase decay. As [FIXa] approaches 0, the quantity
[FIXa-FVIIIa-PL]/[FVIIIa-PL]
, which is
calculated from , approaches 0 and k
`
equals k
. From the above equation the calculated
value for k
= 0.039 ± 0.003
min
(n = 4) was determined over a
range of FIXa levels (5-50 nM) using the mutant
FVIIIa
so that FXase decay was solely attributed to
subunit dissociation.
A final consideration is FXase decay resulting
from proteolysis. Cleavage of FVIIIa at Arg by FIXa (12) inactivates the cofactor by markedly reducing the affinity
of A2 for the truncated A1
/A3-C1-C2 dimer(4) .
Therefore, proteolytic inactivation can be represented
by,
where k is the rate constant for
proteolysis at a given concentration of FIXa.
Thus, the observed rate of FVIIIa-dependent decay of FXase for a given concentration of FIXa can be represented as a sum of the contributing rate constants as follows,
Figure 1:
Time-dependent decay of FXase activity. A, reactions contained 5 nM FIXa, 100 µg/ml
phospholipid, and 0.3 units/ml FVIIIa in the absence (circles) or presence (squares) of 100 nM A2
subunit. Reactions were initiated with addition of FIXa and aliquots
were removed at the indicated times and assayed for FXa generating
activity. Data were fitted to a single exponential decay with offset as
described under ``Materials and Methods.'' Decay rates and
offset values were 0.106 ± 0.017 min
and 6.4
nM FXa/min and 0.084 ± 0.005 min
and
19.5 nM FXa/min for reactions run in the absence and presence
of exogenous A2, respectively. B shows a plot of offset value (circles) and calculated FVIIIa concentration (squares, from ) determined for the indicated
exogenous A2 concentrations.
In order to further assess the role of
FVIIIa subunit dissociation in the decay of FXase, a similar reaction
was performed with a high level (100 nM) of exogenous A2
subunit added at the initiation of the reaction (Fig. 1A,
squares). This condition was predicted to reduce the rate and/or
extent of FVIIIa dissociation, thereby increasing cofactor stability.
While the presence of the added A2 subunit resulted in a similar rate
of decay (0.084 ± 0.014 min), the offset
value was significantly increased (
20 nM/min). This
suggested that while the dissociation rate constant of FVIIIa still
remained much faster than the association rate constant, as the time
course progressed, a new equilibrium was established reflecting the
higher offset in response to the exogenous A2. Fig. 1B shows that the offset, as measured from FXase decay curves in the
absence and presence of several levels of exogenous A2 (primary data
for intermediate A2 concentrations not shown), is linearly related to
the concentration of A2 over the concentrations used. This result would
be expected for all A2 subunit levels below the K
for the A2-dimer interaction (260 nM; (5) and (6) ) if subunit dissociation were a primary reason for the
decay of FXase. Furthermore, the offset values correlated well with the
calculated concentrations of FVIIIa (from ). These results
suggested that at the above FVIIIa and FIXa concentrations employed,
proteolysis was not a factor in loss of FXase activity.
Figure 2:
Western blot of FVIIIa forms following
reaction with FIXa. FVIIIa was prepared from native FVIII (15
units/ml) and the FVIII
mutant (
5 units/ml)
following reaction with thrombin (3 nM) for 2 min and
subsequent inhibition of the thrombin with hirudin. FIXa (100
nM) and phospholipid (50 µg/ml) were added to each
reaction and aliquots were removed at 2 and 30 min, subjected to
SDS-polyacrylamide gel electrophoresis, transferred, and probed with an
anti-A1 subunit monoclonal antibody as described under ``Materials
and Methods.'' Lanes 1-3 and 4-6 represent reactions before and at 2 and 30 min after addition of
FIXa to FVIIIa
and wild type FVIIIa, respectively. The
high molecular weight band likely represents residual heavy chain
(contiguous A1-A2) not cleaved by thrombin. Note that this band also
persists in the mutant FVIIIa preparation, whereas it disappears with
time in the wild type FVIIIa preparation.
Reactions similar to those shown in Fig. 1A were performed using equivalent activity levels (based upon the
one-stage clotting assay) of the FVIII mutant (Fig. 3). Results showed that the decay rates in the two
reactions (0.120 ± 0.044 min
and 0.126
± 0.046 min
for the absence and presence of
100 nM exogenous A2 subunit, respectively) were equivalent and
similar to the values observed with the wild type FVIII. Furthermore,
the offset value in the presence of A2 was about twice that observed in
its absence. Since the A1 subunit of this material is not cleavable by
FIXa, the above results, taken together, indicated that at low
FVIIIa/FIXa concentrations, loss of FXase activity is primarily caused
by dissociation of FVIIIa subunits with little if any contribution of
proteolysis to FXase decay.
Figure 3:
Decay of FXase containing
FVIIIa. Reactions were as described in the legend to Fig. 1and run in the absence (circles) and presence (squares) of 100 nM A2 except that the mutant FVIIIa
replaced the native form. Decay rates (single exponential) and offset
values were 0.120 ± 0.044 min
and 7.1 nM FXa/min and 0.126 ± 0.046 min
and 12.1
nM FXa/min for reactions run in the absence and presence of
exogenous A2, respectively.
An additional series of experiments,
using the same level of FVIIIa as above but with a 10-fold increase in
the concentration of FIXa (50 nM), was evaluated, since the
potential for high ratios of protease to cofactor are physiologically
possible based upon their relative plasma concentrations. Results are
presented in Fig. 4and show decay of FXase composed of either
wild type FVIIIa (circles) or FVIIIa (squares). The rate of decay of FVIII
is
actually reduced by a factor of
3-fold (0.048 ± 0.022
min
) compared with the low FIXa situation (Fig. 3). This reduction in FXase decay rate suggests the high
FIXa level promotes FVIIIa subunit association and verifies the concept
of the stabilization factor described in . On the
other hand, the rate of decay of FXase containing wild type FVIIIa was
evaluated using a double exponential decay curve to extract rate
constants for proteolysis (k
) and subunit
dissociation (k
`) for that concentration of FIXa.
Values of 0.819 and 0.046 min
were obtained. The
latter was equivalent to the above value obtained with the mutant
molecule suggesting this represented the decay rate attributed to
subunit dissociation; whereas the former value was increased by
8-fold compared with that observed with the lower FIXa level (Fig. 1A). These results suggest that at high
stoichiometries of FIXa relative to FVIIIa, proteolysis contributes
significantly to the decay of FXase, outweighing any inter-FVIIIa
subunit stabilizing activity.
Figure 4:
Decay of FXase containing wild type and
mutant FVIIIa in the presence of high FIXa. Reactions contained
0.3 units/ml wild type (circles) or FVIII
mutant (squares) FVIIIa, 100 µg/ml phospholipid, and
50 nM FIXa and were run as described under ``Materials
and Methods.'' Decay rates for the wild type FVIIIa used a double
exponential curve fit to account for contributions of proteolytic
inactivation and subunit dissociation. The fitted constants were 0.819
± 0.181 min
and 0.046 ± 0.007
min
. Decay rate for the mutant FVIIIa was fitted to
a single exponential and yielded a value of 0.048 ± 0.022
min
.
Figure 5: Thrombin activation of reconstituted FVIII forms. FVIII heterodimers (25 nM) isolated from the Mono S column were reacted with thrombin (1 nM). Aliquots were removed at the indicated times and assayed for activity using a one stage assay. Circles represent the authentic FVIII, and squares represent FVIII reconstituted from native heavy chain and FIXa-cleaved light chain.
On the other hand, inclusion of FX had a marked affect on the
cleavage of FVIIIa (Fig. 6). In these experiments, FVIIIa (50
nM), FIXa (70 nM), and phospholipid vesicles (100
µg/ml) were incubated in the presence and absence of FX. Aliquots
were removed at the indicated times, run on a gel, and Western blotted
with the anti-A1 subunit monoclonal antibody. In the presence of FX (A), the A1 subunit was rapidly cleaved to a fragment of size
consistent with cleavage at Arg. However, after a few
minutes, both bands disappeared suggesting that the FXa generated by
FXase further cleaved the A1 to small fragments and/or within the
epitope such that no products were detected. To eliminate the effect of
generated FXa, a similar reaction was performed using the specific FXa
inhibitor, TAP(24) . Thus any FXa generated would be
sequestered from FVIIIa. Sufficient TAP was included to result in a t
of inhibitor-substrate formation of less than
1 s(25) . This concentration of TAP did not effect the rate of
FIXa-catalyzed proteolysis of FVIIIa in the absence of FX (0.519
min
) as shown in B. The bottom panel (C) shows the reaction performed in the presence of FX
plus TAP. Scans of this blot show that the presence of FX reduced the
rate at which FVIIIa A1 subunit was cleaved by about 10-fold (0.050
min
). This result suggested that the
(FXa-independent) damping of FXase via proteolysis is dependent upon
the substrate availability such that FIXa-catalyzed proteolysis of A1
is reduced when FX is present.
Figure 6:
Effect of FX on FIXa-catalyzed proteolysis
of FVIIIa. All reactions contained FVIIIa (50 nM), FIXa (70
nM), and phospholipid (100 µg/ml). Additional components
were: A, FX (400 nM); B, TAP (1
µM), and C, FX plus TAP. Reactions were run and
blotted as described under ``Materials and Methods.'' Lanes 1-9 represent time points at 0, 1, 2, 4, 7, 10,
15, 25, and 40 min after FIXa addition. Data from densitometric scans
were analyzed using a single exponent decay. Rates of conversion of the
A1 subunit to the A1 fragment were 0.519 ± 0.046
min
and 0.051 ± 0.025 min
,
for reactions shown in B and C,
respectively.
In this report, we present a model to evaluate the
instability of the intrinsic FXase complex. A scheme summarizing the
reaction pathways is shown is Fig. 7. The FVIIIa-dependent decay
of FXase arises from both the weak affinity interaction between the
A1/A3-C1-C2 dimer and A2 subunit and FIXa-catalyzed proteolysis of the
A1 subunit. The relative contribution of either mechanism is dependent
upon the level of FIXa present. Rate constants for dissociation of
FVIIIa into subunits (k, k
)
predominate at low reactant concentrations. At high relative FIXa
concentrations, while dissociation of subunits is minimized by a
FIXa-dependent stabilizing activity, FVIIIa inactivation by proteolysis
of A1 subunit (k
) predominates. Thus, the presence
of FIXa has profound effects on FVIIIa stability/activity. These
effects have been controversial and can now be related to the above
model.
Figure 7:
Reaction pathways in the formation and
degradation of FXase. Association of all FVIIIa and A1/A3-C1-C2 dimer
forms with PL is implicit. Rate constants for the FIXa-dimer
interaction (k`, k
`) are
assumed to be equivalent to those for the FIXa-FVIIIa interaction (k
, k
). The constant k
may be severalfold greater than k
` from comparison of cleavage of the two
substrates(12) .
Several years ago, Jesty (14) observed that the rate
and yield of FXa formed by the intrinsic FXase decreased with
increasing FIXa concentration and a constant level of FVIIIa. He
suggested that, while the most probable cause for the damping of FXase
activity was the spontaneous decay of the labile FVIIIa, this was
likely not the only consideration since the rate constant for FXase
decay varied with FIXa concentration. At lower FIXa concentrations
there was a tendency toward a minimum decay rate of 0.2
min
, whereas this value was as high as 0.94
min
at high levels of FIXa. That high concentrations
of FIXa result in an acceleration of the decay of FXase (14, 26) is compatible with FIXa-catalyzed proteolysis
of FVIIIa indeed contributing to the instability of this complex.
Results presented in this study show similar rates of proteolysis of A1
subunit (0.51 min
; Fig. 6B) and the
contribution to FXase decay by proteolysis (0.82
min
, Fig. 4). Furthermore, it has been argued
that because of the proteolytic activity of FIXa toward FVIIIa, FIXa
does not contribute to FVIIIa stabilization(13) . However, this
conclusion was based upon examination of only very high reactant
concentrations (
100-200 nM).
Lollar et al.(11) determined that at low FVIIIa (<1 unit/ml) and FIXa (5 nM) levels and in the presence of a phospholipid surface, porcine FVIIIa was stabilized from spontaneous decay. This effect was also observed with an active site-modified FIXa (27) . Thus, these investigators concluded that association of FVIIIa with the other components of FXase markedly reduced the lability of the cofactor. Furthermore, the observed decay of porcine FVIIIa in that study was slower than that of the human protein (14, 22, present study). The enhanced stability of porcine VIIIa compared with human FVIIIa is consistent with a 3-fold greater dissociation rate constant for the latter(6) .
The effect of FIXa in the enhancement of
FVIIIa reconstitution from isolated subunits clearly supports the role
for a stabilizing activity. Reconstitution analyses typically use
higher FVIIIa and FIXa concentrations (10-40 nM)
compared with assays where FVIIIa is generated from in situ activation of FVIII by thrombin. For this reason, the
FIXa-dependent enhancement of FVIIIa reassociation is transient in the
presence of native enzyme, with decay correlating with proteolysis of
A1 subunit(9) , while active site-modified FIXa yields a
greater -fold enhancement of FVIIIa reconstitution that is relatively
stable.
Use of a mutant form of FVIII, FVIII,
allowed us to assess the degree to which FIXa modulates the stability
of the labile FVIIIa trimer, since FXase containing this FVIIIa
molecule decays via a nonproteolytic mechanism. Comparison of this
decay with the predicted first order dissociation rate constant for
FVIIIa (k
) allowed us to derive an expression for
the extent of added stability conferred by association with FIXa on the
PL surface, based upon the product of mole fraction of FVIIIa complexed
and the ratio of k
/k
.
Saturating FIXa would maximally inhibit this decay by
9-fold (k
/k
= 8.2). Recently,
it was observed that the first order decay of human FVIIIa (2
nM) was reduced 8-fold with saturating FIXa (10
nM)(22) . This -fold reduction would be predicted from
our model as a result of maximal reduction of FVIIIa dissociation and
little or no contribution of proteolysis to decay as a consequence of
low reactant concentration. Furthermore, this value is similar to the
maximal enhancement (8-10-fold) of FVIIIa reconstitution we
observe in the presence of active site-modified
FIXa(9, 28) .
The mechanism by which FIXa stabilizes the labile FVIIIa heterotrimer is not known. A high affinity site for FIXa has been localized to the FVIII light chain, possibly within the A3 domain(28) . Furthermore, a peptide comprised of A2 residues 558-565 inhibits the FIXa-dependent enhancement of FVIIIa reconstitution (29) as well as the A2-dependent contribution to the FVIIIa-dependent fluorescence anisotropy of fluorescein-Phe-Phe-Arg-labeled FIXa(23) . Thus FIXa likely tethers sites within the A1/A3-C1-C2 dimer and the A2 subunit.
Prolonged reaction with FIXa results in cleavage of FVIIIa. Results
from this study suggest that relatively high concentrations of FIXa are
required for efficient proteolysis. The reason for this is not clear
but may suggest that the scissile bonds within the cofactor are not
readily accessible to the bound enzyme and/or that cleavage results
from FIXa molecules other than the one complexed with FVIIIa. The site
cleaved by FIXa that correlated with FVIIIa inactivation was identified
as Arg in A1 domain following N-terminal sequence
analysis of a FIXa-generated FVIII fragment(12) . Previously,
we proposed a mechanism for inactivation as a result of cleavage at
this site(4, 7) . This cleavage would liberate the
acidic C-terminal region of the A1 subunit (residues 337-372),
which is involved in A2 subunit retention (30) following
thrombin cleavage at Arg
(31) in the FVIII heavy
chain.
In a recent study, Neuenschwander and Jesty (32) measured decay of thrombin-activated FVIII in the presence
of FIXa using a continuous FXa generation assay which contained
acetylated-FX, a modified zymogen that is activated to a form of FXa
that does not react with FVIII(a) substrates(33) . It was shown
that a plot of 1/kversus [FIXa] was linear with a positive slope. Thus high
levels of FIXa reduced the apparent decay of cofactor activity from
0.3 to
0.06 min
. This result is consistent
with the results presented in the current study, since we also find
that the presence of substrate (plus an inhibitor of the generated
product) reduced the contribution of k
to FXase
decay by 10-fold, while the high levels of FIXa would modulate (reduce)
the apparent dissociation rate constant (k
`
parameter).
At high FVIIIa/FIXa concentrations and in the presence
of the generated FXa, the FVIIIa A1 subunit was cleaved at
Arg. This site represents a proposed FXa cleavage site (30) as well as a FIXa site (12) and thus would be
inactivating. Additional proteolysis of A1 was observed to be
FXa-dependent and suggested multiple sites in this subunit. This
further proteolysis was observed following several minutes in the FXa
generation time course. Thus, in vivo, FXa could potentially
modulate FXase activity by inactivating the cofactor. Alternatively,
the FXa generated could be sequestered/channeled from FXase to FVa (K
1 nM; (34) ) in forming
the prothrombinase complex. It is of interest to note that the
FVIII
mutant exhibits a 3-fold greater specific
activity than wild type FVIII based upon a Coatest (FXa generation)
assay(17) , whereas similar specific activities are obtained
with the one-stage clotting assay. (
)In the former assay,
FVIII is preincubated with FIXa, FX, phospholipid, and Ca
for several minutes prior to the activity determination. Thus,
the higher activity of the mutant may reflect its resistance to
cleavage by FIXa and/or generated FXa.