(Received for publication, August 22, 1995; and in revised form, February 8, 1996)
From the
Factor IXa, a serine protease of blood coagulation, functions at
least 100,000 times more efficiently when bound to factor VIIIa on a
phospholipid membrane than when free in solution. We have utilized the
catalytic activity of the factor VIIIa-factor IXa complex to report the
effect of phospholipid membranes on binding of factor IXa to factor
VIIIa and on enzymatic cleavage of the product. The apparent affinity
of factor IXa for factor VIIIa was 10-fold lower in the absence of
phospholipid membranes with a K of 46
nMversus 4.3 nM with phospholipid
membranes. The K
for activation of factor
X by the factor VIIIa-factor IXa complex was 1700 nM in
solution, 70-fold higher than the value of 28 nM when bound to
membranes containing phosphatidyl-L-serine,
phosphatidylethanolamine, and phosphatidylcholine at a ratio of
4:20:76. The largest effect of
phosphatidyl-L-serine-containing membranes on the factor
VIIIa-factor IXa complex was the accelerated rate of peptide bond
cleavage, with the k
increased by 1,500-fold
from 0.022 to 33 min
. Membranes in which
phosphatidyl-L-serine was replaced by
phosphatidyl-D-serine, phosphatidic acid, or
phosphatidylglycerol were at least 10-fold less effective for enhancing
the k
. Thus, while membranes containing
phosphatidyl-L-serine enhance condensation of the enzyme with
its cofactor and substrate, their largest effect is activation of the
assembled factor VIIIa-factor IXa enzyme complex.
Factor VIII is a phosphatidyl-L-serine
(Ptd-L-Ser) ()binding cofactor (1, 2) for the vitamin K-dependent serine protease,
factor IXa, that also binds to Ptd-L-Ser containing
membranes(3, 4) . The membrane-bound factor
VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa
which is then responsible for catalyzing prothrombin
activation(5) . The importance of this enzyme complex is
illustrated by hemophilia, a disease in which a deficiency of either
factor VIII or factor IX leads to life-threatening bleeding. Factor IXa
gains more than 100,000-fold greater efficiency in activating factor X
by assembling with factor VIIIa on a Ptd-L-Ser containing
membrane than when free in solution (6) . While prior reports
indicate that Ptd-L-Ser containing membranes decrease the K
of factor IXa for factor X (6, 7) , they do not indicate what effect these
membranes have upon the enzymatic parameters of the factor VIIIa-factor
IXa complex. Therefore, we compared the enzymatic parameters of the
factor VIIIa-factor IXa complex in solution to the parameters of the
membrane-bound complex to determine whether Ptd-L-Ser
containing membranes function to enhance assembly of the enzyme and
cofactor, binding of the substrate to the enzyme, or catalysis of the
substrate.
Bovine brain Ptd-L-ser, phosphatidylethanolamine (PE) synthesized by transphosphatidylation of egg PC, dimyristoyl PE, egg PC, dioleoyl PC, dioleoylphosphatidic acid, and dioleoyl phosphatidylglycerol were from Avanti Polar Lipids (Alabaster, AL). Cholesterol was from Aldrich. Recombinant human factor VIII was a gift from D. Pittman of Genetics Institute, Cambridge, MA. Factor IXa, factor X, and factor Xa were from Enzyme Research Laboratories (Southbend, IN). Thrombin was from Sigma.
We wished to determine whether catalytic enhancement of the
factor VIIIa-factor IXa enzyme complex by Ptd-L-Ser containing
membranes primarily reflects enhanced assembly of factor IXa with
factor VIIIa versus enhanced binding of the substrate, factor
X to the enzyme, factor IXa versus acceleration of peptide
bond cleavage. We first asked whether the apparent affinity of factor
IXa for factor VIIIa is influenced by Ptd-L-Ser-containing
membranes. Saturable binding of factor IXa to 5 nM factor
VIIIa was detected by increased catalytic efficiency of factor IXa
toward factor X (Fig. 1A, lower curve). The
apparent K was 42 nM, approximately
10-fold higher than prior measurements in the presence of phospholipid
membranes. When the factor VIIIa concentration was increased 4-fold to
20 nM the maximum catalytic rate increased 4-fold confirming
that factor IXa assembles with available factor VIIIa to form a complex
with enhanced catalytic activity in the absence of phospholipid. The
average K
obtained from five experiments was 46
nM (Table 1). For comparison we examined the binding of
factor IXa to 1 nM factor VIIIa in the presence of vesicles of
4% Ptd-L-Ser, 20% PE and 25% Ptd-L-Ser (Fig. 1, B and C). The apparent K
was 6.6 nM for vesicles with 4%
Ptd-L-Ser and 4 nM for vesicles with 25%
Ptd-L-Ser. The mean dissociation constants were 4.3 nM for 5 such experiments using vesicles of 4% Ptd-L-Ser and
2.3 for 3 experiments with vesicles of 25% Ptd-L-Ser (Table 1) in agreement with prior measurements under similar
conditions(13) . These results indicate that
Ptd-L-Ser-containing membranes enhance the affinity of factor
IXa for factor VIIIa by 10-20-fold in the presence of the
substrate, factor X.
Figure 1:
Assembly of the factor VIIIa-factor
IXa complex in the absence and presence of phospholipid membranes. A, when increasing quantities of factor IXa were added to 5
nM factor VIIIa () the quantity of factor Xa formed
increased saturably indicating formation of the factor VIIIa-factor IXa
complex. When the factor VIIIa concentration was increased to 20 nM (
) the maximum rate of factor Xa formation increased 4-fold.
Results were corrected for the quantity of factor Xa produced by factor
IXa in the absence of VIII and the correction did not exceed forty
percent of total factor Xa formed at the highest concentration of
factor IXa. A representative experiment is depicted and the line
represents a K
of 42 nM for the
lower curve and 71 nM for the upper curve obtained by
nonlinear least squares analysis of data. When varying concentrations
of factor IXa were added to 1 nM factor VIIIa in the presence
of phospholipid vesicles of 4% Ptd-L-Ser, 20% PE (B)
or 25% Ptd-L-Ser (C) saturable binding of factor IXa
to factor VIIIa was detected with apparent K
values of 6.6 nM and 4.0 nM, respectively.
The reactions were allowed to proceed for 5 min in the presence of 25
µM phospholipid (B and C) or 30 min in
the absence of phospholipid (A). Preliminary experiments
indicated that production of factor Xa was proportional to elapsed
reaction time for 5 min in the presence of phospholipid and for at
least 40 min in the absence of
phospholipid.
We next determined the concentration of
phospholipid vesicles that would enhance cleavage of factor X by the
factor VIIIa-factor IXa complex (Fig. 2). We have recently
observed that PE induces binding sites for factor VIII in membranes
with low mole fractions of Ptd-L-Ser(2) and we
compared these membranes to those containing 25% Ptd-L-Ser
without PE, similar to those used in prior studies of the enzymatic
parameters for this complex. In contrast to our prior studies we
utilized extruded vesicles rather than sonicated vesicles and 1 mM Ca rather than 5 mM Ca
, attempting to better simulate the curvature
of the platelet membrane versus maximally curved sonicated
vesicles and to better approximate the Ca
concentration in plasma. Vesicles of 25% Ptd-L-Ser were
as much as 10-fold more effective than those with 4% Ptd-L-Ser
and PE when the phospholipid concentrations less than 20 µM and the increased activity of the factor VIIIa-factor IXa complex
plateaued at a concentration of 32 µM. When the
phospholipid concentration was increased to 125 µM the
difference between membrane types decreased to 2-fold and the activity
related to vesicles of 4% Ptd-L-Ser with PE had not yet
plateaued. When the Ca
was increased to 5 mM and vesicles were prepared by sonication the two vesicle type were
equivalent at phospholipid concentrations of 2 µM or above
as we previously reported(2) . These results indicate that the
activity of the factor VIIIa-factor IXa complex is greatly increased by
membranes containing Ptd-L-Ser and is modestly influenced by
the curvature of the membranes. They are consistent with prior results
indicating that membrane binding of factors IXa and factor X to
membranes containing Ptd-L-Ser is enhanced by elevating the
Ca
concentrations above the plasma concentration to 5
mM(6, 14, 15) .
Figure 2:
Activation of the factor VIIIa-factor IXa
complex by Ptd-L-Ser-containing membranes. Various quantities
of phospholipid vesicles were added to a mixture of factor IXa, factor
VIIIa and factor X and the quantity of factor X formed was evaluated.
Vesicles contained either 25% Ptd-L-Ser () or 4%
Ptd-L-Ser, 20% PE (
) with the balance as PC. Protein
concentrations were as described in the legend for Fig. 1B.
We next performed
steady state kinetic experiments to determine whether
Ptd-L-Ser-containing membranes primarily affect the Kversus the k
.
Conditions were chosen such that more than 70% of factor IXa would be
bound to factor VIIIa. The rate at which the factor VIIIa-factor IXa
complex activated factor X increased saturably with the factor X
concentration in the presence and the absence of phospholipid vesicles (Fig. 3). The Michaelis constant for the factor VIIIa-factor IXa
complex was 1700 nM in the absence of phospholipid vesicles (Fig. 3A), versus 23 nM in the
presence of vesicles containing 4% Ptd-L-Ser and PE (Fig. 3B and Table 1). In contrast, the k
increased 1,500-fold from 0.022
min
in the absence of phospholipid to 33
min
in the presence of these vesicles (Table 1). Similarly, the k
increased to
136 min
in the presence of membranes containing 25%
Ptd-L-Ser and no PE. These results indicate that
Ptd-L-Ser-containing membranes enhance the efficiency of the
factor VIIIa-factor IX enzyme complex by increasing the affinity for
the substrate and by increasing the rate of peptide bond cleavage.
Figure 3:
Steady state kinetics of the factor
VIIIa-factor IXa complex in the absence and presence of phospholipid
membranes comparable. Factor IXa was incubated with factor VIIIa,
varying concentrations of factor X and no phospholipid (A) or
25 µM phospholipid (B) of 25% Ptd-L-Ser
() or 4% Ptd-L-Ser and 20% PE (
). Lines indicate
best fits of the data sets. Duplicates were averaged and shown as
single points for clarity of presentation in B. The factor IXa
concentrations were 5 nM (A) and 0.01 nM (B); factor VIIIa was 120 nM. The reactions
proceeded for 30 min in the absence of phospholipid (A) or 5
min in the presence (B).
To place our results in context with prior evaluations of the
kinetics of the factor VIIIa-factor IXa complex (6, 7) we also performed steady state kinetics at a
Ca concentration of 5 mM (data not shown).
The apparent affinity of factor IXa for factor VIIIa was affected less
than 2-fold in the absence or presence of both types of vesicles.
Likewise, the K
was affected less than 2-fold in
the presence or absence of vesicles. The k
was
approximately 10-fold higher at 5 mM Ca
for
vesicles of 4% Ptd-L-Ser, 20% PE and 3-fold higher for
vesicles of 25% Ptd-L-Ser with values comparable to prior
reports(6, 7) . The difference between the enhancement
with these two vesicle types may be rationalized by noting that
concentrations of Ca
above 1 mM enhance
binding of factor IXa and factor X to vesicles of low PS content more
than to those with high PS content(14, 16) . In the
absence of phospholipid vesicles the k
was
approximately 3-fold higher at 5 mM Ca
than
at 1 mM Ca
. Thus, elevation of the
Ca
concentration from 1 to 5 mM has modest
effects on the kinetic parameters of the factor VIIIa-factor IXa
complex bound to a membrane site.
To determine whether
Ptd-L-Ser is a specific activator of the factor VIIIa-factor
IXa complex or whether activation could be induced equivalently by
other negatively charged phospholipids we compared membranes containing
4% Ptd-L-Ser to those containing 4% Ptd-D-Ser, 4%
Phosphatidic acid, or 4% phosphatidylglycerol (Fig. 4). We
compared activation at 5 mM Ca (main
graph) and 1 mM Ca
(inset).
While Ptd-L-Ser containing membranes activated the factor
VIIIa-factor IXa complex, the diastereomer, Ptd-D-ser was at
least 10-fold less effective. Likewise, phosphatidic acid and
phosphatidylglycerol-containing membranes were at least 10-fold less
effective than Ptd-L-Ser. Activation by Ptd-L-Ser
occurred at a 5-fold lower phospholipid concentration in the presence
of 5 mM Ca
compared to 1 mM Ca
. Unfortunately, the quantity of dioleoyl
Ptd-L-Ser and dioleoyl Ptd-D-Ser available from our
synthesis prevented experiments at 5-10 fold higher
concentrations apparently necessary to activate all of the factor
VIIIa-factor IXa complexes. These results indicate that when negatively
charged phospholipids are present as a low mole fraction of the
membrane the factor VIIIa-factor IXa complex is activated by a
stereoselective interaction with Ptd-L-ser.
Figure 4:
Ptd-L-Ser is a specific activator
of the factor VIIIa-factor IXa complex. The capacity of membranes
containing 4% of the indicated negatively charged dioleoyl
phospholipid, 20% dimyristoyl PE, with the balance as
phosphatidylcholine were compared for their ability to activate the
factor VIIIa-factor IXa complex at 5 mM Ca.
(
) Membranes containing 4% dioleoyl Ptd-L-Ser activated
the complex efficiently at 120 nM. (
) Dioleoyl
phosphatidic acid, with a negative valence twice that of
Ptd-L-Ser, was at least 25-fold less effective. (
)
Dioleoyl Ptd-D-Ser and (
) dioleoyl phosphatidylglycerol
were at least 50-fold less effective than dioleoyl Ptd-L-Ser.
When the Ca
concentration was 1 mM (inset) the effective phospholipid concentrations were
apparently 5-fold higher. Displayed results are the mean of duplicate
samples from two experiments, representative of six
experiments.
Our results indicate that Ptd-L-Ser-containing membranes primarily activate the assembled factor VIIIa-factor IXa complex, enhancing the chemical step of peptide bond cleavage more than the physical steps of enzyme-cofactor or enzyme-substrate binding. The specificity with which Ptd-L-Ser containing membranes activate the factor VIIIa-factor IXa complex parallels the specificity with which Ptd-L-Ser containing membranes bind factor VIII (1, 17) which suggests that membrane binding of factor VIIIa is a primary determinant of enzyme complex activity.
The
studies described in this report are the first to elucidate the kinetic
properties of the factor VIIIa-factor IXa complex in the absence of
phospholipid membranes or cells. We find that the k for the complex in solution is 0.022 min
compared with values ranging from 0.01 and 0.07 min
previously reported for factor IXa in the absence of factor
VIII(6, 7) . Therefore, formation of the factor
VIIIa-factor IXa complex enhances the k
not more
than 2-fold. However the K
for factor IXa alone is
80-180 µM(6, 7) versus 1.7 µM for the factor VIIIa-factor IXa complex. Thus,
binding to factor VIIIa increases the affinity of factor IXa for its
substrate, factor X, by at least 40-fold. We note that binding of
factor IXa to either factor VIIIa or a Ptd-L-Ser containing
membrane affects the K
for factor X to
approximately the same degree (Table 1)(6, 7) .
This raises the possibility that the mechanism for decreasing the K
in both cases may be a similar conformational
change in factor IXa that increases the affinity for factor X.
The
effect of Ptd-L-Ser-containing membranes on the
prothrombin-activating factor Va-factor Xa complex contrasts with the
effects on the factor VIIIa-factor IXa complex (Table 2).
Ptd-L-Ser-containing membranes do enhance the affinity of the
enzyme, factor Xa, for the cofactor, factor Va by 75-800 fold
while a 10-fold effect is detected for factor IXa in the factor
VIIIa-factor IXa complex(18, 19) . The k for the factor Va-factor Xa complex is
increased by only about 5-fold when bound to a
Ptd-L-Ser-containing membrane compared with 1,500-fold for the
factor VIIIa-factor IXa complex. Thus, although
Ptd-L-Ser-containing membranes increase the catalytic
efficiency of the prothrombinase complex and the factor VIIIa-factor
IXa complex to a similar degree the primary effect on the
prothrombinase complex is condensation of the enzyme, the cofactor, and
the substrate on the same site. In contrast, the primary effect of
Ptd-L-Ser-containing membranes on the factor VIIIa-factor IXa
complex is to increase the k
of the
enzyme-cofactor-substrate complex which assembles with high affinity in
the absence of membranes.
This report helps to explain the importance of Ptd-L-Ser as a component of binding sites for the factor VIIIa-factor IXa complex in that binding to this lipid alters the complex by a 1,500-fold enhancement in catalytic activity. Platelets develop procoagulant activity in parallel with the re-orientation of Ptd-L-ser and PE from the inner to the outer bilayer of the plasma membrane(20) . Under the same conditions that lead to Ptd-L-ser and PE re-orientation, platelets express specific receptors/binding sites for factor VIII and support function of factor VIII in the factor Xase complex(21, 22) . When platelets are stimulated by agonists that induce procoagulant activity, they release small vesicles derived from the plasma membrane(23, 24, 25) . These vesicles, also referred to as microparticles, have a high density of membrane receptors/binding sites for factor VIII(22) . In vitro data support the hypothesis that Ptd-L-Ser on the platelet membrane is a constituent of binding sites/receptors for factor VIII. The affinity of factor VIII binding to activated platelets and to microparticles is equivalent to the affinity of binding to synthetic membranes containing Ptd-L-ser(21, 22, 26) . Binding sites containing Ptd-L-ser, like those of activated platelets, are highly specific for factor VIII(17) . The specificity is mediated by a stereoselective interaction of factor VIII with O-phospho-L-serine, the head group of Ptd-L-ser(1) . Furthermore, inclusion of PE induces high affinity binding sites for factor VIII in synthetic membranes with a low mol fraction of Ptd-L-Ser, comparable to platelet membranes(2) .
High affinity binding of factor IXa to factor
VIII in the absence of a Ptd-L-Ser-containing membrane was
recently reported by Lenting et al.(27) . The binding
energy for this interaction comes primarily from interaction of the
light chain of factor VIII with factor IXa. Prior studies (13) indicated that the affinity of factor IXa for factor VIII
is not affected by proteolytic activation of factor VIII. However,
proteolytic activation releases factor VIII from von Willebrand factor
in plasma, making the binding to factor IXa possible. The studies in
the present report imply a 4-fold lower affinity of factor IXa for
factor VIIIa in the absence of phospholipids than previously reported;
a K of 46 nMversus 11
nM. The difference in the absence of phospholipid may reflect
the different techniques employed or it may indicate that the
interaction of factor X with both factor IXa and factor VIIIa decreases
the apparent affinity of each protein for the other.
The enzyme
constants in this report are in agreement with prior measurements for
the membrane-bound factor VIIIa-factor IXa
complex(6, 7, 28) . Of the three prior
reports, two did not include experiments for the factor VIIIa-factor
IXa complex in the absence of phospholipid membranes (6, 28) and the other reported that factor VIIIa and
factor IXa, in the absence of phospholipid, had parameters equivalent
to factor IXa alone(7) . The experimental design employed in
that study may explain why no significant effect of factor VIIIa on
factor IXa was detected. First, factor VIII, which was used at a 1:5
ratio to factor IXa, was activated by thrombin prior to mixing with
factor IXa. This allowed some time for the unstable factor VIIIa
molecule to decompose prior to mixing and probably further reduced the
factor VIIIa:factor IXa ratio. Second, the lowest concentration of
factor X examined was 5 µM, more than 3-fold greater
than the K
for the factor VIIIa-factor IXa
complex. At this substrate concentration the quantity of factor Xa
generated by free factor IXa which was present at a >4:1 ratio to
the factor VIIIa-factor IXa complex would be substantial. Thus, in the
presence of these factor X concentrations the effect of the factor
VIIIa-factor IXa complex may have been small compared to the effect of
free factor IXa.
We report that the apparent affinity of human factor IXa for human factor VIIIa in the presence of Ptd-L-Ser containing membranes is 5-10-fold higher than previously reported(8) . The discrepancy likely relates to the differences in experimental design and is rationalized by considering the instability of free factor VIIIa which dissociates into inactive subunits with a half-life of two minutes versus factor IXa-bound factor VIIIa which decays with a half-life of 20 min(8) . Our reported values are derived from experiments in which varying factor IXa was added to a fixed concentration of factor VIIIa. This design allows for factor IXa to stabilize factor VIIIa at all concentrations where it is in excess over factor VIIIa. The prior report utilized fixed concentrations of factor IXa and increasing factor VIIIa. In this design all factor VIIIa that is in excess over factor IXa decays rapidly so that the equilibrium process favors a large fraction of dissociated factor VIIIa. The plausibility of this explanation is supported by the reported stoichiometry of human factor VIIIa to factor IXa, 3:1 versus the stoichiometry of the more stable porcine factor VIIIa to factor IXa 0.8:1 associated with a 10-fold higher apparent affinity. To confirm the adequacy of this explanation we performed experiments in which increasing concentrations of factor VIIIa were added to a fixed concentration of factor IXa. This approach yielded an apparent affinity 3-5-fold lower than the results in Table 1and within 2-fold of the previously reported value (data now shown).
There are four possible mechanisms through
which the Ptd-L-Ser containing membranes may activate the
factor VIIIa-factor IXa complex. First, the membranes may have an
allosteric effect on factor IXa. Second, they may work by altering the
configuration of the factor IXa-factor VIIIa complex. Third they may
affect the conformation of the substrate, factor X, which is also a
membrane-binding protein. Finally, membrane lipids may alter the
geometry of the interaction between factor X and the factor
VIIIa-factor IXa complex. The first mechanism, an allosteric effect
upon factor IXa is consistent with two prior reports indicating that
Ptd-L-Ser-containing membranes enhance the k with which factor IXa cleaves factor X in the absence of factor
VIIIa by 15-25-fold(6, 7) . Because factor VIIIa
is also required to achieve the 1,500-fold enhancement in k
it may provide additional constraints upon the
factor IXa conformation. The plausibility of this explanation is
enhanced by a report indicating that modified factor X with an
abbreviated activation peptide has a normal K
for
interaction with the membrane-bound factor VIIIa-factor IXa complex but
a 100-fold reduced k
(28) . The
difference in k
was only 3-fold for
membrane-bound factor IXa in the absence of factor VIIIa. These data
indicate that factor X interacts with a factor IXa exosite which
influences the k
but not the K
and that interaction with the exosite is modified when factor IXa
binds to both factor VIIIa and Ptd-L-Ser containing membranes.
A conformational change of this nature would resemble the change in
factor Xa when bound to factor Va on Ptd-L-Ser containing
membranes (29) that occurs only if the membrane lipids contain
unsaturated acyl chains(30) . Thus, the activation
peptide-binding exosite is probably the best candidate site to undergo
a conformational change enhancing the k
. If this
interpretation is correct then the factor VIIIa-factor IXa complex
should also be activated by Ptd-L-Ser and possibly other
phospholipids in micelles as opposed to bilayers, and possibly by
soluble, short-chain Ptd-L-Ser molecules. We are currently
investigating these possibilities. This explanation may be further
probed by examining the effect of Ptd-L-Ser-containing
membranes upon cleavage of substrates that do not interact with the
membrane.