From the Cardiovascular Biology Research Program, Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma 73104
It is believed that heparin accelerates factor Xa
(FXa) inactivation by antithrombin (AT) by conformationally activating
the inhibitor rather than by bridging AT and FXa in a ternary complex (template effect). This is derived from kinetic studies done in the
absence of Ca2+ or in the presence of EDTA. To test
the possibility that the anionic Gla domain of FXa, when not
neutralized by Ca2+ ions, prevents heparin binding to FXa,
the heparin and pentasaccharide dependence of FXa inactivation by AT in
both the absence (100 µM EDTA) and presence of
Ca2+ (2.5 mM) was studied using wild-type FXa
and a FXa derivative that lacks the Gla domain (GDFXa). AT inactivated
both FXa derivatives similarly in both the absence and presence of
Ca2+ (k2 = 1.7-2.5 × 103 M
1 s
1). The
active AT-binding pentasaccharide also accelerated the inactivation
rates of both derivatives similarly in both the absence and presence of
Ca2+ (k2 = 5.7-8.0 × 105 M
1 s
1).
However, in the presence of an optimum concentration of heparin (~50
nM) the inactivation rate constant of FXa in the presence of Ca2+ (k2 = 4.4 × 107 M
1 s
1) was
13-fold higher than the rate constant in the absence of Ca2+ (k2 = 3.5 × 106 M
1 s
1). Heparin
acceleration of GDFXa inactivation by AT was rapid and insensitive to
the presence or absence of Ca2+ (k2 = 5.1-5.9 × 107 M
1
s
1). The additional cofactor effect of heparin with all
FXa derivatives was a bell-shaped curve, which disappeared if the
ionic strength of the reaction was increased to ~0.4. These results
suggest that although the major effect of heparin in acceleration of
FXa inactivation is through a heparin-induced conformational change in
the reactive site loop of AT, the template effect of heparin,
nevertheless, contributes significantly to rapid FXa inactivation at
physiological Ca2+.
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INTRODUCTION |
The mechanism by which the therapeutic heparin catalyzes the
inactivation of thrombin and factor Xa
(FXa)1 by antithrombin (AT)
has been extensively studied in the past (1-4). The consensus view is
that heparin accelerates thrombin inactivation by AT by facilitating an
initial interaction between the two proteins by a bridging mechanism
(4-6). In FXa inactivation, however, it is believed that a template
mechanism has a minimal role or no role at all in the acceleration and
that a heparin-induced conformational change in the reactive site loop
of AT accounts for all of the accelerating effect of heparin in the
reaction (7-10). These distinct inactivation mechanisms are supported
by the observation that a unique pentasaccharide fragment of heparin, which can bind and activate AT but is not long enough to bridge the
inhibitor and enzyme, specifically promotes FXa but not thrombin inhibition (9, 10). For thrombin inhibition, heparin chains containing
the pentasaccharide plus at least 13 additional saccharides are
required to accelerate the reaction (2, 4).
All the previous heparin-catalyzed protease inactivation studies were
conducted under experimental conditions that either lacked
Ca2+ (11, 12) or contained EDTA in the reaction buffers (9, 13, 14). Under these conditions heparin-catalyzed thrombin inactivation
by AT is at least 10-20-fold faster than the inactivation of FXa (9,
11-13). Unlike thrombin, however, FXa contains a highly anionic
N-terminal domain, which is rich in
-carboxyglutamic acid (Gla)
residues that are involved in Ca2+-dependent
membrane binding. This domain of FXa contains 11
-carboxylated Glu
residues, which are in a random disordered conformation in the absence
of Ca2+ (15). To test the possibility that in the absence
of Ca2+ the Gla domain of FXa antagonizes heparin binding
to FXa and thereby prevents heparin from acting as a template in the
FXa-AT reaction, the heparin and pentasaccharide dependence of FXa
inactivation by AT was studied in both the absence and presence of
Ca2+ using wild-type FXa and a recombinant FXa derivative,
which lacks the Gla domain (GDFXa). It was found that in contrast to a
similar ~300-fold cofactor effect of the pentasaccharide in FXa
inactivation in both the absence and presence of Ca2+, the
cofactor effect of heparin was markedly higher if the reaction was
carried out in the presence of Ca2+. The difference in the
magnitude of the cofactor effect between heparin and the
pentasaccharide was ~5- and ~60-fold for FXa in the absence and
presence of Ca2+, and ~89- and ~74-fold for GDFXa in
the absence and presence of Ca2+, respectively. These
findings can explain why the anti-FXa activity of heparin exhibits a
minimal chain length dependence in ex vivo and
in vitro assays but significant chain length dependence in in vivo assays (16, 17).
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EXPERIMENTAL PROCEDURES |
Proteins and Other Reagents--
Human plasma FXa, GDFXa (18),
and recombinant human thrombin (19) were prepared as described
previously. Human plasma AT was purchased from Hematologic Technologies
Inc., VT. Unfractionated heparin from porcine intestinal mucosa, sodium
salt (169.2 USP units/mg), and Polybrene were purchased from Sigma.
Spectrozymes FXa (SpFXa) and PCa (SpPCa) were purchased from American
Diagnostica, Greenwich, CT. The oligosaccharides, ranging in size from
6 to 18 saccharide units, were generous gifts from Dr. Ingemar
Björk (Swedish University of Agricultural Sciences, Uppsala,
Sweden), and high affinity heparin fragments with 22-64 saccharide
units were generous gifts from Dr. Steven Olson (University of
Illinois, Chicago).
Kinetic Methods--
The rate of inactivation of FXa, GDFXa, and
thrombin in the absence or presence of heparin and the pentasaccharide
fragment of heparin (1 µM) were measured under
pseudo-first order rate conditions by a discontinuous assay as
described previously (19, 20). The heparin concentration dependence of
the inactivation reactions was determined by incubating 0.2 nM FXa or GDFXa with 3-20 nM AT and varying
concentrations of heparin (0-50 µM) in Tris-HCl (pH
7.5), 0.1 M NaCl (TBS buffer, I ~ 0.12), 1 mg/ml BSA, and 0.1% PEG 8000 containing either 2.5 mM
CaCl2 or 100 µM EDTA in 50-µl reactions.
After 10 s to 1 min of incubation at room temperature
(~25 °C), 50 µl of SpFXa in TBS buffer containing 1 mg/ml
Polybrene (to block heparin function immediately) was added to give a
final concentration of 0.4 mM. The remaining activities and
the second-order inhibition rate constants (k2)
for heparin-catalyzed inactivation of FXa derivatives were determined
as described previously (19, 20). The bell-shaped dependence of
k2 on heparin concentration was computer fit to
the following equation as described previously (6),
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(Eq. 1)
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where [AT]o is total antithrombin concentration,
[H]o is total heparin concentration, S is the
stoichiometry of heparin-AT interaction (this value was 1.7 for the
unfractionated heparin used in this study as determined by fluorescence
measurements), KAT,H is the dissociation
constant for heparin-AT interaction, k2,max is
the maximum second-order inhibition rate constant,
k2,penta represents the
pentasaccharide-accelerated second-order rate constant, and
KFXa,H is the dissociation constant for the
FXa-heparin interaction. This equation neglects the uncatalyzed rate
constant because its contribution was found to be negligible within
experimental error. Fitting of the data by this equation revealed that
k2,penta was not well determined. Its value was
therefore fixed at the independently determined value.
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RESULTS |
Fig. 1 shows the heparin
concentration dependence of FXa inactivation by AT in the presence of
either 100 µM EDTA (open circles) or 2.5 mM Ca2+ (closed
circles). The heparin concentration dependence of the second-order rate constant (k2) for FXa
inactivation in the presence of EDTA reaches saturation at ~50
nM heparin (Fig. 1), and further increasing the
concentration of heparin does not result in a decline in
k2. The maximal rate constant achieved under
these conditions was k2 = 3.5 ± 0.7 × 106 M
1 s
1 (Table
I). In the presence 2.5 mM
Ca2+, the accelerating effect of heparin was significantly
increased over that measured in EDTA, and the dependence of
k2 on heparin concentration was bell-shaped,
with an initial increase in k2 followed by a
decrease. The maximal k2 of 4.4 ± 0.4 × 107 M
1 s
1,
reached at ~50-300 nM heparin, was 13-fold higher than
that measured in the absence of Ca2+ (Fig. 1 and Table I).
At higher heparin concentrations, k2 decreased such that at ~20 µM heparin there was essentially no
difference between the inactivation rates in the absence or presence of
Ca2+ (Fig. 1). Thus, only the additional 13-fold cofactor
effect of heparin in the presence of Ca2+ was abolished at
high heparin concentration, suggesting that a ternary complex by
bridging or template mechanism was responsible for this portion of the
cofactor effect of heparin in FXa inactivation.

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Fig. 1.
Heparin concentration dependence of FXa
inactivation by AT in the absence or presence of Ca2+.
FXa (0.2 nM) inactivation by AT (5 nM) was
monitored at varying concentrations of heparin (0-26 µM)
in TBS buffer containing 1 mg/ml BSA and 0.1% PEG 8000 in the presence
of either 100 µM EDTA ( ) or 2.5 mM
CaCl2 ( ). The second-order association rate constants
were determined as described under "Experimental Procedures." The
solid lines represent fits to Equation 1.
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Table I
The second-order inhibition rate constants (k2) for AT
inactivation of FXa, GDFXa, and thrombin in the presence or absence of
pentasaccharide (PS) or heparin
The k2 values for inactivation of each enzyme in the
absence or presence of a saturating concentration of cofactors (1 µM pentasaccharide or 50 nM heparin) were
determined in TBS buffer containing 1 mg/ml BSA, 0.1% PEG 8000, and
either 100 µM EDTA or 2.5 mM Ca2+ as
described under "Experimental Procedures." The kinetic values are
average of at least three independent measurements ± S.D.
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FXa has at least three known Ca2+-binding domains that can
influence the magnitude of the heparin cofactor effect in the FXa-AT reaction: the N-terminal Gla domain, the epidermal growth factor-like domain-1 (EGF-1), or the C-terminal catalytic domain (15, 21). To
determine which one of these domains is responsible for the Ca2+-dependent cofactor effect of heparin in
the reaction, the inactivation studies were carried out with two FXa
derivatives, one lacks the Gla domain (GDFXa) and the other lacks both
the Gla and EGF-1 domains (E2FXa). It was previously demonstrated that
except for the membrane binding properties, all other enzymatic
properties of wild-type FXa are preserved in these two mutants (18).
Unlike the cofactor effect of heparin in FXa inactivation, which was a
bell-shaped curve only in the presence of Ca2+, the
accelerating effect of heparin in GDFXa inactivation exhibited a
bell-shaped dependence on heparin concentration in both the absence and
presence of Ca2+ (Fig. 2). At
the optimum heparin concentration (50-300 nM), AT inactivated GDFXa with k2 = 5.1 ± 0.6 × 107 M
1 s
1 and
k2 = 5.9 ± 0.1 × 107
M
1 s
1 in the absence and
presence of Ca2+, respectively (Table I). The maximal value
and heparin concentration dependence of k2 for
E2FXa inactivation were similar to that for GDFXa inactivation (data
not shown). These results suggest that Ca2+ occupancy of
the EGF-1 domain or the catalytic domain does not alter the cofactor
function of heparin in the reaction; rather the anionic Gla domain of
FXa when not neutralized by Ca2+ ions interferes with
heparin binding to FXa, thereby preventing the bridging effect of
heparin in the acceleration of protease inactivation. The
Ca2+ concentration dependence of the heparin cofactor
effect also correlated well with the Ca2+ occupancy of the
Gla domain (optimal at ~1 mM).

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Fig. 2.
Heparin concentration dependence of GDFXa
inactivation by AT in the absence or presence of Ca2+.
GDFXa (0.2 nM) inactivation by AT (5 nM) was
monitored at varying concentrations of heparin (0-26 µM)
in TBS buffer containing 1 mg/ml BSA and 0.1% PEG 8000 in the presence
of either 100 µM EDTA ( ) or 2.5 mM
CaCl2 ( ). The second-order association rate constants
were determined as described under "Experimental Procedures" and
fitted by Equation 1 (solid lines).
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It has been demonstrated that the activated conformer of AT, whether it
is activated by heparin or the pentasaccharide, contributes similarly
to the acceleration of protease inhibition (9). It may, therefore, be
possible to estimate the extent that the template mechanism contributes
to the heparin rate enhancements observed with FXa inactivation in the
presence of Ca2+. This can be done by dividing the
inhibition rate constants determined in the presence of heparin by the
rate constants determined in the presence of the pentasaccharide. The
ratios of these rate constants in the absence and presence of
Ca2+ are shown in the last column of Table I. For
comparative purposes, the same values for thrombin inhibition were also
determined and presented in the same table. The data of Table I suggest
that AT inactivates both FXa and thrombin at comparable rates in the presence of heparin and at a physiologic concentration of
Ca2+. The template effect of heparin for GDFXa inactivation
in the presence of EDTA is comparable with that in Ca2+
within experimental error (74-90-fold) and similar to that for FXa
inactivation in the presence of Ca2+. The ascending limbs
of the curves represent titration of high affinity AT-heparin
(KAT,H) interaction, and the descending limbs represent titration of the low affinity GDFXa-heparin interaction (KFXa,H) (6). The computer fit of the
k2 values to Equation 1 provided a
KAT,H value of 7.2 ± 2.4 nM
and a KFXa,H value of 2.3 ± 0.7 µM for FXa in the presence of 2.5 mM
Ca2+. For GDFXa inactivation, similar values of 11.6 ± 2.7 nM for KAT,H and 2.8 ± 0.4 µM for KFXa,H were found in
the presence of Ca2+. In this case, lower values of
5.4 ± 2.7 nM for KAT,H and
0.26 ± 0.08 µM for KFXa,H
were obtained in the presence of EDTA, indicating tighter binding of
heparin to GDFXa in the absence of Ca2+.
The heparin chain length dependence of FXa inactivation by AT in the
presence of Ca2+ was studied using oligosaccharides
containing 6, 10, 14, and 18 saccharides and high affinity heparin
fragments containing on average 22, 35, 50, and 64 saccharides. The
cofactor effect of the 6-18-residue long oligosaccharides and the
22-residue long heparin fragment were all simple saturation curves (up
to 50 µM) in both the absence and presence of
Ca2+ with only an ~1.7-fold enhanced cofactor effect for
the 18-residue long oligosaccharide and an ~2.5-fold enhanced
cofactor effect for the 22-residue long heparin fragment in the
presence of Ca2+. A slight improvement in the reactivity of
FXa with AT in the presence of Ca2+ was also observed in
the absence of heparin (Table I). The maximal ~10-13-fold template
cofactor effect of heparin displaying a bell-shaped dependence on
heparin concentration was observed with any of the 35-64-residue long
high affinity heparin fragments used for FXa or GDFXa inactivation
(data not shown). The optimal heparin concentration, k2 values, and the KAT,H
and KFXa,H values for the 50- and 64-residue long heparin fragments were all similar to the values determined for
the unfractionated heparin (data not shown).
It is known that the bridging effect of heparin on the thrombin-AT
reaction is dependent on the ionic strength of buffer, but the cofactor
effect, which is mediated by a conformational change in the reactive
site loop of AT, is relatively insensitive to the ionic strength as
long as AT is saturated with heparin (5). Similarly, the additional
cofactor effect of heparin in FXa inactivation in the presence
Ca2+ or in GDFXa inactivation in both the absence and
presence of Ca2+ decreased as a function of increasing salt
concentration such that the differences in the reactivities of FXa
derivatives was completely abolished at 0.4 M NaCl (Table
II).
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Table II
Salt concentration dependence of the cofactor function of heparin in
FXa and GDFXa inactivation by AT in the presence of EDTA or
Ca2+
The k2 values were determined by incubating each FXa
derivative (0.2 nM) with AT (5 nM) and optimal
concentration of heparin (300 nM) in TBS buffer containing
0.1-0.4 M NaCl, 1 mg/ml BSA, 0.1% PEG 8000, and either
100 µM EDTA or 2.5 mM Ca2+ as
described under "Experimental Procedures." The kinetic values are
the average of at least three independent measurements ± S.D.
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DISCUSSION |
The anticoagulant effect of heparin is primarily mediated through
the acceleration of the rate of inactivation of coagulation proteases
(mainly thrombin and FXa) by AT. It is thought that formation of a
heparin-AT-protease ternary complex by a bridging or template mechanism
accounts for the bulk of the accelerating effect of heparin in thrombin
inactivation (5, 6, 9). In FXa inactivation, however, it is believed
that a heparin-induced conformational change in the reactive site loop
of AT is responsible for virtually all of the accelerating effect and
that heparin bridging AT and protease in a ternary complex has a
minimal or no effect on acceleration of the reaction (4, 7-10).
Results of the current study are consistent with these observations
only when the heparin-catalyzed AT inactivation of FXa was monitored in
the absence of Ca2+, an experimental condition that all
other previous studies have used to compare the extent of the cofactor
effect of heparin and pentasaccharide in the acceleration of FXa
inactivation. In the presence of Ca2+, however, it was
found that heparin-catalyzed FXa inactivation was at least 13-fold
higher than the inactivation in the absence of Ca2+.
FXa belongs to a family of vitamin K-dependent coagulation
proteases, which all contain a highly anionic N-terminal Gla domain with several
-carboxylated glutamic acid (Glu) residues that are
involved in Ca2+-dependent membrane binding
(22, 23). In FXa, this domain contains 11
-carboxylated Glu
residues, which are in random disordered conformation in the absence of
Ca2+ but fold properly to their native conformation in the
presence of Ca2+ (15). The observation that the
accelerating effect of heparin in FXa inactivation in the presence of
Ca2+ and in GDFXa inactivation in both the presence and
absence of Ca2+ was ~13-15-fold higher than FXa
inactivation in the absence of Ca2+ suggests that the
anionic Gla domain prevents the binding of heparin to FXa and thereby
the formation of a ternary bridging complex. The Gla domain can
interfere with heparin binding to FXa in the absence of
Ca2+ by either folding on itself and masking the positively
charged residues of the heparin binding site and/or simply by creating a highly electronegative molecule preventing the approach of heparin by
repulsive interactions. The interference of an unfolded Gla domain with
heparin binding in the absence of Ca2+ may be a universal
phenomenon occurring in other vitamin K-dependent Gla-containing coagulation proteases, because in a recent study it was
also noticed that heparin dramatically accelerates protein C activation
by thrombin and FXa in the presence but not in the absence of
Ca2+ (24). In the absence of Ca2+, however,
heparin accelerated the activation of the Gla-domainless protein C by
thrombin, suggesting that similar to FXa, the Gla domain of protein C
when not stabilized by Ca2+ ions interferes with the
cofactor function of heparin in the activation reaction. The existence
of a heparin-binding site in FXa and protein C homologous to that in
thrombin is suggested by the conservation of the basic site of thrombin
in FXa and activated protein C (25, 26). Interestingly, the observed
affinity of FXa for heparin in the presence of Ca2+ is
comparable with that measured for thrombin, albeit in the absence of
Ca2+ (27).
This is the first study to show that the cofactor effect of heparin in
FXa inactivation by AT at physiologic Ca2+ exceeds the
cofactor effect of heparin in thrombin inactivation and that this
effect is mediated by a combination of an ~300-fold enhancement by
the pentasaccharide-induced conformational change in the reactive site
loop of AT and an ~60-fold enhancement by a template mechanism. The
results of this study further suggest that heparin fragments of 18 saccharides long are required to observe a template effect for heparin
in FXa inactivation in the presence of Ca2+, similar to the
chain length requirement in thrombin inactivation. Low molecular weight
heparins (Mr = 4000-6000) are used as
antithrombotic agents, and a number of studies suggest that the
antithrombotic effect of these types of heparins can be accounted for
primarily by their anti-FXa activity, because oligosaccharides less
than 18 residues long that are devoid of anti-thrombin activity still exhibit significant antithrombotic activity (28). Relative to unfractionated heparin, however, these oligosaccharide fragments show
less antithrombotic activity despite the existence of a comparable ex vivo and in vitro anti-FXa activity
for both types of heparins (17). Based on the oligosaccharide chain
length dependence of the antithrombotic effect it has been concluded
that in addition to the anti-FXa activity, the longer chain heparins in
low molecular weight preparations also provide an additional
anti-thrombin activity. Results of the current study now suggest that
the reduced antithrombotic effect of low molecular weight heparins may
be accounted for solely by their anti-FXa activity, because this
activity is expected to show a chain length dependence in the presence
of Ca2+ like that of thrombin. Because all current in
vitro assays measure the anti-FXa activity of heparin in the
absence of Ca2+, they would miss this additional chain
length-dependent cofactor effect of heparin in FXa
inactivation.
I thank Dr. Steven Olson for critical reading
of this manuscript as well as for invaluable suggestions and
discussions. I also thank Mei Cheng for preparation of the figures.