From the
The role of the sequence-specific pentasaccharide region of high
affinity heparin (HAH) in heparin acceleration of
antithrombin-proteinase reactions was elucidated by determining the
accelerating mechanism of low affinity heparin (LAH) lacking this
sequence. LAH was shown to be free of HAH (<0.001%) from the lack of
exchange of added fluorescein-labeled HAH into LAH after separating the
polysaccharides by antithrombin-agarose chromatography. Fluorescence
titrations showed that LAH bound to antithrombin with a 1000-fold
weaker affinity ( K
Heparin is a naturally occurring glycosaminoglycan which acts as
a potent antagonist of blood coagulation. This anticoagulant effect of
heparin is primarily due to the polysaccharide accelerating the
inactivation of blood clotting proteinases by their principal protein
inhibitor, antithrombin, several thousandfold (for reviews, see Refs. 1
and 2) The accelerating activity results from a sequence-specific
pentasaccharide region, present in about one-third of heparin
polysaccharide chains
(3, 4, 5) , which binds
antithrombin with high affinity and induces an activating
conformational change in the inhibitor
(6, 7, 8, 9, 10, 11) .
This conformational change appears to be primarily responsible for
heparin acceleration when factor Xa is the target proteinase since the
synthesized heparin pentasaccharide region is nearly as active as a
full-length heparin containing this sequence in accelerating the
inactivation of factor Xa by antithrombin
(11, 12, 13) . However, the conformational
change does not appear to be critical for heparin acceleration when
thrombin is the target proteinase, since the pentasaccharide has
negligible ability to accelerate the inactivation of this enzyme by
antithrombin
(11) . Instead, heparin rate enhancement of
thrombin inhibition appears to be mostly due to the polysaccharide
bridging antithrombin and thrombin in a ternary complex in which both
the inhibitor and proteinase are bound to the same polysaccharide chain
(12, 14, 15, 16, 17, 18, 19, 20, 21) .
Whereas heparin molecules containing the pentasaccharide binding
sequence for antithrombin (high affinity heparin) account for the bulk
of the anticoagulant activity of heparin preparations, heparin
molecules lacking this sequence (low affinity heparin) nevertheless
exhibit an anticoagulant effect of 5-10% that of high affinity
heparin molecules. Low affinity heparin has been shown to bind
antithrombin with about a 1000-fold weaker affinity than the high
affinity polysaccharide and appears to induce a conformational change
in the inhibitor, although one that is different in kind or extent
(6, 7, 22, 23) . Low affinity heparin
chains also can accelerate antithrombin-proteinase reactions, but the
magnitude of these accelerations are relatively small and
proteinase-dependent
(24) . It has been suggested that the
observed binding of low affinity heparin to antithrombin and
accelerating effect of the polysaccharide on antithrombin-proteinase
reactions may be accounted for by contaminating high affinity heparin
in the low affinity heparin preparations studied
(22) .
The
purpose of the present study was to determine whether low affinity
heparin possesses an intrinsic ability to accelerate
antithrombin-proteinase reactions, to establish the mechanism of such
an accelerating effect, and thereby to elucidate the role of the
antithrombin-binding pentasaccharide in the heparin accelerating
mechanism. A further goal was to evaluate whether pentasaccharide
activation of antithrombin can potentiate the low affinity heparin
acceleration of the antithrombin-thrombin reaction to an extent that
duplicates the high affinity heparin acceleration, as has been proposed
(25) . The results of our studies provide convincing evidence
for an intrinsic accelerating activity of low affinity heparin on
antithrombin-proteinase reactions and demonstrate a mechanism for this
accelerating effect which resembles that of high affinity heparin. The
reduced accelerating activity of the low affinity polysaccharide is
further shown to be fully explained by the reduced affinity of the
polysaccharide for antithrombin and its inability to induce a full
activating conformational change in the inhibitor. Finally, our studies
show that pentasaccharide binding to antithrombin diminishes rather
than enhances the low affinity heparin acceleration of the
antithrombin-thrombin reaction, in keeping with a predominant bridging
mechanism of heparin rate enhancement of this reaction and a minimal
role of the antithrombin conformational change in the rate enhancement.
These findings suggest that nonspecific antithrombin binding sequences
may contribute to the anticoagulant effects of clinically administered
heparin or of endogenous heparin or heparan sulfate glycosaminoglycans
involved in maintaining normal hemostasis.
A second set of rapid kinetic experiments were
monitored from the decrease in fluorescence accompanying the
displacement of the probe, p-aminobenzamidine, from the
proteinase by the inhibitor, as in previous studies
(15, 35) . Antithrombin-thrombin reactions employed 0.25
µ
M thrombin, 70 µ
M p-aminobenzamidine, and variable antithrombin and heparin
concentrations. Antithrombin-factor Xa reactions were done with 0.5
µ
M factor Xa, 85 µ
M p-aminobenzamidine, and either with 10 µ
M inhibitor and variable heparin or 10 µ
M heparin and
variable inhibitor concentrations. Fluorescence changes were followed
in an SLM 8000 spectrofluorometer equipped with a Milli-flow
stopped-flow attachment with wavelengths set at
The dependence of
k
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
For the heparin-catalyzed antithrombin-factor Xa
reaction, the dependence of k
On-line formulae not verified for accuracy
In fitting kinetic data by the above
equations, second-order rate constants for uncatalyzed
antithrombin-proteinase reactions were fixed at previously measured
values for k
In the case of the heparin-enhanced
antithrombin-factor Xa reaction, a sigmoidal heparin concentration
dependence of k
The purpose of these studies was to determine whether low
affinity heparin lacking the pentasaccharide recognition sequence for
antithrombin was capable of accelerating antithrombin-proteinase
reactions and if so, whether the mechanism of acceleration was similar
to that of high affinity heparin molecules possessing the
pentasaccharide. Several pieces of evidence argue that the acceleration
of antithrombin-proteinase reactions by low affinity heparin is an
intrinsic property of the polysaccharide and not due to the presence of
a small amount of high affinity polysaccharide. First, the extent of
exchange of labeled high affinity heparin into the low affinity heparin
was far too low to account for the observed activity of the low
affinity polysaccharide. Second, dextran sulfate produced
concentration-dependent accelerating effects similar in kind to,
although of lower magnitude than, those of low affinity heparin,
indicating that an anionic polysaccharide lacking the specific
pentasaccharide is capable of producing rate enhancement. Third, the
observation of similar saturation curves for the acceleration of the
antithrombin-factor Xa reaction by low affinity heparin when either the
polysaccharide or the inhibitor concentrations were varied in a
reciprocal fashion indicated the acceleration could not be due to
contaminating high affinity heparin.
Several observations indicate
that low affinity heparin accelerates antithrombin-proteinase reactions
by mechanisms which resemble those previously elucidated for high
affinity heparin. The similar bell-shaped curves characterizing high
affinity heparin, low affinity heparin, and dextran sulfate
accelerations of the antithrombin-thrombin reaction suggest that the
two latter polysaccharides act by the same ternary complex bridging
mechanism previously shown for high affinity heparin acceleration of
thrombin inhibition
(14, 15, 16, 17, 18, 19, 20, 21) .
However, the different relative binding affinities of antithrombin and
thrombin demonstrated for low and high affinity heparins imply a
different order of assembly of the ternary complex for the two
reactions. The greater affinity of antithrombin than of thrombin for
high affinity heparin thus was previously shown to result in ternary
complex assembly primarily through an antithrombin-heparin binary
complex intermediate
(19, 22) . Diagnostic of this mode
of assembly is the correspondence of the K
Rapid kinetic studies
provided direct evidence for a heparin-thrombin-antithrombin ternary
complex intermediate on the low affinity heparin-accelerated reaction
pathway. Such studies demonstrated that the difference in accelerating
effects of low and high affinity heparins arises from a 100-fold weaker
affinity of thrombin for antithrombin in forming the ternary complex
and not from a different rate of reaction of inhibitor with proteinase
within the ternary complex (Fig. S1). Comparison with the
K
Low affinity heparin also appears to accelerate the
antithrombin-factor Xa reaction by a mechanism similar to that
previously shown for high affinity heparin, but different from the
mechanism by which these heparins accelerate the antithrombin-thrombin
reaction. This conclusion is evident from the simple sigmoidal
saturation curve, instead of a bell-shaped curve, which characterizes
the polysaccharide or antithrombin concentration dependence of high
affinity heparin, low affinity heparin and dextran sulfate
accelerations of the antithrombin-factor Xa reaction (Figs. 4 and 6)
(11, 35, 38) . The displacement of the low
affinity heparin saturation curve to 1000-fold higher polysaccharide
concentrations than the high affinity heparin curve and the
correspondence of the apparent K
Previous
studies which compared pentasaccharide and full-length high affinity
heparin accelerations of the antithrombin-factor Xa reaction
demonstrated that an activating conformational change induced in
antithrombin by the pentasaccharide, rather than heparin bridging, was
primarily responsible for the heparin rate enhancement. That activation
of antithrombin by a heparin-induced conformational change is also
responsible for the low affinity heparin rate enhancement of the factor
Xa reaction is supported by observations in this and prior studies.
Binding of low affinity heparin and dextran sulfate to antithrombin
thus induces changes in the intrinsic fluorescence, near ultraviolet
absorbance and circular dichroism spectra of antithrombin, as well as
in the fluorescence of an extrinsic probe covalently linked to the
reactive center P1 residue, which are indicative of the activating
conformational change
(6, 7, 23) . Moreover,
these spectroscopic changes are reduced in magnitude relative to those
produced by pentasaccharide or full-length high affinity heparins to an
extent which correlates with the diminished rate enhancements of the
low affinity polysaccharides. Together, these observations are
consistent with the reduced acceleration of the antithrombin-factor Xa
reaction produced by low affinity heparin being due to both the lower
affinity of the polysaccharide for antithrombin as well as the failure
of the polysaccharide to induce a full activating conformational change
in antithrombin.
NMR difference spectroscopy and the high affinity
heparin pentasaccharide fragment were used to further probe the
mechanism by which low affinity heparin accelerates
antithrombin-proteinase reactions. NMR difference spectra of
antithrombin complexes with low and high affinity heparins and
competitive binding studies together demonstrated that low and high
affinity heparins bind to the same site on antithrombin, in agreement
with the findings of earlier studies
(41) . In keeping with such
findings, blocking the high affinity heparin binding site on
antithrombin with pentasaccharide abolished the acceleration of the
antithrombin-factor Xa reaction, indicating a requirement for low
affinity heparin to bind at this site and induce a conformational
change to mediate its accelerating effect. In the case of the
antithrombin-thrombin reaction, the pentasaccharide markedly reduced
but did not abolish the rate-enhancing effect of low affinity heparin
nor did it affect the bell-shaped dependence of the residual rate
enhancement on polysaccharide concentration. These observations
suggested that low affinity heparin can still bridge thrombin and
antithrombin when antithrombin is complexed with pentasaccharide,
although to a reduced extent. Such bridging can be explained by a
nonspecific electrostatic association of low affinity heparin with
antithrombin outside the pentasaccharide binding region, similar,
although not necessarily identical, to that which occurs with high
affinity heparin
(8, 11, 25) . This explanation
would be consistent with the reduced affinity of
pentasaccharide-blocked antithrombin for low affinity heparin inferred
from the bell-shaped curve.
Our results conflict with a previous
report which suggested that the small pentasaccharide acceleration of
the antithrombin-thrombin reaction could be increased by low affinity
heparin to an extent comparable to that of high affinity heparin
(25) . This observation was taken as evidence for the
pentasaccharide-induced conformational change in antithrombin being
principally responsible for heparin acceleration of the
antithrombin-thrombin reaction, with heparin bridging of antithrombin
and thrombin playing only a minor role. While our results show that low
affinity heparin can bridge pentasaccharide-activated antithrombin with
thrombin, they also demonstrate that this bridging does not increase
the rate enhancement beyond that expected from a bridging effect alone.
Our results thus argue against a significant role for conformational
activation of antithrombin in the mechanism of heparin acceleration of
the antithrombin-thrombin reaction and are in keeping with a
predominant bridging mechanism of rate enhancement for this reaction
(20, 21) .
In summary, the results of this study
indicate that low affinity heparin possesses an intrinsic ability to
accelerate antithrombin-proteinase reactions and that the mechanism of
this accelerating effect is identical to that of high affinity heparin
having a pentasaccharide recognition sequence for antithrombin. Our
studies thus establish that the pentasaccharide is not absolutely
required for heparin anti-proteinase activity but does play an
important role in mediating the high affinity binding of antithrombin
to the polysaccharide and in the polysaccharide's ability to
induce a full activating conformational change in the inhibitor. These
findings suggest that nonspecific heparin sequences may contribute to
the anticoagulant effects of clinically administered heparin, as well
as of heparan sulfate glycosaminoglycans present in the blood vessel
wall. The high levels of low affinity heparin required to produce an
anticoagulant effect do not preclude such a contribution, since such
heparin levels are also required for physiologic activation of heparin
cofactor II
(42, 43) . Although heparin cofactor II
inhibits thrombin with a 10-fold greater bimolecular rate constant than
antithrombin at optimal levels of low affinity heparin, the 2-fold
greater plasma concentration of antithrombin over heparin cofactor II
(42) would imply that one-fifth of the free thrombin generated
in plasma would be inhibited by antithrombin in the presence of low
affinity heparin. The observation that low affinity heparin possesses
significant antithrombotic activity exceeding that of the specific
heparin cofactor II activator, dermatan sulfate
(44) , supports
the possibility that this antithrombotic activity is not only mediated
by activation of heparin cofactor II but also in part by activation of
antithrombin.
Dissociation constants
were measured from fluorescence titrations performed as described under
``Materials and Methods'' in I 0.15, pH 7.4 sodium
phosphate buffer at 25 °C.
We thank Ann Marie Francis-Chmura and Rick Swanson for
technical assistance in these studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
19 ± 6
µ
M) and 5-6-fold smaller fluorescence enhancement (8
± 3%) than HAH. LAH accelerated the antithrombin-thrombin
reaction with a bell-shaped dependence on heparin concentration
resembling that of HAH, but with the bell-shaped curve shifted to
100-fold higher polysaccharide concentrations and with a
100-fold reduced maximal accelerating effect. Rapid kinetic
studies indicated these differences arose from a reverse order of
assembly of an intermediate heparin-thrombin-antithrombin ternary
complex and diminished ability of LAH to bridge antithrombin and
thrombin in this complex, as compared to HAH. By contrast, LAH and HAH
both accelerated the antithrombin-factor Xa reaction with a simple
saturable dependence on heparin or inhibitor concentrations which
paralleled the formation of an antithrombin-heparin binary complex. The
maximal accelerations of the two heparins in this case correlated with
the inhibitor fluorescence enhancements induced by the polysaccharides,
consistent with the accelerations arising from conformational
activation of antithrombin.
H NMR difference spectroscopy
of antithrombin complexes with LAH and HAH and competitive binding
studies were consistent with LAH accelerating activity being mediated
by binding to the same site on the inhibitor as HAH. These results
demonstrate that LAH accelerates antithrombin-proteinase reactions by
bridging and conformational activation mechanisms similar to those of
HAH, with the reduced magnitude of LAH accelerations resulting both
from a decreased antithrombin affinity and the inability to induce a
full activating conformational change in the inhibitor.
Proteins
Antithrombin was purified from outdated
human plasma by heparin-agarose, DEAE-Sepharose and Sephacryl S-200
chromatography as described previously
(19, 26) . Human
-thrombin was a generous gift of Dr. John Fenton, New York State
Department of Health, Albany, NY. Human
-factor Xa was prepared by
activation of purified factor X followed by chromatography on soybean
trypsin inhibitor-agarose as described elsewhere
(27) and, in
part, generously provided by Dr. Paul Bock of Vanderbilt University,
Nashville, TN. All proteins were judged pure by SDS-gel
electrophoresis. Antithrombin concentrations were determined from the
absorbance at 280 nm using a molar extinction coefficient of 37,700
M
cm
(28) .
Proteinase concentrations were determined by active-site titration as
described previously
(11) which indicated >70% active enzyme
preparations.
Polysaccharides
Low affinity heparin was isolated
from purified, size-fractionated heparin with a molecular weight of
7900 ± 10% by repeated antithrombin-agarose affinity
chromatography as previously described
(19) . The anti-factor Xa
activity was measured after each affinity-purification step from the
increase produced by 0.8 µ
M polysaccharide on the pseudo
first-order rate constant for the inactivation of 20 n
M factor
Xa by 200 n
M antithrombin. After three such steps, the
anti-factor Xa activity was reduced to 0.08 ± 0.01% of the
activity of high affinity heparin with no further reduction observed
after a fourth step. The resulting anticoagulant activity of the low
affinity heparin measured from the prolongation of the plasma clotting
time
(29) was 1 ± 0.2% that of high affinity
heparin.(
)
High affinity heparin was a
preparation previously described, having the same molecular weight as
the low affinity polysaccharide
(11) . For NMR difference
spectra, low affinity and high affinity heparins with a molecular
weight of
10,000 and with anti-factor Xa activities comparable to
the 7900 M
heparins were employed. The
-methyl glycoside of the pentasaccharide corresponding to the
antithrombin binding sequence in heparin was synthesized as described
elsewhere
(30) . Dextran sulfate with an average molecular
weight of 8000 was from Sigma. Fluorescein-labeled high affinity
heparin was prepared by reacting intrinsic free amino groups in the
7900 M
high affinity polysaccharide with
fluorescein 5-isothiocyanate (Molecular Probes) and purifying the
labeled from the unlabeled polysaccharide by hydrophobic chromatography
on octyl-Sepharose, as previously described
(31, 32) .
The labeled polysaccharide was further purified by antithrombin-agarose
affinity chromatography. A labeling ratio of 0.90 ± 0.02 mol of
fluorescein/mol of heparin was determined by analysis for heparin by an
Azure A dye binding assay
(33) and for fluorescein from the
absorbance at 493 nm at pH 8.3 using an extinction coefficient of
68,000
M
cm
. The latter
value was determined for fluorescein isothiocyanate reacted with a
200-fold molar excess of ethanolamine under the same conditions.
Titrations of antithrombin with the labeled heparin monitored by
protein fluorescence changes (see below) indicated a binding
stoichiometry and affinity similar to that of unlabeled high affinity
heparin.
Exchange of Fluorescein-labeled High Affinity Heparin
into Low Affinity Heparin
A mixture of 3 mg of low affinity
heparin and 0.03 mg of fluorescein-labeled high affinity heparin or
0.03 mg of labeled high affinity heparin alone in 3.3 ml of
equilibration buffer was applied to an antithrombin-agarose affinity
column (30 mg of antithrombin coupled to 10 ml of Affi-gel 15 from
Bio-Rad; capacity, 2 mg of high affinity heparin) equilibrated in
20 m
M sodium phosphate, 0.25
M NaCl, 0.1 m
M EDTA, pH 7.4. Following elution of low affinity heparin with the
equilibration buffer, high affinity heparin was eluted by increasing
the NaCl concentration in the buffer to 3
M. Fractions (1.8
ml) were assayed for total heparin by the Azure A dye binding method
and for labeled heparin by fluorescence detection at
495 nm (excitation),
515 nm (emission).
Experimental Conditions
All experiments were
conducted at 25 °C in an ionic strength ( I) 0.15, pH 7.4
buffer consisting of 20 m
M sodium phosphate, 0.1
M NaCl, 0.1 m
M EDTA, 0.1% polyethylene glycol 8000 except
where otherwise noted.
Binding Studies
Binding of low affinity and high
affinity heparins and heparin pentasaccharide to antithrombin was
analyzed by fluorescence titrations in which the enhancement in protein
tryptophan fluorescence accompanying polysaccharide binding was used to
monitor the interaction
(6, 9, 22) . High
affinity heparin titrations were done with 10 n
M antithrombin,
whereas low affinity heparin titrations employed either 1 or 10
µ
M inhibitor. Measurements were made on an SLM 8000
spectrofluorometer with fluorescence detection at 280 and
340 nm except for low affinity heparin
titrations of 10 µ
M antithrombin, in which
was set at 295 nm to avoid inner filter corrections. Because the
stoichiometry for low affinity heparin binding to antithrombin was
indeterminate under the conditions of measurement, the 1:1 binding
stoichiometry determined for high affinity heparin binding was assumed
for low affinity heparin binding. Corrections for background
fluorescence of the titrant were required for low affinity heparin
(maximally
10% for titrations of 1 µ
M antithrombin
and <1% for titrations of 10 µ
M inhibitor) and made by
subtracting the signal observed in parallel titrations of
polysaccharide into buffer. Titrations were analyzed by computer
fitting to the quadratic binding equation
(26) .
NMR Spectroscopy
H NMR spectra of
antithrombin and antithrombin-heparin complexes were recorded at 500
MHz on a Bruker AMX500 spectrometer. Difference spectra were generated
by computer subtraction as described previously
(10) .
Kinetic Studies
The kinetics of low affinity
heparin acceleration of antithrombin-proteinase reactions were measured
under pseudo first-order conditions. In one set of experiments, 20
n
M antithrombin was reacted with 1 n
M thrombin or
with 2 n
M factor Xa at increasing concentrations of heparin or
dextran sulfate. In some experiments, 0.5 or 1 µ
M heparin
pentasaccharide was also present. Reaction mixtures of 50 µl were
incubated for increasing times in polyethylene glycol-coated
polystyrene cuvettes
(34) and then quenched with 1 ml of 50
µ
M tosyl-Gly-Pro-Arg-7-amido-4-methyl coumarin (Sigma) in
the case of the thrombin reaction or with 1 ml 100 or 200 µ
M Spectrozyme FXa (American Diagnostica) in the case of the factor
Xa reaction. Both substrates were in a high salt buffer consisting of
20 m
M sodium phosphate, 1.0
M NaCl, 1 m
M EDTA, 0.1% polyethylene glycol 8000, pH 7.4, plus 0.1 mg/ml
Polybrene (Aldrich) (except for dextran sulfate reactions where
Polybrene was absent) to quench the reaction. The residual enzyme
activity was then determined from the initial linear rate of substrate
hydrolysis monitored by the increase in fluorescence of
7-amido-4-methyl coumarin at 380 nm and
440 nm or by the increase in p-nitroaniline absorbance
at 405 nm. p-Aminobenzamidine (2 m
M) was included in
the high affinity heparin-catalyzed antithrombin-thrombin reaction to
reduce the reaction rate sufficiently for analysis by the discontinuous
sampling method. Observed pseudo first-order rate constants
( k
) were obtained from the slope of semilog
plots of the decrease in enzymatic activity with time or by computer
fitting the decay of enzyme activity by an exponential function
(26) .
340
nm and
370 nm and with excitation and emission slits
of 2 nm and 32 nm, respectively. Observed rate constants were obtained
by fitting fluorescence decay curves from four to five averaged
reaction traces by a single exponential function.
on polysaccharide concentration determined
from studies at low antithrombin concentrations (20 n
M) was
computer fit by equations previously shown to describe the two
different mechanisms of polysaccharide acceleration of
antithrombin-proteinase reactions, i.e. either by a mechanism
requiring antithrombin and proteinase to bind to the same heparin chain
or one requiring just antithrombin to bind heparin
(11, 19, 20) . For the former mechanism, which
describes the heparin-catalyzed antithrombin-thrombin reaction, the
equation for k
is given by
on heparin
concentration was fit by a mechanism requiring heparin binding to just
antithrombin. This dependence is given by the equation
(11) ,
and k
given in
. Similarly, k
was fixed at the
value determined for the uncatalyzed reaction, since this parameter was
poorly determined and indistinguishable from the uncatalyzed rate
constant. Reported errors represent ± 2 S.E.
Purity of Low Affinity Heparin
Heparin with low
antithrombin affinity was purified by repeated antithrombin-agarose
affinity chromatography to a constant specific anti-factor Xa activity
(see ``Materials and Methods'') equal to 0.1% that of
high affinity heparin. To determine whether this activity was an
intrinsic property of low affinity heparin or was due to small amounts
of residual high affinity heparin, 30 µg of fluorescein-labeled
high affinity heparin were added to 3 mg of low affinity heparin, and
the exchange of labeled polysaccharide into unlabeled polysaccharide
was measured after separating the polysaccharide mixture by
antithrombin affinity chromatography. Fluorescein high affinity heparin
was quantitatively separated from the low affinity polysaccharide by
this procedure, as indicated by the appearance of 99% of the applied
fluorescence in the high affinity polysaccharide fraction
(Fig. 1). The residual
1% fluorescence, corresponding to
0.3 µg of labeled heparin which coeluted with low affinity
heparin was also found in control experiments in which just the labeled
high affinity polysaccharide was chromatographed (Fig. 1).
Subsequent G-25 chromatography of this nonbinding fluorescent fraction
revealed it to be free fluorescein (>90%) that had hydrolyzed from
the labeled polysaccharide during the experiment. This indicated the
presence of <0.03 µg or <0.001% high affinity heparin in the
low affinity polysaccharide.
(
)
The levels of high
affinity heparin predicted to be present in low affinity heparin in the
above experiment assumed that the anti-factor Xa activity of high
affinity heparin would not be affected by the low affinity
polysaccharide. This assumption was confirmed by the finding that
addition of 0.2% high affinity heparin to low affinity heparin
increased the anti-factor Xa activity of the latter to an extent equal
to that of the high affinity polysaccharide alone. Together, these
observations suggested that the activity of low affinity heparin was
not due to contaminating high affinity heparin.
Figure 1:
Exchange of fluorescein-high affinity
heparin into low affinity heparin after antithrombin-agarose
chromatography of a mixture of the polysaccharides. Experimental
details are given under ``Materials and Methods.''
Chromatography of a mixture of 3 mg of low affinity heparin and 0.03 mg
of high affinity heparin ( circles) or of 0.03 mg of high
affinity heparin alone ( triangles) was monitored by assays for
total heparin ( open circles) and for fluorescent-labeled
heparin ( closed circles and open triangles). The
arrow depicts the change from low to high salt elution
buffer.
Binding of Low Affinity Heparin to Antithrombin
To
characterize the mechanism by which low affinity heparin accelerates
antithrombin-proteinase reactions, binding of the polysaccharide to
antithrombin was quantified by monitoring the protein fluorescence
enhancement previously shown to accompany such binding. Titrations of
antithrombin with low affinity heparin resulted in a saturable 8
± 3% enhancement in protein fluorescence and an average
dissociation constant (from four titrations) of 19 ± 6
µ
M (Fig. 2), in agreement with previous studies
(6, 7, 22) . Similar titrations of antithrombin
with high affinity heparin gave a 47 ± 7% fluorescence
enhancement and a Kof 19 ± 3
n
M (average from four titrations); i.e. 1000-fold
lower than the K
for low affinity heparin
binding to antithrombin (Fig. 2). Contrasting this large
differential affinity of the two heparins for antithrombin, the two
polysaccharides bound thrombin with indistinguishable
K
values of 0.7 ± 0.1 µ
M and 0.9 ± 0.1 µ
M, respectively, as shown by
previous equilibrium binding studies
(19) (Fig. 2). These
results demonstrated marked differences in the relative binding
affinities of antithrombin and thrombin for the two heparins; i.e. a 100-fold higher affinity of antithrombin than of thrombin for
high affinity heparin, but a 20-fold greater affinity of thrombin than
of antithrombin for low affinity heparin.
Figure 2:
Equilibrium binding of low affinity
heparin and high affinity heparin to antithrombin and thrombin.
Titrations of 10 n
M antithrombin with high affinity heparin
() or of 10 µ
M antithrombin with low affinity
heparin (
) were monitored by changes in intrinsic protein
fluorescence as described under ``Materials and Methods.''
Titrations of thrombin with high affinity heparin (
) or with low
affinity heparin (
) are taken from Olson (19) and were monitored
from the 17% quenching of the fluorescence of active site-bound
p-aminobenzamidine. Solid curves are computer fits of
data by the quadratic binding equation.
NMR Difference Spectroscopy of Antithrombin-Heparin
Complexes
H NMR difference spectroscopy was also
used to compare the binding of low and high affinity heparins to
antithrombin. The aromatic/amide region is particularly suited to
making such a comparison, since several of the relatively few
resonances in this region that are perturbed by heparin binding have
been assigned to specific residues that lie in or close to the heparin
binding site. Thus, prominent signals in the difference spectrum arise
from the side-chains of tryptophan 49
(36) , histidines 1, 65,
and 120
(37) , and tyrosine 131.
(
)
The
perturbation of all of these side chains in a similar, though not
identical, manner by both low and high affinity heparins (Fig. 3)
indicates that both heparins bind at the same site and perturb the same
contact residues. The aliphatic region of the difference spectrum (not
shown) is less straightforward to interpret since there are many more
perturbed and overlapping resonances, and no residue-specific resonance
assignments. Perturbations arise not only from surface residues in the
heparin binding site, but also from buried residues, that are only
indirectly perturbed by heparin, through the heparin-induced
conformational change
(6, 7, 8, 9, 10, 11) .
In this region the two heparins produce significantly different
difference spectra, consistent with the conformational changes induced
by the two species differing in kind or extent.
Figure 3:
Comparison of the H NMR
difference spectra between antithrombin and antithrombin-heparin
complex for ( A) low affinity heparin and ( B) high
affinity heparin. Spectra representing the average of 4000 scans were
collected at 300 K for antithrombin alone (110 µ
M) or of
antithrombin-heparin complex (110 µ
M, 1:1 mole ratio of
antithrombin:heparin). The samples were in 20 m
M sodium
phosphate buffer, pH 7.15, containing 0.10
M NaCl. Negative
peaks arise from residues in antithrombin that are perturbed by heparin
binding. Positive peaks are from the same residues, but at the chemical
shifts that obtain in the antithrombin-heparin complex. Assignments
(for negative peaks) are: 8.14 ppm, His-65 C(2); 7.87 ppm, His-1 C(2);
7.74 ppm, His-120 C(2); 7.45 ppm, Trp-49; 7.06 ppm, His-1 C(4); 6.64
ppm, Tyr-131.
Kinetics of Low Affinity Heparin Acceleration of
Antithrombin-Proteinase Reactions
Increasing concentrations of
low affinity heparin progressively enhanced pseudo first-order rate
constants ( k) for the inactivation of thrombin
and factor Xa by antithrombin. However, the maximum rate enhancements
were lower and the polysaccharide concentrations required to reach
these maxima were considerably higher than those obtained with high
affinity heparin (Fig. 4). Thus, low affinity heparin maximally
enhanced k
for the antithrombin-thrombin
reaction 60 ± 10-fold, as compared to 4400 ± 400-fold for
high affinity heparin, and this enhancement required
100-fold
higher concentrations of polysaccharide. Similarly, k
for the antithrombin-factor Xa reaction was maximally enhanced
140 ± 30-fold by low affinity heparin, as compared to the 600
± 50-fold rate enhancement produced by high affinity heparin,
and this reduced rate enhancement required
1000-fold higher levels
of polysaccharide. Dextran sulfate with a chain-length similar to that
of the two heparins also accelerated both antithrombin-proteinase
reactions, although to an extent which was lower than that found for
low affinity heparin; i.e.
10-fold for the
antithrombin-thrombin reaction and
40-fold for the
antithrombin-factor Xa reaction.
Figure 4:
Polysaccharide concentration dependence of
the acceleration of antithrombin-proteinase reactions by high affinity
heparin, low affinity heparin, and dextran sulfate. Pseudo first-order
rate constants ( k) for
polysaccharide-accelerated reactions of 20 n
M antithrombin
with 1 n
M thrombin ( circles) or with 2 n
M factor Xa ( triangles) were measured as a function of
polysaccharide concentration as described under ``Materials and
Methods.'' Solid lines are computer fits of data by
Equations 1 and 2 of the text, with K
,
K
, k
(for high affinity
heparin-accelerated reactions), k
(for low
affinity heparin and dextran sulfate-accelerated reactions), and
k
being the fitted parameters.
k
values given on the left- and
right-hand axes for the high affinity heparin-accelerated
reactions refer to thrombin and factor Xa reactions, respectively. The
former k
values are 32.2-fold greater than
measured values due to their correction for the competitive effect of
p-aminobenzamidine.
Notably, the dependence of
kon polysaccharide concentration was similar
when the effects of the two heparin species or dextran sulfate on the
same antithrombin-proteinase reaction were compared. However, this
dependence differed for the two antithrombin-proteinase reactions,
consistent with the different mechanisms of heparin acceleration of
these two reactions demonstrated previously
(11) . The
polysaccharide concentration dependence of k
was
thus bell-shaped for the antithrombin-thrombin reactions, suggesting
that low affinity heparin and dextran sulfate act by bridging
antithrombin and thrombin in a ternary complex, as previously shown for
the high affinity heparin-accelerated reaction
(14, 15, 16, 17, 18, 19, 20, 21) .
Fitting of the low affinity heparin data by this mechanism suggested
that the ascending portion of the bell-shaped dependence reflected the
assembly of a ternary complex from a binary protein-heparin complex
with a K
(2 ± 1 µ
M)
similar to that measured for the thrombin-heparin interaction. The fit
further indicated that the descending portion of the bell-shaped curve
corresponded to antagonism of ternary complex formation by a
protein-heparin binary complex with a K
(70 ± 40 µ
M) approaching that determined for
the antithrombin-heparin interaction, given the error associated with
the fitted parameters. The reverse situation was apparent for the high
affinity heparin-accelerated reaction; i.e. the ascending
portion of the bell-shaped curve corresponded to ternary complex
assembly via an intermediate antithrombin-heparin binary complex
( K
6 ± 4 n
M), while the
descending portion reflected the antagonism of this assembly by a
thrombin-heparin binary complex ( K
1.1
± 0.4 µ
M).
, reflecting a simple saturation
curve, was evident for the reactions accelerated by both low- and high
affinity heparin, as well as dextran sulfate. For the two
heparin-enhanced reactions, the K
for
this saturation curve (8 ± 3 n
M for high affinity
heparin and 30 ± 10 µ
M for low affinity heparin)
corresponded closely to that directly measured for the
antithrombin-heparin binary complex interaction. The data are thus
consistent with prior studies which have indicated that heparin
accelerates factor Xa inhibition primarily by conformational activation
of antithrombin, with no significant role for a proteinase-heparin
interaction
(11, 13, 23, 25, 38, 39) .
summarizes the second-order rate constants obtained from
computer fits of the data of Fig. 4for the
polysaccharide-accelerated antithrombin-proteinase reactions at
saturation of the appropriate binary protein-polysaccharide complexes
(see``Materials and Methods'').
Rapid Kinetic Evidence for Low Affinity Heparin Bridging
of Antithrombin and Thrombin in a Ternary Complex
Rapid kinetic
studies were performed to provide direct evidence for the assembly of
the putative ternary complex in the low affinity heparin-accelerated
antithrombin-thrombin reaction and to determine the rate at which
thrombin and antithrombin react in this complex. The kinetics were
analyzed as a function of low affinity heparin concentration in a range
where thrombin-heparin but not antithrombin-heparin binary complexes
would be saturated (10 µ
M polysaccharide) and at
increasing, fixed concentrations of antithrombin, over which ternary
complex saturation was anticipated
(15, 19) . In these
studies, antithrombin binding to proteinase was monitored by the
decrease in fluorescence accompanying the displacement of the probe,
p-aminobenzamidine, from the proteinase active-site as the
inhibitor is bound. A saturable dependence of k
on heparin concentration was observed for each antithrombin
concentration, and this dependence appeared to parallel the saturation
of thrombin by the low affinity polysaccharide in each case, similar to
the data of Fig. 4(Fig. 5). However, the limiting rate
constant reached for each saturation curve increased in a
nonproportional manner with increasing inhibitor concentration,
suggesting progressive saturation of an intermediate
thrombin-heparin-antithrombin ternary complex. A global fit of the data
by equations describing the ternary complex mechanism (see
``Materials and Methods'') indicated a dissociation constant
for ternary complex formation of 10 ± 4 µ
M and
first-order rate constant for reaction of antithrombin and thrombin in
the ternary complex of 5 ± 1 s
. Comparison
with the kinetic parameters previously measured for the high affinity
heparin-accelerated reaction (Fig. S1) indicate a 100-fold
increased ternary complex K
but a similar
rate constant for antithrombin-thrombin reaction within the ternary
complex for the low affinity heparin accelerated reaction.
Figure 5:
Heparin and antithrombin concentration
dependence of the acceleration of the antithrombin-thrombin reaction by
low affinity heparin. kvalues for reactions of
antithrombin with 0.25 µ
M thrombin were measured as a
function of the low affinity heparin concentration at different fixed
antithrombin concentrations of 10 µ
M (
), 20
µ
M (
), 50 µ
M (
), and 100
µ
M (
) by stopped-flow fluorometry as detailed under
``Materials and Methods.'' Solid lines are fits to
equations describing the ternary complex bridging model for heparin
action with k
, K
, and
K
the fitted parameters. K
was fixed at the value of 19 µ
M determined by
equilibrium binding.
Figure S1:
Rapid Kinetic Evidence for Acceleration of Factor Xa
Inactivation by Antithrombin-Low Affinity Heparin Binary
Complex
Rapid kinetic studies were also used to verify that the
acceleration of the antithrombin-factor Xa reaction by low affinity
heparin is mediated by an antithrombin-heparin binary complex. The
analyses were done as a function of low affinity heparin concentration
at a fixed inhibitor concentration of 10 µ
M and also as a
function of antithrombin concentration at a fixed heparin concentration
of 10 µ
M. Reactions were monitored as above from the
decrease in fluorescence accompanying the displacement of
p-aminobenzamidine from the factor Xa active site. In each
case, a similar saturable dependence of kwas
found, reflecting a K
(38 ± 17
µ
M) in reasonable agreement with the value directly
measured for the antithrombin-low affinity heparin binary complex
interaction (Fig. 6). The 70 ± 20-fold rate enhancement obtained
from these data was also in fair agreement with the kinetic data of
Fig. 4
, considering the smaller data set analyzed in this case.
These data confirmed that the reaction proceeded as a second-order
association between antithrombin-heparin binary complex and free factor
Xa, with no evidence for factor Xa binding to heparin being involved in
the rate enhancement.
Effect of the Antithrombin-binding Heparin
Pentasaccharide Fragment on Low Affinity Heparin Rate
Enhancements
To confirm that the acceleration of
antithrombin-proteinase reactions by low affinity heparin involved
binding of the polysaccharide to the same site on antithrombin that
mediates high affinity heparin binding, we examined the effect on these
accelerations of the sequence-specific pentasaccharide responsible for
high affinity heparin binding. Equilibrium binding titrations of
antithrombin with pentasaccharide in the absence and presence of low
affinity heparin (140-160 µ
M), monitored by changes
in protein fluorescence, demonstrated that low affinity heparin
increased the apparent Kfor
pentasaccharide binding to antithrombin to an extent compatible with
low affinity heparin competing with the pentasaccharide for binding to
the inhibitor (). Saturation of antithrombin with
pentasaccharide levels sufficient to block low affinity heparin binding
(0.5 µ
M) nevertheless did not prevent low affinity heparin
from accelerating the antithrombin-thrombin reaction in a
concentration-dependent manner above the 1.7-fold acceleration produced
by the pentasaccharide alone (Fig. 7). This rate enhancement
exhibited a bell-shaped curve similar to that observed when low
affinity heparin was varied in the absence of pentasaccharide. However,
the rate enhancement was considerably reduced from that of low affinity
heparin alone, i.e. from 60-fold to 16-fold ().
Doubling of the pentasaccharide concentration minimally affected
measured k
values, suggesting that the maximum
pentasaccharide effect on the low affinity heparin acceleration was
being observed. Fitting of the data by the ternary complex mechanism
indicated a similar K
for a
thrombin-heparin binary complex intermediate mediating ternary complex
assembly (0.7 ± 0.3 µ
M), but higher
K
for antagonism of ternary complex
formation by antithrombin-heparin complexes (200 ± 100
µ
M), although there was considerable uncertainty in the
latter value. These data are consistent with pentasaccharide binding to
antithrombin reducing its affinity for the full-length heparin and
thereby the ability of the full-length polysaccharide to bridge
antithrombin and thrombin in a ternary complex.
Figure 7:
Effect of the heparin pentasaccharide on
the acceleration of the antithrombin-thrombin reaction by low affinity
heparin. kvalues for the reaction of 20 n
M antithrombin with 1 n
M thrombin were measured as a
function of the low affinity heparin concentration in the absence
(
) or presence of 0.5 µ
M (
) or 1 µ
M (
) pentasaccharide. Data obtained in the absence of
pentasaccharide are reproduced from Fig. 4. Solid curves are
fits of data by the ternary complex-bridging model described under
``Materials and Methods'' in which K
,
K
, and k
were the
fitted parameters.
The effect of the
pentasaccharide on the low affinity heparin-accelerated
antithrombin-factor Xa reaction was similarly studied. Saturation of
antithrombin with the pentasaccharide produced a 230 ± 30-fold
enhancement of the antithrombin-factor Xa reaction rate constant, in
fair agreement with earlier reports
(11, 13) . Addition
of low affinity heparin to the pentasaccharide-saturated antithrombin
at levels as high as 50 µ
M had no significant effect on
k(), suggesting that the
pentasaccharide blocks the low affinity heparin acceleration of this
reaction. These results support a requirement for low affinity heparin
binding to the pentasaccharide site on antithrombin for enhancing the
rate of the antithrombin-factor Xa reaction.
values for the ascending and descending limbs of the bell-shaped
curve characterizing the high affinity heparin accelerated reaction to
those of antithrombin-heparin and thrombin-heparin binary complex
interactions, respectively. It follows that the greater affinity of
thrombin than of antithrombin for low affinity heparin should result in
a reverse order of ternary complex assembly primarily through a
thrombin-heparin binary complex intermediate. This expectation is borne
out by the observations that 1) the bell-shaped curve for the low
affinity heparin-accelerated reaction is displaced to higher
polysaccharide concentrations and 2) the ascending and descending limbs
of this curve correspond to K
values
similar to those measured for thrombin-heparin and antithrombin-heparin
binary complex interactions, respectively.
of
1 m
M for the initial
encounter complex interaction of thrombin with antithrombin and rate
constant of 10 s
for the subsequent reaction of
inhibitor with proteinase in this complex measured in the absence of
heparin
(15) , indicates that both heparins act by promoting the
encounter of enzyme and inhibitor in the ternary complex without
significantly affecting the rate of conversion of this complex to a
stable complex. The 10,000-fold enhancement of the enzyme-inhibitor
encounter complex interaction by high affinity heparin was previously
shown to arise from a thrombin-heparin bridging interaction increasing
the affinity of thrombin for antithrombin in the ternary complex
(20) . The 100-fold promotion of the enzyme-inhibitor encounter
by low affinity heparin can similarly be accounted for by an
antithrombin-heparin bridging interaction stabilizing the association
of antithrombin with heparin-bound thrombin in the ternary complex. The
antithrombin-heparin binary complex K
suggests more than sufficient binding energy to produce a
100-fold enhancement in the ternary encounter complex
K
. The excess binding energy may arise
from the need to correct the binary complex K
for the anticipated large number of overlapping interaction sites
on the polysaccharide for the inhibitor and/or to different numbers of
ionic interactions involved in binary and ternary complexes
(20, 40) . Our results are thus consistent with the
reduced rate-enhancing effect of low affinity heparin relative to high
affinity heparin resulting from the reduced affinity of the
polysaccharide for antithrombin diminishing the ability of heparin to
bridge antithrombin and thrombin in the ternary complex intermediate.
values
describing these saturation curves to the K
values for the binding of the two heparins to antithrombin
indicate the rate enhancements produced by both heparins arise from an
interaction of the polysaccharide with antithrombin. Moreover, the
failure to observe any diminution of the accelerating effect of either
heparin at the highest concentrations of polysaccharide examined
implies that a factor Xa-heparin bridging interaction contributes
minimally or not at all to heparin rate enhancement.
Table: Second-order rate constants (k) for
polysaccharide-accelerated antithrombin-proteinase reactions
Table: Effect of low
affinity heparin on the apparent dissociation constant for
pentasaccharide binding to antithrombin
3 or 2.7 µg
of labeled high affinity heparin into the low affinity heparin,
assuming the affinity separation procedure results in 0.1% residual
high affinity heparin in the low affinity heparin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.