From the Center for Molecular Biology of Oral
Diseases, College of Dentistry, University of Illinois at Chicago,
Chicago, Illinois 60612 and § Department of Haematology,
Hammersmith Hospital Campus, Faculty of Medicine, Imperial College,
London W12 0NN, United Kingdom
Received for publication, January 3, 2003, and in revised form, February 12, 2003
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ABSTRACT |
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A novel variant of antithrombin, the major serpin
inhibitor of coagulation proteases, has been identified in a patient
with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one
of the two alleles was abnormal due to an in-frame deletion of the
codon for the P1 arginine residue. The abnormal antithrombin was
separated from the normal inhibitor by complexing the latter with
thrombin followed by heparin-agarose affinity chromatography. The
purified variant, antithrombin London, was completely inactive as a
thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a KD
reflecting an ~10-fold greater affinity than the normal inhibitor.
Stopped-flow kinetic analysis showed that this was almost entirely due
to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction.
These results suggest that deletion of the antithrombin P1 residue
partially activates the serpin by inducing strain in the reactive
center loop, which destabilizes the native loop-buried state and favors
the activated loop-exposed state with high heparin affinity. The
unusually severe thrombosis associated with the heterozygous mutation
may be explained by the ability of antithrombin London to bind
endogenous heparan sulfate or heparin molecules with high affinity and
to thereby block activation of the normal inhibitor.
Antithrombin, a member of the serpin family of protein proteinase
inhibitors, is the principal regulator of blood coagulation proteinases
in plasma (1, 2). The essential role of this protein in hemostasis and
in sustaining life is clear from the association of inherited or
acquired deficiencies of the serpin in humans with an increased risk of
thrombosis (2) and from the finding that complete deficiency in mice
results in embryonic lethality due to a consumptive coagulopathy (3).
Efficient regulation of clotting proteinases by antithrombin requires
the polysaccharide cofactors heparin and heparan sulfate, which act to
increase the moderate rates of inhibition of clotting proteinases by
the serpin several thousand-fold (1). Only a fraction of heparin
molecules are functional in this regard due to their possessing a
specific pentasaccharide sequence capable of binding antithrombin with
high affinity and inducing activating conformational changes in the
serpin (4-7). Endothelial cell surface heparan sulfate molecules are
thought to be to be the natural activator of antithrombin, but only a
limited fraction (~1%) of such molecules contain the pentasaccharide
sequence and are anticoagulantly active (8). The crystal structures of
antithrombin free and complexed with the heparin pentasaccharide reveal
that pentasaccharide binding to an allosteric site on the inhibitor
transmits conformational changes to a reactive proteinase binding loop
on the inhibitor surface that enhances the loop accessibility to
proteinases (9, 10). However, mutagenesis studies suggest that not only
the proteinase binding loop common to serpins but also a unique exosite region adjacent to the loop are made accessible for proteinase interaction as a result of heparin activation (11).
Natural variants of antithrombin have provided valuable insights into
the anticoagulant function of the serpin. Such variants initially led
to the identification of the pentasaccharide binding site as well as
verification of the proteinase binding site of the inhibitor before the
crystal structures became available (1, 2). Antithrombin variants have
additionally provided significant insights into the relative
physiologic importance of antithrombin interactions with clotting
proteinases and heparin (2). Individuals expressing variant
antithrombins with abnormal proteinase binding are thus typically
heterozygous for the genetic defect and are usually at risk for
thrombosis. However, those individuals expressing a variant
antithrombin with a defect in heparin binding may be heterozygous or
homozygous, and only homozygotes are typically more susceptible to thrombosis.
In the present report we describe a novel dysfunctional variant of
antithrombin associated with early onset thrombosis in the
proband and a clear family history of thrombosis in affected individuals. Sequencing of the gene for the variant revealed an in-frame deletion of the P1 Arg of the serpin. Purification of the
variant antithrombin confirmed the complete absence of inhibitory activity but also revealed an abnormally high affinity for heparin. The
latter property was shown to result in the variant acting as an
effective antagonist of the heparin-catalyzed inhibition of clotting
proteinases by normal antithrombin, providing an explanation for the
unusually severe thrombosis associated with this mutation. The
abnormally high heparin affinity of the variant suggests that the
deletion of the reactive loop residue causes activation of the variant
without heparin and provides new insights into the structural basis of
heparin activation.
Coagulation Tests--
Patient antithrombin activity was
measured as heparin cofactor activity using a commercially available
kit (Dade-Behring, Deerfield, IL). Antithrombin antigen was measured
using antibody-coated microlatex particles in a commercially available
kit (Diagnostica Stago, Parsippany, NJ). The reference plasmas used
were 100% reference plasma from Technoclone for antithrombin activity
and the 7th British standard supplied by the National Institute for
Biological Standards and Control for antithrombin antigen.
Routine testing did not identify any other thrombophilic trait in the
proband (protein C and protein S were normal, and the proband tested
negative for the genetic lesions, Factor V Leiden, and prothrombin
20210A). Crossed immunoelectrophoresis of antithrombin with heparin
included in the gel was performed according to Sas et al.
(12) as described (13).
Sequencing of the Variant Antithrombin Gene--
Genomic DNA was
extracted from peripheral blood leukocytes by standard methods. The 7 exons (and their flanking regions) were amplified by the polymerase
chain reaction using primers described in full elsewhere (14).
Amplified products were sequenced using an ABI 377 sequencer.
Purification of Normal and Variant Antithrombins--
Normal
antithrombin was purified from outdated human plasma by chromatography
on heparin-Sepharose, DEAE-Sepharose, and Sephacryl S-200 columns as
previously described (15). The variant antithrombin was isolated from
~200 ml of plasma of the proband by adsorption to 20 ml of
heparin-Sepharose gel equilibrated in 0.1 M Tris-Cl, 0.15 M NaCl, pH 7.4, washing the gel with equilibrating buffer containing 0.40 M NaCl to remove unbound protein, and then
step-eluting the bound antithrombin with buffer containing 2.5 M NaCl. SDS-PAGE of the purified protein revealed a major
antithrombin band in addition to a minor protein contaminant. After
dialysis into 0.1 M NaHCO3, 0.15 M
NaCl, pH 8.3, the protein was repurified by binding to
heparin-Sepharose and eluting with a NaCl gradient according to the
procedure used to purify normal antithrombin from plasma. Protein
fractions eluting at the end of the gradient were pooled, concentrated,
and dialyzed into 20 mM sodium phosphate, 0.1 M NaCl, 0.1 mM EDTA, pH 7.4. The concentration of normal
functional antithrombin in the purified inhibitor was assessed by
titrating 100 nM thrombin with 0-2 molar eq of purified
inhibitor in I 0.15 buffer. After overnight incubation at 25 °C, the
residual thrombin activity was determined from the initial rate of
hydrolysis of the substrate, S-2238, monitored from the change in
absorbance at 405 nm. The amount of active antithrombin was
determined from the abscissa intercept of a plot of the linear decrease
in activity versus the molar ratio of inhibitor to enzyme
added. A 10% molar excess of thrombin over the normal antithrombin
found in the preparation (5.8 µM) was then added, and the
mixture was incubated for 20 min at 25 °C, a time sufficient to
fully complex the functional inhibitor based on the measured inhibition
rate constant (>99.9%). Any remaining thrombin was inactivated by the
addition of 30 µM D-Phe-Pro-Arg-chloromethyl
ketone (Calbiochem), and the mixture was chromatographed in ~5-mg
batches on a 5-ml Hi-Trap Heparin-Sepharose column (Amersham
Biosciences) equilibrated in dialysis buffer. After loading the sample
at 0.5 ml/min, the column was washed with equilibrating buffer for 10 min, a gradient from 0.1 to 3 M NaCl in the same buffer was
applied over the next 35 min, and elution was then continued with 3 M NaCl limit buffer for 15 min, all at a flow rate of 1 ml/min. Two incompletely resolved peaks detected by protein
fluorescence eluted between 18 and 28 min followed by a well resolved
peak eluting between 35 and 50 min. Fractions containing the high
heparin affinity variant antithrombin peak were pooled, concentrated,
and dialyzed into equilibration buffer. Concentrations of normal and
variant antithrombins were determined from the absorbance at 280 nm
using an extinction coefficient of 37,700 M Heparins--
The pentasaccharide corresponding to the
antithrombin binding sequence in heparin and a variant of this sequence
having higher affinity for antithrombin (compound 83 in van Boeckel and
Petitou (17)) were synthesized by Sanofi Recherche and generously
provided by Dr. Maurice Petitou. A full-length heparin of ~26
saccharides containing the pentasaccharide binding sequence for
antithrombin was isolated from commercial heparin by size and
antithrombin affinity fractionation as described (15). Concentrations
of all heparins were determined by stoichiometric fluorescence
titrations of plasma antithrombin with the saccharides as described (7, 15).
Proteinases--
Human SDS and Native PAGE--
SDS-PAGE and nondenaturing
electrophoresis of normal and variant antithrombins and of their
interactions with thrombin, heparin, and neutrophil elastase (Athens
Research and Technology) were done with the Laemmli discontinuous
buffer system (21). Samples containing thrombin or neutrophil elastase
were treated with a >100-fold molar excess of
D-Phe-Pro-Arg-chloromethyl ketone or methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (Bachem), respectively, before denaturation and electrophoresis. For
reactions of antithrombin with neutrophil elastase, 5 µM
normal or variant antithrombin was mixed with 0.1 µM
elastase with or without 5 µM heparin pentasaccharide at
25 °C, and samples were taken after 5-80 min and quenched with
chloromethyl ketone inhibitor before electrophoresis.
Experimental Conditions--
All experiments were performed at
25 °C in 20 mM sodium phosphate buffers containing 0.1 mM EDTA and 0.1% polyethylene glycol 8000 adjusted to pH
7.4. NaCl was either absent or added to 0.10 or 0.25 M to
give ionic strengths of 0.05, 0.15, and 0.30.
Heparin Binding to Antithrombin--
Normal or variant
antithrombins were titrated with heparin, and binding of the saccharide
was monitored from the increase in tryptophan fluorescence that
accompanies the binding interaction with an SLM 8000C
spectrofluorometer (SLM Instruments, Rochester, NY) as in previous
studies (15, 22). Titrations to assess heparin binding stoichiometry
were done at antithrombin concentrations (200-800 nM) much
higher than KD (>10-fold) and in I 0.05 (pentasaccharide) or 0.15 (full-length heparin) buffer, whereas titrations to determine the equilibrium dissociation constant were done
at inhibitor concentrations closer to KD in I 0.3 buffer (50-200 nM). Fluorescence data were computer-fit by
nonlinear regression analysis to the equilibrium binding equation for a
1:1 binding interaction (7, 15). Stoichiometries measured in titrations
at lower ionic strength were assumed in titrations at higher ionic strength.
Fast Protein Liquid Chromatography Analysis of Heparin
Binding--
Normal or variant antithrombins (10 µg) were applied to
a 5-ml Hi-Trap heparin-Sepharose column equilibrated in I 0.15 buffer, washed, and eluted with the program used to purify the variant inhibitor given above.
Heparin Binding Kinetics--
Normal or variant antithrombins
were reacted with at least a 5-fold molar excess of heparin to achieve
pseudo-first-order conditions, and the kinetics of heparin binding were
continuously monitored from the increase in protein fluorescence in an
SX-17MV stopped-flow instrument (Applied Biophysics, Leatherhead, UK) as in previous studies (7, 22). The rise in fluorescence was well fit
by a single exponential function in all cases, which yielded the
observed pseudo-first-order binding rate constant (kobs). kobs from 10-20
reaction traces were averaged for each heparin concentration. Heparin
concentrations ranged from 0.1 to 1 µM for determinations
of overall association and dissociation rate constants at ionic
strengths of 0.15 and 0.3. Polysaccharide concentrations were extended
up to 32 µM for resolution of the two steps of the
binding process for the full-length heparin in I 0.15 buffer.
Proteinase Inactivation Kinetics--
The proteinase inhibitor
activity of the variant antithrombin was assessed by reacting 100 nM variant with 10 nM thrombin or factor Xa in
the absence or presence of 300-500 nM heparin in I 0.15 buffer in a total volume of 0.1 ml. Control reactions were done in the
absence of inhibitor to establish the activity of the uninhibited
enzyme and in the presence of normal antithrombin to determine the
normal rate of enzyme inhibition. After varying intervals of time up to
60 min, reactions were quenched by the addition of 0.9 ml of
chromogenic substrate, either 100 µM S-2238 (Chromogenix)
for thrombin reactions or 100 µM Spectrozyme FXa (American Diagnostica) for factor Xa reactions, containing 50-100 µg/ml Polybrene to neutralize heparin. Residual enzyme activity was
then determined by monitoring the initial linear rate of absorbance increase at 405 nM for several minutes. The effect of the
variant antithrombin on the kinetics of normal antithrombin inhibition of thrombin was determined under pseudo-first-order conditions by
reacting 100 nM normal antithrombin with 10 nM
thrombin with or without 1 nM full-length heparin in the
presence of 0-500 nM variant antithrombin. Reactions in
the absence of heparin additionally contained 50 µg/ml Polybrene.
Reactions were quenched after varying times with 0.9 ml of 100 µM S-2238 containing 50 µg/ml Polybrene, and the
residual thrombin activity was measured from the initial rate of
substrate hydrolysis. Progress curves of the loss in thrombin activity
were fit by a single exponential function with a zero activity end
point to obtain kobs. In some cases,
inactivation was allowed to proceed for a fixed time of 3 or 5 min, and
kobs was calculated from the expression for a
first-order reaction, (ln(vo/vt))/t, where
vt and vo represent the enzyme
activities after reaction for time t and for unreacted
enzyme, respectively. The dependence of kobs on the variant antithrombin concentration was fit by the following equation for competitive binding of heparin to normal and variant antithrombins,
Identification of a Novel Variant Antithrombin in a Patient with
Early Onset Thrombosis--
The 16-year-old proband presented in the
clinic with deep vein thrombosis. Coagulation test screening identified
an abnormality in the antithrombin of the patient, as indicated from
the 100% antigen levels but ~50%
activity (Fig. 1 and Table I ), suggesting the presence of an
inactive antithrombin variant. No other
functional abnormality could be detected and the genetic risk factors,
factor V Leiden and prothrombin 20210A, were not present. Further
analysis of the patient antithrombin by crossed immunoelectrophoresis
in the presence of heparin indicated a normal profile, consistent with
the variant antithrombin binding heparin with an affinity at least as
high as that of the normal protein. Sequencing of the coding region of
the two antithrombin alleles confirmed that one of the alleles
contained an in-frame deletion of 3 base pairs corresponding to the
codon for the P1 Arg-393 residue (results not shown but available on
request to the authors). Such a mutation has not been previously
described. Investigation of the family medical history revealed several
cases of thrombosis, including pulmonary embolism, mesenteric
thrombosis, and still births. Thrombosis was correlated with
antithrombin deficiency in several cases that were tested (Fig. 1).
Purification of the Variant Antithrombin--
Antithrombin was
purified from the plasma of the proband by heparin-agarose
chromatography as in previous studies (15, 24). Comparison of the
specific activity of the isolated proband antithrombin with that
purified from normal pooled plasma showed that about twice as much of
the former protein than of the latter was required to neutralize a
fixed amount of thrombin (Fig. 2). This
observation was consistent with the presence of an inactive
antithrombin variant in the purified antithrombin. To separate the
anticipated variant and normal antithrombins present in the patient
antithrombin, thrombin was added in slight molar excess over the amount
of normal antithrombin measured in the preparation. After
allowing sufficient time to fully complex the normal inhibitor, the
protein mixture was subjected to heparin-agarose affinity
chromatography (see "Experimental Procedures" for details). A peak
with high heparin affinity anticipated to be the unreacted variant
antithrombin was well separated from two low heparin affinity peaks
whose elution positions corresponded to those expected for normal
antithrombin-thrombin complex and residual free thrombin. SDS-PAGE
showed that the isolated antithrombin variant was pure and comigrated
with normal antithrombin but was unable to form an SDS-stable complex
with thrombin (Fig. 3A) or
with factor Xa (not shown) either in the absence or presence of
heparin. The variant, termed antithrombin London or Heparin Binding to
Because of the high heparin affinity of the normal inhibitor and
anticipated higher affinity of the variant under these conditions, measurements of the dissociation equilibrium constants were done at an
ionic strength higher than physiologic to reduce the affinity and
thereby allow accurate determination of KD (Fig. 4). The variant antithrombin bound
pentasaccharide and full-length heparins with dissociation constants of
43 ± 4 and 23 ± 4 nM, whereas the corresponding
values for binding of these heparins to normal antithrombin were
400 ± 30 and 150 ± 30 nM. A similar difference
in KD values of 45 ± 7 and 290 ± 10 nM was measured for the binding of variant and normal
antithrombins to a second preparation of the full-length heparin. The
variant antithrombin thus bound the two heparins with 7-9-fold higher
affinities than normal antithrombin. The lower affinity of the
pentasaccharide than of the full-length heparin for both normal and
variant antithrombins indicates that the additional ionic interaction,
which is made by the full-length heparin just outside the
pentasaccharide binding site in normal antithrombin, is similarly made
in the variant inhibitor (7, 10).
Rapid Kinetics of Heparin Binding--
Rapid kinetic studies of
heparin binding to normal and variant antithrombins were performed to
characterize the basis for the abnormally high heparin affinity of the
We have identified a novel antithrombin variant in a patient with
early onset thrombosis and a family history of thrombosis that
segregates with antithrombin deficiency in affected individuals. The
mutation in the proband was identified by sequencing of the two
antithrombin alleles and showing an in-frame deletion of the codon for
the P1 Arg residue of antithrombin in one of the two alleles. Such a
natural mutation has not been previously reported and was expected to
produce a variant antithrombin that lacked inhibitor function. Natural
and engineered mutations of the P1 Arg thus cause dramatic losses in
inhibitor activity (20, 26, 27), in keeping with earlier studies
showing that the P1 Arg residue is the principal reactive site for
recognition of the serpin by the trypsin-like proteinases of the blood
clotting cascade (28). The presence of an abnormal antithrombin in the
proband was indicated from the normal antigen levels but 50%
functional levels of the serpin in the plasma and confirmed from the
50% reduced specific activity of the antithrombin purified from plasma by heparin-agarose affinity chromatography. The inactive
antithrombin variant was separated from the normal inhibitor by
complexing the active antithrombin fraction with thrombin and
rechromatographing the mixture on a heparin affinity matrix, taking
advantage of the large reduction in heparin affinity that accompanies
the binding of the serpin to proteinases (24, 29). The electrophoretic properties of the mutant inhibitor and the lack of inhibitor function were consistent with deletion of the charged P1 Arg residue.
Although lacking in inhibitor activity toward either thrombin or factor
Xa, the mutant antithrombin was surprisingly found to bind either
full-length or pentasaccharide heparins with an affinity substantially
higher than that of the normal inhibitor. This was shown to be due to a
decreased rate constant for reversal of the conformational activation
step of the two-step heparin binding mechanism that shifted the
conformational equilibrium more in favor of the activated high heparin
affinity state. Such findings imply that the variant antithrombin is
already partially activated without heparin. Thus, partial activation
would explain the higher heparin affinity of the variant because the
small equilibrium fraction of the inhibitor normally in the activated
high heparin affinity state would be increased (6, 7, 30, 31).
Moreover, the observation that the heparin-induced conformational
activation of the serpin is more favorable for the variant than for the
normal inhibitor implies that the energy cost for conformational
activation of the variant is lower, and therefore, the conformational
activation equilibrium in the absence of heparin will also favor the
activated state more for the variant than for the normal inhibitor.
Because activation is associated with a ~40% increase in tryptophan
fluorescence, it would be expected that the variant inhibitor might
show a larger basal fluorescence and smaller fluorescence enhancement
upon binding heparin than the normal inhibitor (32). However, no
significant differences were noted in either the basal or the
heparin-enhanced fluorescence of the two antithrombins. Nevertheless,
these observations are still consistent with a partial activation of
the variant. Thus, assuming that in the normal inhibitor there is less
than 1% activated antithrombin in equilibrium with unactivated
antithrombin, a reasonable assumption based on the 300-fold increased
reactivity of the fully activated inhibitor toward factor
Xa1 and the inability to
detect direct binding of heparin to a preequilibrium fraction of
activated antithrombin (6, 27), full activation of the inhibitor is
predicted to enhance the inhibitor affinity greater than
100-fold.2 The observed
7-9-fold increase in affinity would thus represent less than 10%
inhibitor activation and, thus, is unlikely to be detectable by the
anticipated <4% increase in basal fluorescence given the experimental
error of our measurements. Such a small extent of inhibitor activation
would be consistent with the inability to also detect any
preequilibrium fraction of activated antithrombin in kinetic studies of
heparin binding to the variant antithrombin (27, 30). The native
inhibitor conformation must, therefore, still dominate over the
activated conformation in the variant antithrombin despite some partial activation.
The structural basis for partial activation of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1cm
1 (16).
-thrombin was prepared by purifying
prothrombin from plasma followed by activation of the zymogen and
purification of the enzyme as described (18, 19). Human factor Xa
(predominantly
-form) was purchased from Enzyme Research
Laboratories (South Bend, IN). Concentrations of active proteinases
were determined by comparison of the activities in standard enzyme
assays with those of active-site titrated preparations of the enzyme as
in previous studies (20).
where kuncat and kH
are the second order rate constants for reactions of free and
heparin-complexed normal antithrombin with thrombin, [AT]o
and [AT*]o are the total concentrations of normal and variant
antithrombins, respectively, [H]o is the total heparin
concentration, and KAT,H and
KAT*,H are the dissociation constants
for heparin binding to normal and variant antithrombins. The expression
multiplying kH represents the concentration of
normal antithrombin-heparin complex in the presence of the competitor
variant antithrombin. The expression derives from the cubic equation
defining the concentration of antithrombin-heparin complex in the
presence of a competitor antithrombin under the conditions,
[AT]o, [AT*]o
(Eq. 1)
[H]o, in which the
concentrations of the free antithrombins can be equated with their
total concentrations (23). Values for kobs
measured in the absence of the variant antithrombin, denoted
kobs,o, and also in the absence of
heparin, denoted k'obs,o, enabled
simplification of this equation to the form
Data were fit by this equation by fixing
KAT,H at the measured value of 19 nM with KAT*,H a fitted parameter.
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pedigree of proband. The proband is
indicated by the arrow, family member affected with
thrombosis is indicated by , and those documented with antithrombin
deficiency are indicated by
. Individuals affected with thrombosis
and antithrombin deficiency are indicated by
. Table I provides
further relevant clinical information on family members.
Family history of propositus
P1 antithrombin to indicate that the P1 Arg residue is deleted, also showed no detectable ability to inhibit thrombin or factor Xa enzymatic activity
with or without added heparin (<0.001% normal antithrombin activity,
data not shown). Nondenaturing PAGE showed an increased mobility of
P1 antithrombin as compared with normal inhibitor (Fig.
3B), consistent with deletion of the positively charged P1
Arg residue in the variant. Both normal and variant bands also showed a
similar large mobility shift upon the addition of a molar excess of a
super high affinity pentasaccharide (17), demonstrating that the
variant retained high heparin affinity. Neutrophil elastase cleaved the
variant inhibitor at a rate comparable with that of the normal
inhibitor in the absence or presence of heparin pentasaccharide (not
shown), indicating a similar accessibility of the loop to the enzyme in
both inhibitors whether they were in the native or activated
conformation.
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Fig. 2.
Reduced functional activity of antithrombin
purified from the proband. 0-2 molar eq of purified normal or
patient antithrombin was mixed with 100 nM thrombin, and
residual thrombin activity was measured after ~16 h as described
under "Experimental Procedures." The residual activity expressed as
the % of the uninhibited activity is plotted versus the
molar ratio of antithrombin (AT) to thrombin and fit by
linear regression (solid lines).
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Fig. 3.
SDS and nondenaturing PAGE of purified normal
and variant antithrombins. A, SDS-PAGE of purified normal
(lanes 1-3) and variant (lanes 5-7)
antithrombins (5 µM) alone (lanes 1 and
5) or reacted with 0.5 molar eq of thrombin in the absence
of heparin for 5 min (lanes 2 and 6) or in the
presence of 0.1 µM full-length heparin (lanes
3 and 7) for 1 min. Unreacted thrombin is shown in
lane 4. B, purified normal (lanes 1,
3, and 5) and variant (lanes 2 and
4) antithrombins (3 µg) were electrophoresed in a
nondenaturing gel in the absence (lanes 1, 2, and
5) or presence of a 2-fold molar excess of the super high
affinity pentasaccharide (lanes 3 and 4).
P1 Antithrombin--
The heparin affinity of
the P1 Arg deletion variant of antithrombin was initially compared with
that of normal antithrombin by analyzing the NaCl concentration
required to elute the protein from a heparin-agarose
matrix. Interestingly, the variant protein eluted at a much higher salt
concentration of 1.9 M than that required to elute the
normal inhibitor (1.3 M), indicating that the variant bound
heparin with a significantly higher affinity than normal. Direct
equilibrium binding studies were conducted by titrating variant and
normal antithrombins with both pentasaccharide and full-length high
affinity heparins and monitoring polysaccharide binding from the 40%
increase in tryptophan fluorescence that signals this binding (15). The
stoichiometry of heparin binding was first analyzed by titrating normal
and mutant inhibitors with heparin at inhibitor concentrations much
higher than the anticipated KD and at low ionic
strengths (I 0.05-0.15) where binding was tight. Although the purified
variant antithrombin bound 0.62 ± 0.03 mol of heparin/mol of
protein (n = 3) when initially tested, the heparin
binding stoichiometry increased to 0.92-0.95 (n = 3)
after rechromatography of the inhibitor on heparin-agarose, values
experimentally indistinguishable from those observed with normal plasma
antithrombin. The variant inhibitor thus appeared to be fully
functional in binding heparin after removal of some nonfunctional,
presumably latent, inhibitor formed during the initial three-step
purification (25). The intrinsic protein fluorescence of uncomplexed
normal and variant antithrombins was the same within experimental error
and both inhibitors underwent similar maximal fluorescence enhancements
of ~40% upon binding heparin.
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Fig. 4.
Higher affinity binding of heparin to
P1 antithrombin than to normal antithrombin.
Normal (circles) or variant antithrombins (AT)
(triangles) (60-200 nM) were titrated with
pentasaccharide (panel A) or full-length heparins
(panel B) in I 0.3 buffer and heparin binding to the protein
was monitored from increases in tryptophan fluorescence as described
under "Experimental Procedures." Data were fit by the quadratic
equilibrium binding equation assuming functional antithrombin
concentrations determined in stoichiometric heparin binding titrations
at lower ionic strength.
P1 Arg antithrombin variant. The observed pseudo-first-order rate
constant for binding of the full-length heparin, measured at
physiologic ionic strength (I 0.15) by continuously monitoring the
protein fluorescence change that signals binding, increased as a
function of heparin concentration in a saturable manner for both
antithrombins (Fig. 5). This behavior reflects the two-step nature of the binding interaction established in
previous studies (6, 7), which is depicted in the following reaction
scheme.
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Fig. 5.
P1 antithrombin binds heparin
with a lower dissociation rate constant than normal antithrombin.
Observed pseudo-first-order rate constants
(kobs) for the binding of heparins to normal
(circles) or variant (triangles) antithrombins
(AT) were measured as a function of the heparin concentration by
continuously monitoring the rate of protein fluorescence changes in a
stopped-flow spectrofluorometer as described under "Experimental
Procedures." A, experiments done in I 0.15 buffer over a
wide range of full-length heparin concentrations where
saturation of kobs occurred.
B, experiments conducted at low heparin concentrations with
both pentasaccharide (open symbols) and full-length heparins
(filled symbols) in I 0.3 buffer to better determine overall
association and dissociation rate constants. Solid lines
indicate nonlinear regression fits of data in panel A by the
hyperbolic equation in the text or linear regression fits of data in
panel B.
According to the scheme, heparin initially forms a low affinity
interaction with antithrombin with dissociation constant, K1, in a rapid equilibrium binding step, which
then induces the inhibitor to undergo an activating conformational
change with a forward rate constant, k+2, and
reverse rate constant, k
(Eq. 3)
2, which results in a
high affinity interaction. The expected dependence of
kobs on heparin concentration ([H]o)
for this scheme is given by the following equation.
Notably, the kinetic data for heparin binding to normal and mutant
inhibitors displayed an indistinguishable dependence of kobs on heparin concentration that was well fit
by this equation (Fig. 5). Values for K1 of
21 ± 2 µM and for k+2 of 290 ± 10 s
(Eq. 4)
1 obtained from fitting the normal antithrombin data
were thus the same within experimental error as the values
obtained by fitting the variant antithrombin data. However, the value
of k
2 given by the intercept on the ordinate
axis was indistinguishable from zero for both inhibitor interactions,
in keeping with the small value previously measured for this parameter
at this ionic strength (7). These results suggested that the greater
heparin affinity of the variant than of normal antithrombin was due to an effect of the mutation on the rate constant for reversal of the
conformational activation step, k
2. To verify this possibility, the kinetics of heparin binding were measured at low
heparin concentrations and at a higher ionic strength to increase the
value of k
2 and better determine its value. In
the range of low heparin concentrations well below the value of
K1 ([H]o
K1), the
equation for kobs simplifies to the linear
function,
The intercept and slope of the limiting linear variation of
kobs at low heparin concentrations thus yields
k
(Eq. 5)
2 and k+2/K1, respectively,
which represent the overall dissociation and association rate
constants, koff and kon,
for the two-step binding process. kobs showed
the expected linear dependence on heparin concentration for the binding
of pentasaccharide and full-length heparins to the two antithrombins at
low heparin concentrations (Fig. 5). Whereas the plots were virtually
indistinguishable for full-length heparin binding to normal and variant
inhibitors, they were clearly distinguishable for pentasaccharide
binding. Similar slopes corresponding to kon
were found for the binding of either heparin to normal and variant
antithrombins, but the intercept representing
koff was significantly lower for the
P1 variant than for normal antithrombin, this difference being most marked
for the pentasaccharide interaction. These results confirmed that the
higher heparin affinity of the variant antithrombin was due to a marked
reduction in the dissociation rate constant with no significant effect
on the association rate constant. Table II summarizes kon
and koff values obtained from these experiments and other experiments at I 0.15.
Association and dissociation rate constants for heparin binding to
normal and variant antithrombins
P1 Antithrombin Effects on the Heparin-catalyzed Normal
Antithrombin-Thrombin Reaction--
The substantially higher heparin
affinity of
P1 antithrombin relative to normal antithrombin
suggested that the variant could have a deleterious effect in
individuals heterozygous for the mutation because of its ability to
block activation of normal antithrombin by heparin. To test this
possibility, we examined the ability of the mutant antithrombin to
antagonize the heparin-catalyzed reaction of normal antithrombin with
thrombin.
P1 Arg antithrombin was found to progressively reduce the
heparin-catalyzed rate of thrombin inhibition by antithrombin to that
of the uncatalyzed rate as the concentration of the mutant inhibitor
was increased in the reaction (Fig. 6).
Moreover, substoichiometric levels of the mutant inhibitor were
sufficient to cause the bulk of the rate decrease. The mutant
antithrombin showed no ability to inhibit thrombin itself in the
presence of heparin and had no effect on the uncatalyzed
antithrombin-thrombin reaction, consistent with the effect of the
mutant inhibitor being solely to compete with the normal inhibitor for
binding the limiting heparin component. Analysis of the decrease in
kobs for the heparin-catalyzed
antithrombin-thrombin reaction as a function of the added
P1 Arg
antithrombin concentration according to a simple competitive heparin
binding model (Fig. 6, inset) indicated that the variant
inhibitor bound heparin with a dissociation constant of 1.6 ± 0.1 nM, representing a ~12-fold higher affinity than the
normal inhibitor at the lower physiologic ionic strength of these
experiments.
View larger version (21K):
[in a new window]
Fig. 6.
Antagonism of the heparin-catalyzed
antithrombin-thrombin reaction by P1
antithrombin. Reactions of 100 nM normal antithrombin
with 10 nM thrombin were done in I 0.15 buffer in the
absence (
,
) or presence (
,
,
,
,
) of 1 nM full-length heparin without variant antithrombin (
,
) or with 50 nM (
), 100 nM (
,
),
200 nM (
), or 500 nM (
) variant
antithrombin as described under "Experimental Procedures." Control
reactions of 370 nM variant antithrombin and 10 nM heparin with 10 nM thrombin are shown by
×'s. Inset, kobs for the
heparin-catalyzed antithrombin-thrombin reaction in the presence of
P1 antithrombin, corrected for the uncatalyzed reaction and
expressed relative to the corrected kobs for the
reaction in the absence of variant antithrombin, is plotted as a
function of the ratio of variant to normal antithrombins. Data are
shown for fixed-time assays with variant antithrombin concentrations
ranging from 5-300 nM (
). The solid line
shows a nonlinear regression fit by the competitive binding equation
given under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
P1 variant can be
understood from the established mechanism by which heparin activates
antithrombin as has been deduced from the structures of native and
activated inhibitors (9, 10) and from structure-function analyses of
mutant inhibitors (32, 33) (Scheme 1). In
this mechanism, the serpin is maintained in a low heparin affinity, low
activity state by partial burial of the N-terminal P15 and P14 residues
of the reactive proteinase binding loop in
-sheet A of the protein
core. Binding of the heparin pentasaccharide to helix D and surrounding
regions comprising the heparin binding site of the serpin on one side
of the
-sheet induces the inhibitor into the high heparin affinity
activated state by extending the D helix and expelling the portion of
the proteinase binding loop buried in sheet A to increase the exposure
of the loop and its central P1 Arg residue (10, 20). The activated
state is accessible in the absence of heparin through a conformational
equilibrium, but the equilibrium highly favors the low activity state.
Heparin shifts the equilibrium to the high activity state only because of the preferential binding of heparin to the activated state (30). The deletion of the P1 Arg residue in
P1
antithrombin could be expected to influence the conformational
equilibrium of the native inhibitor because this deletion would shorten
the length of the loop. The shortened loop would thus induce strain in
the native buried loop conformation that could be relieved in the
activated exposed loop conformation.
View larger version (31K):
[in a new window]
Scheme 1.
Proposed mechanism of partial activation of
the P1 antithrombin variant. Antithrombin
exists predominantly in a native low heparin affinity state, in which
the reactive loop (solid black curve) is partially inserted
in
-sheet A (arrows) and the P1 Arg (the side chain
depicted in stick representation) interacts with the serpin
body when heparin is not bound. However, the native state is in
equilibrium with a minor fraction of an activated high heparin affinity
state in which the reactive loop is expelled from sheet A and the P1
Arg is exposed. Preferential binding of heparin to helix D (depicted by
a coil) in the activated inhibitor state shifts the equilibrium in
favor of this state and causes helix D to extend and to thereby
contract sheet A and result in expulsion of the reactive loop from the
sheet. Deletion of the P1 Arg in the loop is proposed to induce strain
in the partially inserted loop of the native state that can be relieved
in the activated state by expelling the loop from sheet A. The
conformational activation equilibrium would, thus, be shifted in favor
of the activated conformation in the variant antithrombin.
Support for this proposal has recently been provided by a study which
noted that antithrombin has a three-residue insertion on the primed
side of the reactive loop when aligned with other serpins (34).
Progressive deletion of these residues corresponding to the P6'-P8'
positions resulted in partial activation of the inhibitor as judged
from an enhanced basal rate of factor Xa inhibition, which is a
sensitive indicator of activation. However, only small effects of the
mutations on heparin affinity of at most 2-fold were noted. Moreover,
single residue deletions resulted in modest 2-fold increases in factor
Xa inhibition, and a double-residue deletion increased the factor Xa
inhibition rate constant ~10-fold, i.e. <5% of the
~300-fold maximal inhibition rate enhancement produced by activation
of the wild-type inhibitor. Deletion of the P1 residue, thus, appears
to have a more marked activating effect on the serpin than the deletion
of any single residue in the unique loop insertion. This finding can be
rationalized from the x-ray structure of antithrombin, which reveals
that two of the three inserted residues in the antithrombin loop reside
in strand 1 of -sheet C, which anchors the reactive loop to the serpin body on the primed side (9, 10), rather than being a part of the
exposed, flexible loop. Deletion of the P1 residue may thus more
accurately reflect the effect of loop shortening on the conformational
activation equilibrium than deletion of the P6'-P8' residues, since the
latter deletions may shorten the loop less effectively and cause
perturbations of C-
-sheet structure. Interestingly, the expected
strain in the reactive loop produced by deletion of the P1 residue did
not noticeably affect accessibility of the loop to proteinase in either
the native or activated inhibitor states, as judged from the comparable
rate of cleavage by neutrophil elastase of the P4-P3 Ile-Ala bond of
the loop in normal and mutant inhibitors. The exposure and flexibility
of the loop, thus, appear to be less affected by the single residue deletion.
It is thought that individuals with heterozygous type II antithrombin
deficiency resulting from the presence of an abnormal antithrombin with
defective proteinase binding function show evidence of thrombotic
disease later in life than that seen in family members carrying the
P1 Arg variant unless there is coinheritance of additional genetic
risk factors such as factor V Leiden or the presence of strong
environmental risk factors (2, 35). Presumably this reflects the fact
that 50% levels of the inhibitor are just sufficient to maintain the
balance of hemostasis. However, in the case of the
P1 Arg variant,
the higher than normal heparin affinity provides a reasonable
explanation for the unusually severe thrombotic phenotype observed in
this family, characterized by mesenteric thrombosis, still births,
pulmonary embolism, and early onset spontaneous venous thrombosis, all
associated with inhibitor deficiency. This is because the inactive
mutant inhibitor can interfere with the function of the normal
inhibitor by blocking activation of the latter by endogenous heparan
sulfate molecules on the endothelium. The mutant inhibitor is, thus,
predicted to preferentially bind to the limited heparan sulfate
activators estimated to comprise ~1% of cell surface heparan sulfate
molecules (8). As a consequence the effective levels of functional
inhibitor in vivo would be less than the 50% measured
in vitro and would thereby be expected to compromise the
regulation of coagulation proteinase activity.
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ACKNOWLEDGEMENT |
---|
We thank Peter Gettins for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01-HL-39888 and grants from the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Rm. 530E Dentistry (M/C 860), 801 S. Paulina St., Chicago, IL 60612. Tel.: 312-996-1043; Fax: 312-413-1604.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M300062200
1 If the reactivity of native antithrombin with proteinases is all due to a preequilibrium fraction of activated antithrombin, then at most 0.03% of the inhibitor would be activated since inhibitor fully activated with heparin is 300-fold more reactive toward factor Xa than the native inhibitor. If native antithrombin reactivity partly reflects an intrinsic reactivity of the unactivated inhibitor, then the preequilibrium fraction of activated antithrombin would be <0.03%.
2 Heparin pentasaccharide binding to native antithrombin causes the conformational equilibrium to favor the activated over the native conformation by 700-fold (7,19). If one assumes that the conformational equilibrium in the absence of heparin favors the native over the activated conformation >100-fold, then binding of pentasaccharide to the activated conformation is predicted to be >70,000-fold higher affinity than to the native conformation because of the thermodynamic linkage between binding and conformational activation (30). Because conformational activation enhances the measured heparin pentasaccharide affinity for native antithrombin by 700-fold, full activation of the inhibitor would be expected to increase the measured affinity by >70,000/700 = 100-fold.
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