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INTRODUCTION |
Antithrombin is a member of the serpin family of serine proteinase
inhibitors whose primary physiologic function is to inhibit and thereby
regulate several serine proteinases in the blood coagulation pathway
(1, 2). Inhibition of these proteinases results from the serpin
trapping the enzymes as stable acyl-intermediate complexes of a regular
substrate reaction through a major conformational change. A unique
feature of antithrombin is that it requires activation by the
polysaccharide, heparin, to inhibit its main target enzymes, thrombin
and factor Xa, at a physiologically significant rate. Heparin activates
antithrombin through two distinct mechanisms whose relative
contribution depends on the proteinase inhibited (3, 4). In both
mechanisms, antithrombin binds to a sequence-specific pentasaccharide
in heparin (5) which induces an activating conformational change in the
reactive center loop of the serpin (6-9). Such conformational changes
are alone sufficient to accelerate antithrombin inactivation of factor
Xa through an allosteric mechanism. By contrast, the conformational
changes negligibly affect the rate of thrombin inhibition, heparin
accelerating antithrombin inhibition of this enzyme instead through a
bridging mechanism wherein thrombin binding to a bridging site on
heparin next to bound antithrombin enhances thrombin recognition of the
serpin (3, 4, 10).
Although it is well established that conformational activation of
antithrombin by the heparin pentasaccharide specifically enhances the
reactivity of the serpin with factor Xa, the basis for this enhanced
reactivity has not been determined. The x-ray crystal structures of
antithrombin (11, 12) and its complex with the heparin pentasaccharide
(9) have shown that heparin induces an increased exposure of the
reactive center loop which may allow unhindered access of the
proteinase to the loop and also permit optimal proteinase interaction
by increasing loop flexibility. The factor Xa specificity of the
conformationally activated serpin suggests that the loop sequence must
also be critical for discrimination between factor Xa and thrombin.
Supporting this idea, the preferred P4 to P1 substrate sequence for
factor Xa recognition in the natural substrate prothrombin, IEGR or
IDGR, does resemble the P4-P1 IAGR sequence found in antithrombin,
whereas the proposed FPRSFR P3-P3' optimal thrombin recognition
sequence (13) deviates significantly from the AGRSLN sequence in
antithrombin. The factor Xa specificity of the antithrombin reactive
loop sequence is further supported by our finding that mutation of the
factor Xa-preferred P2 Gly of the loop to the thrombin-preferred Pro enhances antithrombin specificity for thrombin at the expense of
decreasing specificity for factor Xa in a manner dependent on
allosteric activation of the serpin (14).
Together, these considerations have strongly suggested that the
presence of a factor Xa recognition sequence in the reactive center
loop and the need for conformational activation of the loop to make
this sequence accessible to factor Xa is the basis for allosteric
activation of antithrombin by heparin pentasaccharide. To rigorously
test this hypothesis, we sought to determine whether changes in the
antithrombin reactive center loop sequence to an optimal thrombin
recognition sequence would increase the thrombin specificity of the
serpin in a manner that was dependent on pentasaccharide activation and
comparable to the activation-dependent increase in factor
Xa specificity of the wild-type serpin. To accomplish this goal, we
evaluated 12 antithrombin variants with single or multiple mutations in
the putative P6-P3' proteinase binding region in the loop other than
P1-P1' to a more favorable thrombin recognition sequence. The results
of analyzing the effects of these mutations on antithrombin specificity
for thrombin and factor Xa revealed that such reactive center loop
changes significantly increased the specificity of antithrombin for
thrombin, but the specificity enhancements were only modestly dependent
on allosteric activation of the serpin and were greatly attenuated with
a bridging heparin activator. Remarkably, the effects of the reactive
center loop sequence changes on factor Xa specificity were quite
modest, and all variants showed a large inhibition rate enhancement
upon allosteric activation comparable to that of the wild-type serpin
and independent of the loop sequence. Together, these results strongly
argue against our initial hypothesis that exposure of a factor
Xa-specific reactive center loop sequence in antithrombin by a
pentasaccharide-induced conformational change in the loop is the basis
for allosterically activating antithrombin for rapid factor Xa
inhibition. Rather, our findings suggest that the specificity of
antithrombin for factor Xa arises from the exposure of still unknown
recognition determinants outside the P6-P3' reactive center loop
sequence which are only made accessible upon heparin activation.
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MATERIALS AND METHODS |
Construction, Expression, and Purification of Antithrombin
Variants--
The cDNA for N135Q antithrombin was used as the
template for mutating reactive center loop residues to eliminate
glycosylation heterogeneity resulting from incomplete glycosylation at
Asn-135 and to mimic the high heparin affinity
-form of plasma
antithrombin that lacks the Asn-135 carbohydrate chain (15, 16).
Site-directed mutagenesis of human antithrombin cDNA was carried
out in the single-stranded M13mp19 vector as described (7) using an
antisense oligonucleotide encoding the desired mutation. This method
was used for the construction of the G392P, A391H, and N396H mutations. Other mutants were made by annealing complementary oligonucleotides carrying the desired mutant codons to pAlter (Promega) or pMAStop (7)
plasmids in which the antithrombin N135Q cDNA was preinserted and
generating mutant plasmids by polymerase chain reaction. cDNAs for
antithrombin-protease nexin-1 loop chimeras were generated in pAlter
plasmids and then excised and ligated into the pMAStop expression
plasmid. The chimeras were generated by first mutating the antithrombin
P6-P2 sequence and then the P2'-P3' sequence to that of protease
nexin-1. Variants containing G392P, A391F, and L395F mutations in all
possible combinations were generated by stepwise mutation.
Oligonucleotides used for generating mutations (antisense strand only)
were as follows (mutant codons underlined): A391F, 5'-GTT TAG CGA ACG
GCC GAA AAT CAC AAC AGC GG-3'; G392P, 5'-GTT TAG CGA ACG
GGG AGC AAT CAC AAC-3'; L395F, 5'-CCT GTT GGG GTT
AAA CGA ACG GCC AGC-3'; A391F/G392P, 5'-GG GTT TAG CGA ACG GGG GAA AAT CAC AAC AG-3'; A391F/L395F, 5'-GTT
AAA CGA ACG GCC AAA AAT CAC AAC AGC-3';
G392P/L395F, 5'-GTT AAA CGA ACG CGG AGC AAT CAC
AAC-3'; A391F/G392P/L395F, 5'-C CCT GTT GGG GTT AAA CGA ACG
GGG AAA AAT CAC AAC AGC-3'; PN1 P6-P2, 5'-CAC
CCT GTT GGG GTT TAG CGA ACG CGC GAT
GAG GAT GGC AGC GGT ACT TGC AGC TGC
TTC ACT-3'; PN1 P6-P3', 5'-GGC CTT GAA AGT CAC CCT GTT GGG
CGG CGA GCT TCT
GGC GAT GAG GAT
GGC AGC GGT ACT TGC-3'; V388A/V389A/I390A, 5'-G CGA ACG GCC
AGC AGC GGC AGC AGC GGT ACT TGC
AG-3'; A391H, 5'-TAG CGA ACG GCC ATG AAT CAC AAC AGC-3';
N396H, 5'-CAC CCT GTT GGG ATG TAG CGA ACG GCC-3'. All
mutations were confirmed by DNA sequencing.
Baby hamster kidney cells were cotransfected with the expression vector
carrying the reference or mutant cDNA together with selection
plasmids and stably transfected cell lines obtained as described
previously (7, 15). Recombinant antithrombins were isolated from
serum-free cycles of medium collected from the stably transfected baby
hamster kidney cells grown to confluence in roller bottles, with
expression levels routinely reaching 15-40 mg/liter (7, 15).
Recombinant antithrombins were purified by heparin-agarose
chromatography to resolve the high heparin affinity glycoform
corresponding in affinity to plasma
-antithrombin followed by
DEAE-Sepharose and Sephacryl S-200 chromatography (15, 17).
Concentrations of recombinant antithrombins were determined from the
absorbance at 280 nm using a molar absorption coefficient of 37,700 M
1 cm
1
(18).
Proteinases--
Human
-thrombin was a gift of Dr. John
Fenton (New York State Department of Health, Albany, NY). Human factor
Xa (predominantly
) was obtained by activation of purified factor X
followed by purification on soybean trypsin inhibitor-agarose as
described (19) or generously provided by Dr. Paul Bock (Vanderbilt
University, Nashville, TN). Proteinase concentrations were based on
active-site titrations that indicated >90 and >70% active enzyme for
thrombin and factor Xa, respectively (20). Human neutrophil elastase was purchased from Athens Research and Technology (Athens, GA). Bovine
-trypsin was isolated from commercial enzyme (Sigma) by soybean trypsin inhibitor-agarose chromatography as described (21).
Heparins--
The
-methyl glycoside of a synthetic heparin
pentasaccharide corresponding to the antithrombin-binding sequence in
heparin was generously provided by Dr. Maurice Petitou (Sanofi
Recherche, Toulouse, France). A full-length heparin containing the
pentasaccharide with an average molecular weight of ~8000 (~26
saccharides) was isolated from commercial heparin by size and
antithrombin affinity fractionation (17). Concentrations of
pentasaccharide and full-length heparins were determined by
stoichiometric titrations of antithrombin with the
saccharides monitored by changes in protein fluorescence (4, 17).
Experimental Conditions--
All experiments were done at 25 or
37 °C as noted in buffers consisting of 20 mM sodium
phosphate, 0.1 mM EDTA, 0.1% (w/v) polyethylene glycol
8000 containing either 0.1 M NaCl (I 0.15) at pH
7.4, 0.25 M NaCl (I 0.3) at pH 7.4, or 0.125 M NaCl (I 0.15) at pH 6.0. An exception was
experiments with trypsin that were done in 100 mM Hepes
containing 0.1 M NaCl, 0.01 M
CaCl2, and 0.1% polyethylene glycol 8000, pH 7.4.
Stoichiometry of Inhibition--
Thrombin (5-100
nM), factor Xa (10-100 nM), or trypsin (100 nM) were incubated with increasing molar ratios of
recombinant antithrombin to enzyme in the absence or presence of
heparin levels equimolar with the maximum inhibitor concentration
(0.1-2 µM) for times sufficient to complete the reaction
based on measured second-order rate constants (17). Residual enzyme
activity was then measured by 50-100-fold dilution of an aliquot of
the reaction mixtures into 100 µM S2238 (Chromogenix) for
thrombin, 200 µM Spectrozyme FXa (American Diagnostica)
for factor Xa, or 100 µM S-2222 (Chromogenix) for
trypsin, followed by monitoring the initial rate of substrate
hydrolysis from the linear absorbance increase at 405 nm. In some cases
thrombin or trypsin activity was measured by dilution of reaction
mixtures into 50 µM
tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin (Sigma) and substrate
hydrolysis monitored from the linear fluorescence increase at
excitation and emission wavelengths of 380 and 440 nm, respectively.
All substrates contained 50-100 µg/ml Polybrene (Aldrich) to
neutralize any heparin present. The inhibition stoichiometry was
obtained by extrapolating linear least squares fits of the decrease in
enzyme activity with increasing molar ratio of inhibitor to enzyme to
the ratio yielding complete enzyme inhibition.
Electrophoresis--
SDS-polyacrylamide gel electrophoresis
analysis was performed using the Laemmli discontinuous buffer system
and a 10% polyacrylamide gel under nonreducing conditions (22). The
products of antithrombin-proteinase reactions were analyzed after
reacting 2-5 µM proteinase with a 1-3-fold molar excess
of antithrombin in the absence or presence of full-length heparin
equimolar with the inhibitor for 5-30 min in I 0.15 buffer.
Reactions were quenched with 250 µM
Phe-Pro-Arg-chloromethyl ketone (Calbiochem) for thrombin or 500 µM Glu-Gly-Arg-chloromethyl ketone (Bachem) for factor Xa
prior to addition of SDS sample buffer and boiling. Neutrophil elastase
cleavage of wild-type and V388A/V389A/I390A variant antithrombins was
analyzed by incubating 3 µM serpin with 0.3 nM enzyme and 10 µM unfractionated heparin (Sigma) for varying times up to 60 min in I 0.15 buffer at
37 °C followed by quenching with SDS sample buffer and boiling for 3 min. Protein bands were detected by Coomassie Blue R-250 staining.
Heparin Binding--
The KD for heparin
binding to recombinant antithrombins was measured by titrating 50 nM antithrombin with at least a 10-fold molar excess of
polysaccharide in I 0.3 buffer, and monitoring heparin
binding from the intrinsic protein fluorescence enhancement at
excitation and emission wavelengths of 280 and 340 nm, respectively (4,
17). Stoichiometries of heparin binding were determined by similar
titrations in I 0.15 buffer where binding is considerably
tighter (4). Titrations were fit by the quadratic equilibrium binding
equation with KD, the stoichiometry and the maximal
relative fluorescence change being the fitted parameters (17).
Stoichiometries determined at I 0.15 were assumed in fitting
titrations at I 0.3 where KD was best determined.
Kinetics of Antithrombin Inhibition of
Proteinases--
Second-order rate constants for the association of
variant antithrombins with proteinases were measured under pseudo
first-order conditions by using a molar excess of inhibitor over enzyme
of at least 10 times the inhibition stoichiometry (16). Reactions contained 10-300 nM antithrombin and 0.1-10
nM proteinase with or without catalytic levels of
pentasaccharide or full-length heparins ranging from 0.25 to 20 nM. For pentasaccharide-accelerated antithrombin-thrombin
reactions or for pentasaccharide or full-length heparin-accelerated
antithrombin-trypsin reactions, a 1.5-4-fold molar excess of heparin
over antithrombin was used to saturate the inhibitor (
92%). The
time-dependent decrease in enzyme activity was measured by
quenching samples at varying times into substrate and determining the
initial rate of chromogenic or fluorogenic substrate hydrolysis as in
determinations of reaction stoichiometry. Enzyme inhibition progress
curves were computer-fitted by a single exponential function with a
zero end point to obtain the observed pseudo first-order rate constant,
kobs. Apparent second-order association rate
constants for uncatalyzed and heparin-catalyzed reactions were
determined from the least squares slope of the linear dependence of
kobs on the antithrombin or the heparin
concentration, respectively, in accordance with Equation 1 that
applies when [AT]o
[H]o,
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(Eq. 1)
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where kuncat and kH
are the second-order rate constants for uncatalyzed and
heparin-catalyzed reactions, respectively, and [AT]o and
[H]o represent the total antithrombin and heparin
concentrations, and KAT, H is the dissociation
constant for the antithrombin-heparin interaction (16). The expression
multiplying kH represents the antithrombin-heparin
complex concentration which under the conditions of the experiments
([AT]o
KAT,H) was closely approximated by [H]o. Association rate constants for reactions of thrombin with antithrombin-pentasaccharide complex or of
trypsin with antithrombin complexes with either pentasaccharide or
full-length heparins were obtained by dividing the measured or fitted
kobs at saturating heparin by the antithrombin
concentration. Apparent second-order rate constants were corrected for
the different fractions of wild-type and variant antithrombins reacting
through the inhibitory pathway by multiplying by the measured
stoichiometry of proteinase inhibition (1).
Rapid Kinetics of Antithrombin-Proteinase
Reactions--
Resolution of the noncovalent and covalent reaction
steps of the reaction of wild-type and variant antithrombin-heparin
complexes with thrombin was done by monitoring the reaction kinetics
with a reporter fluorogenic substrate in an SX-17MV Applied
Photophysics stopped-flow instrument under pseudo first-order
conditions in I 0.15 buffer as in previous studies (14).
Reactions contained 0.06-75 nM thrombin and a 40-400-fold
molar excess of antithrombin-heparin complex generated by saturating
0.025-2 µM full-length heparin with a 1.1-2-fold molar
excess of antithrombin. Reactions were continuously monitored from the
exponential decrease in the rate of hydrolysis of the fluorogenic
substrate, tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin, present at 5 µM. kobs values were averaged for
each inhibitor-heparin complex concentration and corrected for the
uncatalyzed reaction due to excess free inhibitor. The dependence of
kobs on the antithrombin-heparin complex
concentration ([AT·H]) was fit by the hyperbolic Equation 2,
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(Eq. 2)
|
where k represents the limiting rate constant for
conversion of the noncovalent heparin-antithrombin-thrombin ternary
complex to a covalent serpin-proteinase complex with release of
heparin, KT,ATH is the dissociation constant for
formation of the ternary complex, [S]o is the concentration
of fluorogenic substrate, and KM is the Michaelis
constant for substrate hydrolysis by thrombin. KM
was fixed at the measured value of 4.5 ± 0.4 µM,
and KT,ATH and k were the fitted parameters.
Kinetics of Dissociation of Antithrombin-Proteinase
Complexes--
Rate constants for dissociation of covalent
antithrombin-proteinase complexes were measured as in previous studies
by continuously monitoring the recovery of proteinase activity in the
presence of a reporter chromogenic substrate (14, 23, 24). Briefly, complexes were formed by reacting 5-10 µM antithrombin
with 0.5-1 µM proteinase and 1 µM
full-length heparin for 10 min at 25 °C. In the case of the triple
P3/P2/P2' AGL
FPF variant and protease nexin-1 loop swap variants,
heparin was either omitted or the complex formed at higher ionic
strength to minimize any substrate reaction (14). Complexes were
diluted to 0.3-6 nM in 400 µM S2238 or 400 µM Spectrozyme FXa in I 0.15 buffer containing
100 µg/ml Polybrene at 37 °C, conditions which effectively block
reassociation of residual inhibitor with dissociated enzyme (17), and
the initial rate of complex dissociation was continuously monitored from the parabolic increase in the rate of substrate hydrolysis at 405 nm for ~80 min (<1% complex dissociation). The
time-dependent absorbance changes were fit by a
second-order polynomial equation to obtain the initial rate of complex
dissociation using the independently measured turnover number for
enzyme hydrolysis of the substrate under these conditions to relate
absorbance changes to changes in enzyme concentration (14, 23, 24).
Initial rates of complex dissociation were plotted against the
concentration of complex and the first-order dissociation rate constant
determined from the least squares slope of this linear plot.
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RESULTS |
Design of Thrombin-specific Reactive Center Loop Variants of
Antithrombin--
Table I summarizes the
reactive center loop mutations that were made in antithrombin to
improve its specificity for thrombin. Such mutations were confined to
the P6-P3' residues since the proteinase recognition sequence is
typically contained in this region (25). P3 and P3' residues were
individually changed to basic His side chains due to the reported
thrombin preference for basic residues in these positions (26, 27), and
specificity changes were analyzed under pH conditions where the His
side chain was expected to be either uncharged or charged. The critical
P3, P2, and P2' specificity-determining residues were substituted with
the proposed thrombin consensus residues, Phe, Pro, and Phe, respectively (26), by stepwise mutation. The entire P6-P3' sequence was replaced with the corresponding sequence of the serpin, protease nexin-1, since the latter serpin is an ~100-fold faster inhibitor of
thrombin than antithrombin in the absence of a cofactor (28). The
contribution of P6-P4 residues to specificity was evaluated by
mutating these residues simultaneously to Ala. For this particular mutant, the mutation was verified at the protein level by showing that
the rate of cleavage of the wild-type P4-P3 Ile-Ala bond by
neutrophil elastase (29) was greatly curtailed in the mutant (data not
shown). All variants were homogeneous following purification and were
functional in forming SDS-stable complexes with thrombin and in having
such complex formation accelerated by heparin.
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Table I
Engineered reactive center loop variants of antithrombin
Reactive center loop residues are designated according to the
nomenclature of Schechter and Berger (38) with mutated residues in bold
and underlined.
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Stoichiometries of Proteinase Inhibition by Variant
Antithrombins--
In the absence of heparin, all antithrombin
variants behaved like the wild-type serpin in showing an ~1:1
stoichiometry for inhibiting thrombin, factor Xa, and the prototype P1
Arg-specific proteinase, trypsin (Table
II). Most variant antithrombins also resembled the wild-type inhibitor in showing similar increases in
stoichiometry of inhibition of thrombin and factor Xa of greater than 1 mol of inhibitor/mol of proteinase in the presence of pentasaccharide or full-length heparins. In the case of the factor Xa reaction, both
pentasaccharide and full-length heparins elevated the stoichiometry to
similar extents, whereas in the case of the thrombin reaction, only the
full-length heparin increased the stoichiometry, an effect that
paralleled the ability of both types of heparin to catalyze factor Xa
inhibition but only the full-length heparin to catalyze thrombin
inhibition (4).
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Table II
Association rate constants and stoichiometries for variant
antithrombin-proteinase reactions
Second-order association rate constants (kassoc in
units of M 1 · s 1) and
stoichiometries of inhibition (SI given as mol of inhibitor/mol of
proteinase) were determined in the absence or presence of
pentasaccharide (H5) or full-length heparins (H26) at 25 °C,
I 0.15, pH 7.4, as described under "Materials and
Methods." Multiplication of kassoc by SI corrected
kassoc for the different extents of substrate
reaction. Reported values are averages of either two
determinations ± range or three or more determinations ± S.E. except for trypsin reactions in which case results from a single
inhibition progress curve are reported.
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The heparin-dependent increases in inhibition stoichiometry
result from heparin promoting an alternative reaction of antithrombin as a substrate of proteinases in competition with the inhibitory reaction when thrombin and factor Xa are the target enzymes (3, 4).
This heparin-dependent substrate reaction was evident from the appearance of cleaved antithrombin on SDS-polyacrylamide gel electrophoresis in the presence but not in the absence of the polysaccharide. The substrate reaction was much more pronounced in the
case of those variants containing a P2 Gly
Pro mutation or protease
nexin-1 loop residues when thrombin was the proteinase and could be
stimulated by both pentasaccharide and full-length heparins, although
much more by the full-length heparin (Table II). Full-length
heparin-catalyzed reactions of P2 Pro variants and of protease nexin-1
loop variants with thrombin thus showed stoichiometries in the range of
20-30 and 9-12, respectively, in contrast to the value of ~2 for
the wild-type and all other variants under these conditions. Such
variants thus reacted preferentially as substrates of thrombin rather
than inhibitors of the enzyme in the presence of a full-length heparin
activator. Inhibition stoichiometries were not significantly elevated
by heparin when trypsin was the target enzyme.
Heparin Binding and Conformational Activation of the Variant
Antithrombins--
To determine whether the mutations affected the
ability of antithrombin to bind heparin and be conformationally
activated, variant and wild-type antithrombins were titrated with a
full-length high affinity heparin and heparin binding, and
conformational activation of the serpins was followed by monitoring the
progressive appearance of the ~40% tryptophan fluorescence
enhancement that reports these events up to saturation of the inhibitor
with the polysaccharide (4, 6). Similar KD values of
~10-30 nM were determined at I 0.3, and
binding stoichiometries of ~1:1 were measured at I 0.15 for wild-type and variant antithrombin interactions by fitting
titration curves by the quadratic equation for equilibrium binding
(Table III). Similar fluorescence
enhancements of 40-50% were also found to be induced by heparin at
saturation in these titrations (Table III). These results indicated
that none of the reactive center loop mutations affected heparin
binding to the serpin or the ability of the serpin to be
conformationally activated to any significant extent.
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Table III
Interaction of heparin with antithrombin variants
Dissociation constants and relative maximal fluorescence enhancements
for heparin binding to antithrombin variants were determined from
titrations of antithrombin with full-length heparin at 25 °C,
I 0.3, pH 7.4, whereas heparin binding stoichiometries were
determined from separate titrations at 25 °C, I 0.15, pH
7.4, as described under "Materials and Methods." Reported
values ± S.E. are from global fits of at least two titrations.
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Association Rate Constants for Variant Antithrombin-Proteinase
Reactions--
The changes in antithrombin specificity produced by the
reactive center loop mutations were analyzed by measuring second-order rate constants for the association of mutant and wild-type
antithrombins with the target enzymes, thrombin and factor Xa, and in
some cases also with the less specific enzyme, trypsin. To resolve the
effects of conformational activation of antithrombin from the effects of heparin bridging antithrombin and proteinase on specificity, second-order rate constants were measured in the absence of heparin, in
the presence of the heparin pentasaccharide and in the presence of a
full-length bridging heparin containing the pentasaccharide (Table II)
(4). Apparent second-order rate constants were corrected for the
different extents of substrate reaction of the variants to provide
valid comparisons of the association rates along the inhibitory pathway
and thereby changes in specificity (1). This correction involved
multiplication of the apparent rate constant by the measured
stoichiometry of inhibition (Table II). The relative changes in
antithrombin specificity for thrombin and factor Xa produced by the
mutations for both the native and heparin-activated inhibitor are
summarized in Fig. 1.

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Fig. 1.
Effect of reactive center loop mutations on
antithrombin specificity for thrombin and factor Xa. The relative
specificity of mutant versus wild-type antithrombins for
inhibiting thrombin (top panel) or factor Xa (bottom
panel) is indicated by the log of the ratio of corrected
second-order association rate constants for the inhibitor-proteinase
reactions (Table II) in the case of unactivated antithrombin
(gray bars), pentasaccharide-activated antithrombin
(black bars), or full-length heparin-activated antithrombin
(white bars).
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Of the single reactive center loop mutations, the P2 Gly to Pro change
produced the largest increase in thrombin specificity over wild-type
antithrombin, and this specificity enhancement was greatest when the
variant inhibitor was activated by pentasaccharide (15-fold) as
compared to when the variant was not activated (3.6-fold) or activated
by full-length heparin (2.1-fold). A more modest specificity
enhancement was produced by the P2' Leu to Phe change that was again
maximal for the pentasaccharide-activated serpin (5.5-fold) relative to
the unactivated (2.5-fold) or full-length heparin-activated inhibitor
(1.6-fold). Other single mutations of P3 Ala to Phe or to His, or of
P3' Asn to His only marginally increased or slightly decreased thrombin
specificity with or without heparin activation. Converting the largely
neutral His side chain at pH 7.4 in the P3 and P3' His variants to a
mostly positively charged side chain by lowering the pH to 6 modestly
increased or did not affect thrombin specificity, respectively,
independent of heparin activation (Table
IV).
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Table IV
Association rate constants and stoichiometries for reactions of P3H and
P3'H antithrombin variants with proteinases at pH 6
Rate constants (kassoc in units of
M 1 s 1) and stoichiometries of
inhibition (SI) ± S.E. were measured at 25 °C, I
0.15, pH 6.0, as detailed under "Materials and Methods."
kassoc was corrected for flux along the substrate
pathway by multiplying by SI.
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Stepwise mutations of P3, P2, and P2' residues of antithrombin to the
proposed consensus residues preferred by thrombin, i.e. P3
Phe, P2 Pro, and P2' Phe (26), resulted in the largest enhancement in
antithrombin specificity for thrombin. The effects of these successive
mutations on thrombin specificity depended on the sequence in which the
P3, P2, and P2' residues were mutated, indicating that the specificity
changes caused by each residue were not additive and thus depended on
cooperative interactions between the substituted residues. An example
of the cooperative effects of these substitutions is exemplified by the
effects of the P2 Pro and P2' Phe mutations alone and in combination on
the specificity of the pentasaccharide-activated inhibitor. The P2 Pro
mutation alone enhanced thrombin specificity 15-fold, whereas when this
mutation was made subsequent to the P2' Phe mutation, the specificity
enhancement was reduced to 6.3-fold. Likewise, the P2' Phe change alone
enhanced thrombin specificity 5.5-fold but when made subsequent to the
P2 Pro change enhanced specificity a lesser 2.3-fold. Similar analysis
of double mutant cycles leading to the triple mutant (30) showed that
each mutation contributed to specificity enhancement in a manner that
depended on the presence or absence of the other two residues. The
variant antithrombin containing all three mutations showed the highest increase in thrombin specificity relative to the wild-type inhibitor of
10-fold for the unactivated inhibitor, 55-fold for the
pentasaccharide-activated inhibitor, and 4.8-fold for the full-length
heparin-activated inhibitor.
Replacement of the entire P6-P3' region with the corresponding
sequence of the serpin, protease nexin-1, produced a maximal 4.2-fold
increase in thrombin specificity for the pentasaccharide-activated variant serpin, slight decreases in specificity for the unactivated variant, and a smaller 1.7-fold enhanced specificity for the
full-length heparin-activated variant. Most of this specificity
enhancement was achieved by replacement of just the P2-P6 residues of
antithrombin with those in protease nexin-1. Substitution of the P6 to
P4 Val-Val-Ile sequence with Ala-Ala-Ala produced only small decreases
in thrombin specificity with or without activation by either heparin.
A notable feature of the thrombin specificity enhancements of most
variants was that a significant fraction of the enhancement (as much as
10-fold) was realized already with the unactivated inhibitor. Thus,
although maximal increases in thrombin specificity were found when the
variants were activated by heparin pentasaccharide, such activation
increased thrombin specificity at most ~9-fold over that of the
unactivated variant inhibitor. Whereas this increase exceeded the
1.6-fold enhancement in thrombin specificity of wild-type antithrombin
due to allosteric activation, it did not come close to approaching the
~200-fold increase in factor Xa specificity resulting from allosteric
activation of the wild-type inhibitor. Also of note were the smaller
increments in thrombin specificity of the antithrombin variants over
that of the wild-type serpin of at most ~5-fold observed when the
inhibitors were activated by a full-length bridging heparin. Thus,
heparin bridging of antithrombin and thrombin appeared to attenuate the
recognition of the antithrombin reactive center loop by thrombin.
Because variants containing the P2 Pro mutation or the protease nexin-1
P6-P2 or P6-P3' residues preferentially reacted as a substrate of
thrombin when activated by the full-length heparin, the apparent rate
constants for thrombin inhibition by such variants in the presence of
the full-length heparin were actually decreased as much as 7-fold
relative to the corresponding wild-type inhibitor reaction.
The effects of the reactive center loop mutations on factor Xa
specificity were surprisingly very different. Instead of the progressive decreases in factor Xa specificity expected to accompany the stepwise increases in thrombin specificity of the variant antithrombins, the reactive center loop mutations produced relatively small changes in factor Xa specificity with or without heparin activation of the inhibitor. For the unactivated inhibitor, the mutations either had no significant effect (P3 Phe, P3 His, P3' His,
P6-P2 PN1) or decreased factor Xa specificity up to 9-fold (all other
variants) relative to wild-type antithrombin with the greatest decrease
associated with the P2 Pro replacement. Activation by either
pentasaccharide or full-length heparins resulted in smaller decreases
in factor Xa specificity of maximally 2.5-fold, and in several cases
specificity was increased as much as 3-fold (P3 His, P3 Phe, P2' Phe,
and P3 Phe/P2' Phe) compared with the wild-type inhibitor. Evaluation
of the specificity changes of the P3 and P3' His variants at pH 6 showed similar modest increases or decreases in specificity,
respectively, with or without heparin activation (Table IV). The
insensitivity of reactive center loop changes in antithrombin to its
specificity for inhibiting factor Xa was particularly evident from the
modest effects on specificity resulting from the swapping of the
antithrombin P6-P3' region with that of protease nexin-1. Minimal
decreases in factor Xa specificity no greater than 2-fold were thus
observed for either the unactivated or heparin-activated variant
despite the seven amino acid changes made in this variant. Most
striking of all was the finding that for every one of the reactive
center loop changes, the pentasaccharide rate enhancement observed for
the wild-type serpin of ~200-fold was unchanged or modestly increased up to ~600-fold for the variant serpins. As with the wild-type inhibitor, the full-length heparin further increased factor Xa specificity ~2-fold in most cases due to a small bridging effect of
the larger heparin, although for mutants containing either a P3 Phe or
P2' Phe, the increase was somewhat greater (3-6-fold).
For the stepwise mutation of antithrombin P2, P3, and P2' residues that
resulted in the largest increment in thrombin specificity, the effects
of the mutations on trypsin inhibition with and without heparin
activation were additionally studied. The mutations produced either no
change or only small decreases (maximally 3-fold) in the rate constants
for trypsin inhibition both for the unactivated as well as for the
heparin-activated variant antithrombins. The ~7-fold maximal rate
enhancement of the wild-type inhibitor reaction produced by the
full-length heparin was consequently similar (3-7-fold) for the
variants. These findings are in keeping with trypsin being relatively
nonspecific for substrate amino acids flanking the primary P1 Arg
residue (31).
Reaction Step Affected by the P3/P2/P2' AGN
FPF Mutation in
Antithrombin--
For the P3/P2/P2' AGN
FPF triple mutant
antithrombin that produced the largest increase in thrombin
specificity, it was of interest to evaluate which step or steps of the
multistep serpin inhibitory pathway were responsible for the
specificity enhancement. To address this question, the kinetics of
thrombin inhibition by triple mutant and wild-type antithrombins
complexed with the full-length heparin were compared by stopped-flow
fluorometry using a fluorigenic substrate to monitor proteinase
inhibition. Comparison was also made with the reaction of the variant
containing just the P2 Gly
Pro mutation using data obtained from a
previous study (14). Fig. 2 compares the
dependence of the pseudo first-order inhibition rate constant for
wild-type and variant inhibitor reactions measured by the continuous
assay as a function of the antithrombin-heparin complex concentration.
Saturation of kobs was evident in all cases, but
the limiting rate constants at saturation for the mutant inhibitors were decreased relative to the wild-type inhibitor, and saturation was
achieved at much lower antithrombin-heparin complex concentrations for
the triple mutant than for the wild-type or single mutant inhibitors.
Fitting of the data by the hyperbolic equation given under "Materials
and Methods" indicated dissociation constants of 300 ± 30, 220 ± 40, and 17 ± 7 nM for formation of the
initial heparin-antithrombin-thrombin ternary complex and limiting rate constants of 3.1 ± 0.1, 0.43 ± 0.03, and 0.15 ± 0.01 s
1 for reaction of antithrombin and thrombin
in the ternary complex to form a covalent complex for wild-type, single
mutant, and triple mutant inhibitor reactions, respectively. The
apparent decrease in limiting rate constant for the mutants as compared
with the wild-type inhibitor could be ascribed to the greater fraction of the mutants that reacted as a substrate (Table II) (14). Correction
of the limiting rate constant for the different extents of substrate
reaction by multiplying by the stoichiometry of inhibition resulted in
values of 6.8 ± 0.5, 11 ± 2, and 4.8 ± 1.1 s
1 for wild-type, single mutant, and triple
mutant inhibitor reactions, respectively. These results indicate that
the single P2 Gly
Pro mutation enhances the specificity of
antithrombin for thrombin mostly by increasing the rate constant for
the covalent reaction step without affecting the noncovalent
serpin-proteinase interaction, whereas the triple mutation enhances
serpin specificity for thrombin primarily by increasing the affinity of
the noncovalent complex with minimal effect on the covalent step. Such
findings, taken together with the cooperativity of antithrombin
reactive center loop interactions with thrombin revealed by double
mutant cycles, suggest that antithrombin has a flexible reactive center
loop whose conformation can adapt to produce an optimal active-site interaction either in the Michaelis complex or in the transition state
leading to the covalent acyl-enzyme complex.

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Fig. 2.
Rapid kinetics of full-length
heparin-catalyzed reactions of wild-type antithrombin and P2 Gly
Pro and P3/P2/P2' AGL FPF variant antithrombins with thrombin. Shown is
kobs for reactions of thrombin with wild-type
( ), P2 P ( ), or P3/P2/P2' FPF ( ) variant antithrombins
complexed with full-length heparin as a function of the
antithrombin-heparin complex concentration corrected for the
competitive effect of a reporter fluorogenic substrate used to monitor
inhibition. Rate constants were measured by stopped-flow fluorometry as
described under "Materials and Methods." Solid lines are
fits to the hyperbolic equation in the text. The top and
bottom panels show the data on two different scales.
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Effect of Reactive Center Loop Mutations on Serpin-Proteinase
Complex Stability--
To determine whether the mutations made in the
reactive center loop of antithrombin affected the stability of the
covalent antithrombin-proteinase complex, the rate constants for
dissociation of complexes of P3/P2/P2' AGN
FPF, P6-P4 VVI
AAA,
and protease nexin-1 P6-P3' loop swap variants as well as wild-type
antithrombin with thrombin and factor Xa were measured over a range of
complex concentrations as in previous studies (14, 17, 23, 24) (Table
V). Whereas P3/P2/P2' AGN
FPF and
protease nexin-1 loop variant inhibitor complexes were dissociated with
rate constants up to ~2-fold slower than those of the wild-type
inhibitor complexes with thrombin and factor Xa as proteinase, similar
to the effect of the single P2 Gly
Pro mutation reported previously
(14), the P6-P4 VVI
AAA mutant inhibitor complexes were
dissociated with rate constants up to ~2-fold greater than wild-type
complexes. These results suggest that changes in P6 to P3' reactive
center loop residues of antithrombin other than P1 and P1' have only minimal effects on complex stability.
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Table V
Dissociation rate constants for variant antithrombin-proteinase
complexes
Dissociation rate constants (kdiss) were measured at
37 °C, I 0.15, pH 7.4, from the slopes ± S.E. of
linear plots of the initial rates of complex dissociation as a function
of complex concentration as described under "Materials and
Methods."
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DISCUSSION |
Reactive Center Loop Contribution to Antithrombin
Specificity--
Our studies were designed to test the hypothesis that
the enhanced factor Xa specificity of allosterically activated
antithrombin arises from a factor Xa-specific sequence in the
antithrombin reactive center loop that becomes accessible to the
proteinase only after activation. Such an hypothesis predicts that
changes in the putative factor Xa recognition sequence in the reactive center loop to a sequence preferred by thrombin would convert antithrombin to an allosterically activated inhibitor of thrombin and a
poor inhibitor of factor Xa. The results of testing this prediction
have shown that the thrombin specificity of antithrombin can be
significantly enhanced in a manner dependent on allosteric activation
by introducing reactive center loop changes more favorably recognized
by thrombin. In particular, introducing the proposed optimal P3-P2'
sequence for thrombin (26) into the antithrombin reactive center loop
resulted in the largest increase in specificity (55-fold), whereas
substitution with the protease nexin-1 P6-P3' loop sequence or the P3
and P3' residues with a positively charged histidine at pH 6 produced
more modest enhancements in thrombin specificity. Substituting the
protease nexin-1 P6-P3' sequence into the reactive center loop of
1-antichymotrypsin similarly produced only modest
increases in thrombin specificity of this serpin (32), suggesting that
the high reactivity of protease nexin-1 with thrombin may involve
recognition by elements in the serpin outside the P6-P3' region.
Changes in the Val-Val-Ile P6-P4 sequence did not significantly affect
thrombin recognition in native or activated states of the serpin,
suggesting that these residues are not important for binding thrombin.
The maximal increase in thrombin specificity achieved with the triple
P3/P2/P2' AGN
FPF mutation exceeds that produced in the serpin,
1-proteinase inhibitor, by similar changes in the
reactive center loop to a thrombin-preferred sequence (30) and is
comparable to the thrombin specificity of protease nexin-1 in the
absence of heparin (28). The reactive center loop sequence of this
variant antithrombin may thus be near optimal for interacting with the
active-site of thrombin, in keeping with recent studies of thrombin
specificity assessed by screening a combinatorial library of synthetic
substrates (33).
Surprisingly, a significant fraction of the thrombin specificity
enhancements achieved in the variant antithrombins were realized without allosteric activation of the serpin. The reactive center loop
sequence thus plays a role in antithrombin recognition of thrombin, but
this recognition appears to be only marginally dependent on allosteric
activation of the inhibitor. Trypsin inhibition by antithrombin was
similarly only modestly affected by allosteric activation with either
wild-type or mutant serpins, suggesting that the reactive center loop
of native antithrombin is largely accessible to thrombin and trypsin
and does not depend on allosteric activation for its rapid association
with these enzymes. The structural basis for allosteric activation of
antithrombin is known to involve the expulsion of the reactive center
loop from
-sheet A of the protein core in which the loop is
partially buried at the P15-P14 hinge in native antithrombin (8, 9,
34). The expulsion results in the exposure of the loop in a manner
similar to that seen in the structures of other native serpins (35,
36), and it has been presumed that this expulsion is important for
increasing accessibility of the loop to target proteinases. However,
our findings suggest that the loop is already accessible to thrombin and trypsin when the hinge region is buried and that the modest increases in association of the loop with these enzymes upon exposure of the hinge can be ascribed to the increased flexibility of the loop
when it is not constrained by burial of the hinge region. Such an
increased flexibility could improve a substrate-like interaction between the loop and the enzyme which is characteristic of
serpin-proteinase interactions (1) by enabling the loop to adopt a
conformation most optimal for proteinase interaction.
It was also surprising to find that the reactive center loop changes
produced relatively small effects on the specificity of antithrombin
for factor Xa whether or not the serpin was allosterically activated by
heparin pentasaccharide, the largest specificity changes being
associated with the P2 Pro substitution. These findings are consistent
with the broad specificity of the S4 and S3 subsites of factor Xa and
more restricted specificity of the S2 subsite of the enzyme for Gly
indicated by x-ray crystallography (37) and combinatorial substrate
screening (33). Most interesting was the finding that allosteric
activation of any of the variant antithrombins still enhanced factor Xa
specificity to the same or a greater extent than the wild-type serpin.
These findings imply that the reactive center loop residues flanking
the P1-P1' scissile bond including the P4 and P2 mimics of the
prothrombin sequence are not strong determinants of antithrombin
specificity for factor Xa in either native or activated states of the
serpin and do not appear to mediate the enhanced specificity of
allosterically activated antithrombin for the enzyme. Previous studies
suggest also that the P1' residue of the serpin is not important for
factor Xa specificity and that replacement of P2' and P3' residues with those in prothrombin do not significantly enhance reactivity (27), implying that the P1 Arg is the most critical specificity-determining residue in this region, a conclusion supported by our recent P1 Arg
mutagenesis studies (51).
Our initial hypothesis that the reactive center loop sequence and its
differential accessibility in native and activated states is
responsible for the factor Xa specificity of activated antithrombin is
therefore not supported by our findings. It would instead appear that
the enhanced factor Xa specificity of activated antithrombin is
minimally dependent on the reactive loop sequence and arises from the
unique ability of factor Xa, unlike thrombin and trypsin, to
discriminate between the constrained and flexible loop conformations of
native and activated antithrombin. In support of factor Xa having such
an unusual ability, a similar level of discrimination between the two
antithrombin loop conformations could be engineered in thrombin by
changing the active-site to be more factor Xa-like (39). However, the
finding that swapping the reactive center loop of antithrombin into
antichymotrypsin resulted in a slow rate of factor Xa inactivation
comparable to that of native, unactivated antithrombin (32) argues that
the reactive center loop sequence of antithrombin has an intrinsically
low reactivity with factor Xa even in a serpin in which the loop
is normally fully exposed (35).
We believe that a more plausible explanation for the enhanced factor Xa
specificity of activated antithrombin is that it does not involve
enhanced factor Xa recognition of the P6-P3' region at all, but
instead involves the recognition of an antithrombin exosite outside of
this region that is made accessible through conformational activation.
According to this idea, factor Xa resembles thrombin and trypsin in
accessing and recognizing the native and activated loop conformations
of antithrombin similarly, with only modest enhancements in recognition
of the activated loop due to its increased conformational flexibility.
Other serpins appear to utilize such nonreactive center loop
determinants to recognize their target proteinases (32, 40, 41).
Examination of the x-ray crystal structures of two specific protein
inhibitors of factor Xa, leech antistasin (42) and tick anticoagulant
peptide (43) and the modeled or elucidated structures of complexes of these inhibitors with factor Xa support the idea that the enhanced factor Xa specificity of allosterically activated antithrombin may
result from an interaction between an antithrombin exosite and a
complementary exosite on factor Xa. Such structures thus reveal the
importance of a basic exosite sequence in factor Xa, Lys-222
Lys-223
Arg-224, located adjacent to the active site and constituting part of the sodium-binding site of the enzyme (44), in
mediating the interaction of the enzyme with both antistasin and tick
anticoagulant peptide. Whether this enzyme exosite is involved in
recognizing a complementary exosite in antithrombin that becomes
accessible through heparin activation remains to be determined.
Contribution of Heparin Bridging to Antithrombin
Specificity--
All antithrombin variants showed large increases in
thrombin specificity when complexed with a full-length heparin due to the longer heparin providing a bridging site for thrombin to bind adjacent to the bound inhibitor (3, 4). However, the thrombin specificities of the mutant antithrombins were not much greater than
that of the wild-type inhibitor when bound to the full-length heparin
(maximally 5-fold) as compared to when they were bound to the
pentasaccharide (maximally 55-fold). Thrombin recognition of the
reactive center loop of the variant antithrombins thus appears to be
poorer when thrombin is constrained to also bind heparin in the ternary
bridging complex. Reactive center loop interactions and heparin
bridging interactions with the proteinase are thus not additive and
hence cooperative. Consistent with this view, heparin binding to the
thrombin exosite that mediates heparin bridging of thrombin with
antithrombin has been shown to affect allosterically active-site
interactions of thrombin with its inhibitors and substrates (45).
Factor Xa reactivity with the antithrombin variants complexed with the
full-length heparin was only slightly greater than when the variants
were activated by pentasaccharide, varying from 2- to 3-fold to as much
as 4- to 6-fold higher. The small enhancement in specificity results
from heparin bridging (4), with the variability in this enhancement
again indicating cooperativity between heparin bridging and reactive
center loop interactions with factor Xa. The relatively modest bridging
effect observable with factor Xa results from the heparin-binding
exosite in this enzyme being inaccessible to heparin due to an
intramolecular interaction between the exosite and the acidic
-carboxyglutamic acid domain of the enzyme (46).
Heparin bridging thus appears primarily responsible for achieving large
enhancements in the thrombin specificity of wild-type antithrombin with
changes in the reactive center loop sequence only modestly affecting
specificity. The overriding effect of heparin-bridging interactions in
determining thrombin specificity suggests that the reactive center loop
sequence of antithrombin need not be optimal for interaction with
thrombin and may have evolved to allow the inhibitor to be selective
for procoagulant proteinases and limit its reaction with the
anticoagulant proteinase, activated protein C (30). A poor thrombin
recognition sequence in wild-type antithrombin may also have been
favored based on our finding that sequence changes that enhance
thrombin specificity also resulted in variants that preferentially
reacted as substrates rather than inhibitors of the enzyme when
complexed with a physiologic bridging heparin (14).
Reactive Center Loop Contribution to Complex Stability--
The
finding that mutations in the P6-P3' reactive center loop sequence had
small or no effects on complex stability despite the marked effects of
many of the mutations on the rate of formation of the stable complex
with thrombin and to a lesser extent with factor Xa supports a unique
mechanism for stabilization of these complexes that is different from
that of lock-and-key inhibitors. A hallmark of the lock-and-key mode of
complex stabilization is the destabilizing effect of amino acid
substitutions in reactive center loop residues of such inhibitors on
complex formation, as reflected by increases in the rate constants for
complex dissociation (47). Our finding that reactive center loop
mutations in antithrombin minimally affect the rate constants for
dissociation of antithrombin-proteinase complexes thus supports the
idea that reactive center loop interactions with the proteinase active
site do not significantly contribute to stabilizing the complexes.
These findings agree with the recent x-ray structure of a
serpin-proteinase complex (48) that supports a model for
serpin-proteinase complex stabilization in which reactive center loop
interactions with the enzyme are disrupted as a consequence of loop
cleavage at the acyl-intermediate stage allowing the loop to fully
insert into
-sheet A of the serpin and translocate the tethered
proteinase to the distal end of sheet A. Complex stabilization arises
according to this model from the reactive center loop burial in sheet A
eliminating reactive center loop interactions with the acyl-linked
proteinase and inducing a distortion of the proteinase catalytic
apparatus (49, 50).
Summary--
In summary, our studies have provided new insights
into the origin of antithrombin specificity for thrombin and factor Xa and how heparin enhances specificity for these enzymes. We have shown
that antithrombin specificity for thrombin depends on thrombin recognition of the reactive center loop sequence but that this sequence
is not optimal for thrombin recognition in either the native or
heparin-activated serpin. Heparin enhances antithrombin recognition by
the enzyme instead by providing a secondary interaction site on heparin
adjacent to the bound serpin for the enzyme to bind. By contrast, our
findings suggest that antithrombin specificity for factor Xa is
minimally dependent on the reactive loop sequence other than the P1
residue in either native or heparin-activated states due to a broader
sequence specificity of this enzyme. Heparin enhances antithrombin
recognition by the enzyme in this case through exposure of an
antithrombin exosite outside the P6-P3' loop region capable of
interacting with a complementary factor Xa exosite. Heparin
additionally provides a bridging site for factor Xa, but optimal
utilization of this site requires the presence of physiologic levels of
calcium to expose a complementary heparin-binding exosite in factor Xa
(46). Importantly, our findings rule out alterations in loop
accessibility as an important mechanism for heparin activation of the
serpin, a conclusion that is supported by the antithrombin P1 residue
mutagenesis studies we have recently reported (51).