Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational
Activation*
Mohamad Aman
Jairajpuri
,
Aiqin
Lu
,
Umesh
Desai§,
Steven T.
Olson¶,
Ingemar
Bjork
, and
Susan C.
Bock
**
From the
Departments of Medicine and Bioengineering,
University of Utah, Salt Lake City, Utah 84132, the
§ Department of Medicinal Chemistry, Virginia Commonwealth
University, Richmond, Virginia 23298, the ¶ Center for
Molecular Biology of Oral Diseases, University of Illinois, Chicago,
Illinois 60612, and the
Department of Veterinary Medical
Chemistry, Swedish University of Agricultural Sciences, Uppsala SE-751
23, Sweden
Received for publication, December 4, 2002, and in revised form, January 28, 2003
 |
ABSTRACT |
The dissociation equilibrium constant for heparin
binding to antithrombin III (ATIII) is a measure of the cofactor's
binding to and activation of the proteinase inhibitor, and its salt
dependence indicates that ionic and non-ionic interactions contribute
~40 and ~60% of the binding free energy, respectively. We now
report that phenylalanines 121 and 122 (Phe-121 and Phe-122)
together contribute 43% of the total binding free energy and 77% of
the energy of non-ionic binding interactions. The large contribution of
these hydrophobic residues to the binding energy is mediated not by
direct interactions with heparin, but indirectly, through contacts
between their phenyl rings and the non-polar stems of positively
charged heparin binding residues, whose terminal amino and
guanidinium groups are thereby organized to form extensive and
specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that
Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and
Lys-114 act cooperatively through non-ionic interactions to promote
P-helix formation and ATIII binding to the pentasaccharide. In summary,
although hydrophobic residues Phe-122 and Phe-121 make minimal contact
with the pentasaccharide, they play a critical role in heparin binding
and activation of antithrombin by coordinating the P-helix-mediated
conformational change and organizing an extensive network of ionic and
non-ionic interactions between positively charged heparin binding site
residues and the cofactor.
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INTRODUCTION |
The sulfated polysaccharide heparin functions as an
anticoagulant by binding to antithrombin III
(ATIII)1 and greatly
accelerating its rates of thrombin and factor Xa inhibition. Heparin
binding to ATIII is a two-step process consisting of an initial weak
interaction that induces a protein conformational change leading to the
formation of a high affinity binary complex with the cofactor (AT*H)
and ATIII activation (1, 2). Functional investigations of chemically
modified, naturally occurring mutant and recombinant antithrombins have
identified Arg-47, Lys-114, Lys-125, and Arg-129 as the most important
positively charged amino acid residues in the heparin binding site of
ATIII (3-12). Direct interactions of these basic residues with
negatively charged groups of heparin are observed in the crystal
structure of an ATIII-pentasaccharide complex (13). Studies of heparin
binding kinetics indicate that Lys-125 is the most important amino acid in the initial docking with heparin and that Arg-129, Lys-114, and
Arg-47 are critical for the protein conformational change step leading
to the high affinity, activated AT*H complex. Heparin binding leads to
elongation of ATIII helix D by 1.5 turns at its carboxyl-terminal end
as well as the formation of a new alpha helix, the P-helix, at its
amino-terminal end (13, 14). These structural changes are associated
with expulsion of the P14 residue from central beta sheet A and
increased reactivity with factor Xa (15, 16).
Protein-heparin interactions are comprised of ionic and non-ionic
components whose energies and relative strengths can be measured using
polyelectrolyte theory. For example, in the case of thrombin, 86% of
the free energy of heparin binding results from ionic interactions,
whereas 14% derives from non-ionic interactions (17). In contrast, for
brain natriuretic peptide, non-ionic interactions contribute 94% of
the heparin binding energy and only 6% is from ionic interactions
(18). Antithrombin III heparin binding involves five to six ionic
interactions that contribute only 40% of the binding energy (2, 17,
19). Non-ionic interactions are responsible for the remaining 60% of
the binding energy. Although non-ionic interactions contribute more to
the strength of ATIII heparin binding than do ionic interactions, work
to date has focused mostly on arginines and lysines of the heparin
binding site and ignored potentially important non-polar residues.
The present study was designed to address the roles of ATIII
phenylalanines 121 and 122 (Phe-121 and Phe-122) in heparin binding and
activation. Phe-121 and Phe-122 were selected for investigation based
on their proximity to positively charged residues of the pentasaccharide binding site and because, although not conserved across
different branches of the serpin family, these phenylalanines are
conserved in antithrombins from different vertebrate species (20),
which also conserve the capacity for heparin binding and activation
(21).
The results of our work demonstrate that, although phenylalanines
121 and 122 make minimal direct contact with heparin, they nevertheless
contribute significantly to antithrombin affinity for its cofactor and
are responsible for 43% of the total binding energy and 77% of the
free energy of non-ionic interactions. An F121A substitution decreased
antithrombin affinity for heparin 13-fold, and an F122L substitution
reduced heparin affinity >2000-fold through exclusively non-ionic
effects. The large decrease in F122L affinity was due to a moderate
decrease in the forward rate of the activating conformational change
and greatly reduced stability of the AT*H complex. Binding equilibrium
and kinetic data collected during the course of this work and
information from the structure of an antithrombin-pentasaccharide
complex also together suggest that Phe-122 and Lys-114 function
cooperatively to coordinate the P-helix-mediated activating
conformational change and to organize an extensive network of ionic and
non-ionic interactions between positively charged antithrombin residues
and heparin.
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EXPERIMENTAL PROCEDURES |
Materials--
The Bac-to-Bac baculovirus expression system,
Sf9 Spodoptera frugiperda cells, and Sf900II
growth medium were obtained from Life Technologies, Inc. S2238 and
S2765 were from Chromogenix. Human
-thrombin and pentasaccharide
were the generous gifts of Drs. William Lawson and Maurice Petitou.
Human factor Xa was purchased from Enzyme Research Laboratories. High
affinity heparin (molecular mass, ~20,000 Da) was purified in
our laboratory as previously described (22). High affinity heparin and
pentasaccharide concentrations were determined by stoichiometric
fluorescence titrations versus plasma antithrombin. Hi-Trap
heparin and Sephacryl HR-300 columns were from Amersham Biosciences,
and Econopak Q columns from Bio-Rad.
Buffers and Experimental Conditions--
PNE buffer is 20 mM sodium phosphate, pH 7.4, 0.1 mM EDTA, 100 mM NaCl. QPNE buffer is PNE with a lower NaCl concentration (20 mM). PE buffer is PNE with no NaCl. Inhibition
studies were conducted at 25 °C in PNE-PEG (PNE plus 0.1% PEG
6000). The ionic strength of pH 7.4 PNE-PEG is 0.15. Additional NaCl
was added to PNE or PE to achieve different ionic strengths for binding studies as indicated.
Mutagenesis--
F121A and F122L substitutions were made
on a beta-ATIII background (N135A) to reduce heparin binding
heterogeneity and facilitate purification (23). PCR-based megaprimer
mutagenesis was as previously described (24) using the
oligonucleotides 5'-CATCTGATCAAATCCACGCTTTCTTTGCCA-3' for F121A
and 5'-CATCTGATCAAATCCACTTCCTGTTTGCCAAAC-3' for F122L and
BstYI digestion to identify mutant clones. Following
sequence verification of the PCR-generated DNAs,
NheI-SacI fragments containing the substitutions
were used to replace the corresponding portion of the human ATIII.N135A
cDNA sequence cloned in pFastbac1, followed by transposition and
virus production in the Bac-to-Bac expression system.
ATIII Purification--
Supernatants from baculovirus-infected
Sf9 cultures were harvested and cleared of cells and debris at
4-5 days post infection, when the trypan blue viability of the host
cells had dropped to ~80%. 20-ml Hi-Trap heparin affinity columns
were loaded with 0.22-µm filtered supernatants diluted 1:1 with PE,
washed with PE, and eluted with a 100 ml of 0.02-2.4 M
NaCl gradient. Fractions containing the peak of inhibitory activity
against thrombin were pooled, concentrated, and buffer-exchanged in
tangential flow centrifugal concentrators. The sample in ~1 ml was
applied to a 2.6- × 60-cm Sephacryl S-300 column. Fractions containing
the peak of thrombin inhibitory activity were pooled, concentrated, and
buffer-exchanged into PE. The sample was subjected to another round of
heparin affinity chromatography as described above, but using a 5-ml
column. The fractions with peak thrombin inhibitory activity were
pooled, concentrated, and buffer-exchanged into QPNE. The sample was
applied to a 5-ml Econopak Q column in QPNE and eluted with a 45-ml
0.02-0.6 M NaCl gradient. The peak of thrombin inhibitory
activity was concentrated and buffer-exchanged into PNE. SDS-PAGE was
also used to monitor purification steps and to assess the purities of
the recombinant antithrombins. Protein concentrations of the purified
F121A/N135A and F122L/N135A were determined from absorbance at 280 nm
using the molar extinction coefficient of plasma ATIII (25).
Stoichiometries, Affinities, and Ionic Strength Dependence of
Heparin Binding--
Stoichiometries and dissociation equilibrium
constants (Kd) for pentasaccharide and full-length
heparin binding to ATIII variants were determined by titrations
monitored by the tryptophan fluorescence enhancement that accompanies
the binding interaction as previously described (22, 26).
Stoichiometric titrations were performed with full-length heparin
at an ionic strength of 0.10, using antithrombin concentrations based
on absorbance measurements that were more than 10 times the
Kd. Pentasaccharide and full-length heparin binding
to antithrombin for Kd titrations used active ATIII
concentrations obtained from the heparin binding stoichiometries. Ionic
and non-ionic components of heparin binding to the F121A/N135A,
F122L/N135A, and N135A antithrombins were obtained from the ionic
strength dependence of the dissociation equilibrium constant for
full-length heparin at pH 7.4 and for heparin pentasaccharide at pH
6.0. Derivation of Z, the number of ionic interactions
involved in the binding, and Kd', which
indicates the strength of non-ionic interactions, is discussed in
detail under "Results."
Kinetics of Heparin Binding--
Heparin binds to antithrombin
according to the two-step, induced-fit mechanism depicted in Scheme I
(19, 27, 28) as follows.
In this scheme, K1 is the dissociation
equilibrium constant for the initial binding of antithrombin III (AT)
and heparin (H), which leads to the formation of a low affinity
complex, AT-H. k+2 is the forward rate constant
for the subsequent rapid conformational change leading to the high
affinity activated complex, AT*H, and k
2 is
the reverse rate constant for this step, and is equivalent to
koff for the overall reaction. The two-step binding mechanism leads to a hyperbolic dependence of
kobs, the pseudo-first-order rate constant, on
total heparin concentration ([H]o), as described by
Equation 1,
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(Eq. 1)
|
kon and koff (=
k
2) for the overall reaction can be obtained
from the initial slope and y-intercept of a linear
regression of a kobs versus
[heparin] plot at low heparin concentrations, whereas
K1 and k+2 can be
determined by non-linear regression of hyperbolic plots of
kobs versus [H]o
extending to higher heparin concentrations that approach saturation.
The kinetics of full-length heparin and pentasaccharide binding to
F122L/N135A were analyzed at 25 °C under two conditions of pH and
ionic strength (pH 7.4, 0.15 I and pH 6.0, 0.075 I). Binding was performed under pseudo-first-order
conditions by maintaining a heparin to antithrombin molar ratio of
5:1, and the binding reactions were followed kinetically by
monitoring the protein fluorescence increase with a stopped-flow
fluorometer (Applied Photophysics SX-17MV). Progress curves from 5 to
12 binding reactions were fit to a single-exponential function to
obtain the average kobs at different cofactor
concentrations, ranging from 0.2 to 25 µM for full-length
heparin, and from 0.2 to 100 µM for pentasaccharide. kon and koff
(k
2) for the overall bimolecular reaction and
K1 and k+2 of Scheme 1 were determined by linear and non-linear regression fitting,
respectively, of the initial linear and overall hyperbolic plots of
kobs versus [H]o to
Equation 1 using GraphPad Prism and Kaleidagraph software. Calculated
Kd values for heparin binding to F122L/N135A were
obtained by dividing the measured koff value by
the measured kon value.
Thrombin and Factor Xa Inhibition--
Inhibition
stoichiometries (SI) for thrombin and factor Xa and apparent second
order rate constants (kapp) for the inhibition of these enzymes by F121A/N135A, F122L/N135A and their N135A parent were measured in the absence and presence of pentasaccharide and full-length heparin as previously described (22). Association rate
constants (kassoc) were obtained by
correcting kapp values for the content of
inactive material in the sample and/or substrate pathway partitioning
by multiplying the average of 2-3 kapp
measurements by the SI value obtained for the same ATIII-target
enzyme-heparin combination.
Structural Analysis--
The I chains from a 2.62-Å
resolution structure of human plasma
-antithrombin (Protein Data
Bank, pdb.1E05) and a 2.90-Å resolution structure of human plasma
-ATIII bound to pentasaccharide (pdb.1E03) were viewed and analyzed
using QUANTA software.
 |
RESULTS |
Expression and Purification--
F121A/N135A and F122L/N135A
recombinant antithrombins and their N135A parent were expressed in the
baculovirus system. The parental N135A molecule corresponds to the
naturally occurring
isoform of antithrombin III, which is not
glycosylated on asparagine-135. The N135A
isoform background
facilitates investigation of antithrombin-heparin interactions by
eliminating the heparin binding heterogeneity resulting from partial
modification of Asn-135 and the associated production of
and
glycoforms when the wild type N-glycosylation consensus
sequence (asparagine-proline-serine) is present at residues 135-137
(23, 29). The higher heparin affinity of the
antithrombin isoform,
relative to the
antithrombin isoform, is experimentally advantageous with respect to purification and increased heparin binding
affinity of the control. The pentasaccharide binding site of ATIII
(pdb.1E04) resembles that of ATIII
(pdb.1E05), and previous studies
have shown that conclusions about the mechanism of heparin binding and
activation obtained from investigation of
antithrombin mutants
(N135A background) apply to the antithrombin
isoform as well (31).
The purification of F121A/N135A and F122L/N135A from supernatants of
baculovirus-infected Sf9 cells involved heparin affinity, gel
exclusion, and anion exchange chromatography steps. Purified
F121A/N135A, F122L/N135A, and N135A comigrated on SDS-PAGE and were
>95% homogeneous.
Affinities of F121A/N135A and F122L/N135A
Binding to Full-length Heparin and Pentasaccharide--
Heparin
affinity column elution behavior during chromatographic purification of
the antithrombins suggested that the F121A substitution moderately
reduced affinity for heparin and that the F122L substitution greatly
decreased heparin binding. In contrast to control N135A antithrombin,
which eluted from immobilized heparin at ~2.2 M NaCl,
F121A/N135A eluted at 1-1.3 M NaCl, and F122L/N135A at
0.2-0.3 M NaCl.
Binding stoichiometries and affinities of the mutant and control
antithrombins for heparin and pentasaccharide were measured by
titrations monitored by the fluorescence enhancement resulting from
protein conformation-dependent changes in tryptophan
environment (1, 30). Compared with the 40% increase reported for the N135A control and plasma-derived antithrombin (23), full-length heparin
induced an intrinsic fluorescence enhancement of ~10% for
F121A/N135A, and ~30% for F122L/N135A. Heparin binding
stoichiometries were 0.8 for two preparations of F121A/N135A, 0.8 and
1.0 for two preparations of F122L/N135A, and 0.6-0.8 for several
preparations of N135A.
Table I presents dissociation equilibrium
constant data for N135A, F121A/N135A, and F122L/N135A binding to
full-length high affinity heparin and pentasaccharide under ionic
strength conditions where the binding could be experimentally measured.
Full-length heparin titrations were conducted at pH 7.4, and
pentasaccharide titrations at pH 6.0. The Kd
obtained for high affinity heparin binding to the N135A control at pH
7.4 and 0.3 I was similar to previously reported values (23,
31). Kd values for heparin binding to the two mutant
inhibitors revealed that, although both bound heparin less tightly, the
magnitude of their affinity losses differed significantly. Measurements
at 0.3 and 0.4 I demonstrated an overall affinity loss of
~13-fold for F121A/N135A compared with its N135A parent. Based on
measurements made at 0.3 and 0.15 I, the F122L substitution
induced a more substantial ~2000-fold decrease in affinity for
full-length heparin. Comparison of Kd values for
F122L/N135A binding to pentasaccharide at pH 6.0 and 0.025 and 0.075 I with previously published Kd values for
the N135A control extrapolated to these ionic strengths (11) revealed a
3000- to 4000-fold loss in F122L/N135A affinity for the core sequence
of anticoagulant heparin.
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Table I
Dissociation equilibrium constants for full-length heparin and
pentasaccharide binding to F121A/N135A,
F122L/N135A, and N135A antithrombin variants at 25 °C
Dissociation equilibrium constants (Kd) for
full-length heparin (HAH) binding were determined from fluorescence
titrations at pH 7.4 and the indicated ionic strengths.
Kd for pentasaccharide (H5) binding to F122L/N135A
were measured at pH 6.0 and the indicated ionic strengths.
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Ionic and Non-ionic Components of F121A/N135A and
F122L/N135A Binding to Full-length Heparin and
Pentasaccharide--
The strengths of the ionic and non-ionic
components of the overall binding interaction between antithrombin and
heparin can be quantified by Z, the number of ionic
interactions involved in the binding, and Kd',
the dissociation constant in 1 M Na+,
which reflects the strength of non-ionic interactions (2, 17). Hydrogen
bonds, although electrostatic in nature, are classified as non-ionic
interactions in this analysis, because the ionic interactions measured
by the technique include only ion pair-type interactions. For a given
antithrombin-heparin combination, values of Z and
Kd' were determined from Equation 2, which
describes the sodium ion dependence of the observed overall
dissociation constant Kd,obs, with
Z and Kd' defined as above, and
being the fraction of Na+ bound per heparin charge and
released upon binding to antithrombin (estimated to be 0.8 (17)).
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(Eq. 2)
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Fig. 1 shows that, in accordance
with Equation 2, the logarithms of Table I Kd values
(and additional Kd values obtained from titrations
at other ionic strengths) varied linearly with the logarithms of the
sodium concentrations at which they were measured. Z and log
Kd' values presented in Table
II were derived from the slopes and
intercepts of linear regression fits for each antithrombin-heparin pair
in Fig. 1. At pH 7.4, approximately six ionic interactions participate
in the binding of the full-length heparin to the N135A control, whereas
at pH 6, approximately five ionic interactions participate in binding of the pentasaccharide, in agreement with measurements from previous work (2, 19). Z values for F121A/N135A and F122L/N135A
binding to full-length heparin and pentasaccharide were not
significantly different from corresponding Z values for the
N135A parent, indicating that the observed 13-fold and >2000-fold
binding affinity losses are occurring without changing the number of
ionic interactions between the mutants and the heparins. Table II also
shows 30-, 1600-, and 5000-fold losses in the strengths of non-ionic
interactions for the binding of F121A/N135A to full-length heparin,
F122L/N135A to full-length heparin, and F122L/N135A to pentasaccharide,
based on increases of 1.5, 3.2, and 3.7 in the
log(Kd'), respectively. Both the moderate loss
of heparin binding affinity due to substitution of phenylalanine 121 with alanine and the large loss of heparin binding affinity due to
substitution of phenylalanine 122 with leucine were entirely accounted
for by reductions in the non-ionic components of the binding
interactions.

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Fig. 1.
Ionic strength dependence of the dissociation
equilibrium constant for full-length heparin binding to N135A,
F121A/N135A, and F122L/N135A at pH 7.4, 25 °C and for
pentasaccharide binding to N135A and F122L/N135A at pH 6.0, 25 °C. Each point represents the average ± S.E. for 2-5 titrations at the indicated ionic strength. Not visible
are error bars that lie within the dimensions of the
symbols. Lines show the linear regression fits used to
determine Z and Kd' values (Table II)
for antithrombin interactions with full-length heparin at pH 7.4 (dotted lines) and pentasaccharide at pH 6.0 (solid
lines). The data for the interaction of pentasaccharide with the
N135A control were taken from previously published work (11) and are
provided for comparison. Open circles, HAH-N135A; open
triangles, HAH-F121A/N135A; open squares,
HAH-F122L/N135A; solid circles, H5-N135A; solid
squares, H5-F122L/N135A.
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Table II
Ionic and non-ionic components of heparin binding to the
F121A/N135A, F122L/N135A, and N135A
antithrombin variants at 25 °C
The number of ionic interactions (Z) involved in the binding
of pentasaccharide (H5) or full-length heparin (HAH) to the
antithrombin variants, and the non-ionic contribution
(log(Kd')) to the overall binding interaction
were determined from the slopes and intercepts, respectively, of the
Fig. 1 log(Kd) versus log[Na+]
plots. Errors represent ± S.E. obtained by linear regression.
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The contributions of phenylalanines 121 and 122 to the free energy of
full-length heparin binding to ATIII were calculated from Table I and
Fig. 1 pH 7.4 dissociation equilibrium constant data. The
Kd for F122L/N135A was measured at 0.15 I, whereas those for N135A and F121A/N135A were extrapolated
from measurements made at higher ionic strengths. Using
G0 =
RT
ln(Kd), total binding energies were calculated and
yielded values of 59.0, 53.4, and 39.0 kJ/mole for N135A, F121A/N135A,
and F122L/N135A, respectively. The magnitudes of the ATIII variant
binding energy losses indicate that Phe-121 and Phe-122 together
contribute 43% of the total free energy of full-length heparin binding
to ATIII at physiological pH and ionic strength. A similar analysis
using log Kd' values obtained at pH 7.4 (Table
II) yielded values of 36.3, 27.4, and 17.3 kJ/mole, respectively, for
the non-ionic binding energies of N135A, F121A/N135A, and F122L/N135A
with full-length heparin. The associated binding energy losses indicate
that Phe-121 and Phe-122 together contribute 77% of the non-ionic
binding energy.
Kinetic Analysis of Full-length Heparin and Pentasaccharide Binding
to F122L/N135A--
Rapid kinetic analysis of full-length
heparin and pentasaccharide binding to F122L/N135A was performed to
determine which step or steps of the two-step induced fit binding
mechanism (Scheme I) are affected by the F122L mutation and responsible
for the >2000-fold reduction in binding affinity. For the binding of
F122L/N135A to 0.2-25 µM HAH at pH 7.4, 0.15 I and for its binding to 0.2-12 µM H5 at pH
6.0, 0.075 I, kobs exhibited a
hyperbolic dependence on heparin concentration as illustrated in
panels A and B of Fig. 2. However, kobs
for F122L/N135A binding to pentasaccharide at pH 7.4 and 0.15 I was linear through at least
100 µM H5 (Fig. 2C). Table III presents
kinetic constants for the binding reactions derived by fitting the data
of Fig. 2 to Equation 1 as described under "Experimental
Procedures" and in the figure legend. Calculated Kd values were obtained by dividing
koff by kon and were in
general agreement with measured Kd values for
binding to full-length heparin at pH 7.4 and 0.15 I, and
binding to pentasaccharide at pH 6.0 and 0.075 I. The
similarity of the Kd values determined by different
methods supports the validity of data obtained by stopped-flow and
fluorescence titration experiments. The somewhat larger difference
between the calculated and measured Kd values for
pentasaccharide binding at pH 6.0, 0.075 I may reflect a
small contribution of the pre-equilibrium pathway in this case (9).

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Fig. 2.
Kinetics of full-length heparin and
pentasaccharide binding to F122L/N135A. Plots show the linear and
hyperbolic dependence of kobs on heparin
concentration under various conditions. A, full-length
heparin (HAH) binding to F122L/N135A at pH 7.4, 0.15 I, 25 °C. B, pentasaccharide (H5)
binding to F122L/N135A at pH 6.0, 0.075 I, 25 °C.
C, pentasaccharide (H5) binding to F122L/N135A at
pH 7.4, 0.15 I, 25 °C. Average values ± S.E. for
5-12 measurements at each HAH or pentasaccharide concentration are
plotted. Not visible are error bars that lie within the
dimensions of the symbols. The kon and
koff values in Table III were obtained by linear
regression fitting of data points in A and B
insets and panel C to Equation 1. The
K1 and k+2 values in
Table III were obtained by non-linear regression fitting of data from
the full range of heparin concentrations in A and
B to Equation 1 using koff values
determined at low concentrations for k 2.
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Table III
Measured kinetic constants, bimolecular association rate constants,
dissociation rate constants, and calculated dissociation equilibrium
constants for full-length heparin and pentasaccharide binding to
F122L/N135A at 25 °C
K1 and k+2 were obtained by
non-linear regression fitting of the hyperbolic curves in Fig. 2 to
Equation 1. kon and koff were
obtained by regression using 5-8 points from the initial linear
regions of the Fig. 2 kobs versus [HAH]
or [H5] plots. Errors are ± S.E. of the fits. Calculated
Kd values were obtained by dividing
koff by kon. N135A control data
are from previously published studies as indicated in the footnotes and
are provided for facile comparison.
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Analysis of Table III data shows that the overall effect of the F122L
substitution is to both reduce the kon values
and increase the koff values for heparin and
pentasaccharide binding. However, the extent to which
kon and koff are altered
varies with the type of heparin and the binding conditions. For the
interaction of F122L/N135A with full-length heparin at pH 7.4 and 0.15 I, the overall association rate constant
kon was reduced ~15-fold, and the overall
dissociation rate constant koff was increased by
~235-fold. The reduction in kon could be
entirely accounted for by a similar reduction in
k+2, the forward rate constant for the
conformational change step leading to the activated AT*H complex, and
there was no evidence for an increase in K1, the
dissociation equilibrium constant for the initial binding step leading
to the AT-H intermediate. Under the same conditions (pH 7.4 and 0.15 I), F122L/N135A binding to pentasaccharide exhibited an
~120-fold decrease in kon and a ~120-fold
increase in koff. Although the magnitude of the
koff increase was similar to that observed with
full-length heparin, the magnitude of the kon
decrease was almost one log higher than with HAH. Moreover, the
linearity of the dependence of kobs on [H5] in
the concentration range studied (Fig. 2C) indicates that the
more severe kon defect for pentasaccharide
binding is due to an increase in K1 that is not
observed with full-length heparin.
K1 and k+2 contributions
to kon for the binding of F122L/N135A and
pentasaccharide were measured under pH 6.0 and 0.075 I
conditions, where binding is tighter and kobs
displayed a hyperbolic dependence on [H5] at reasonable
concentrations of pentasaccharide (Fig. 2B). Under these
conditions, the loss of binding affinity is overwhelmingly due to a
very large ~1600-fold increase in koff. However, there is also a modest 7-fold decrease in
kon, and it was possible to discern from the
non-linear regression fit that this kon
reduction derived from a 3-fold increase in K1
(the dissociation equilibrium constant for the weak AT-H intermediate)
and a 3-fold decrease in k+2 (the forward rate
constant for the conformational change leading to AT*H). The
identification of a K1 defect for F122L/N135A
binding to pentasaccharide, considered in conjunction with the normal
K1 for F122L/N135A binding to full-length
heparin, suggests that the extra oligosaccharide residues of
full-length heparin compensate for the decreased ability of the
pentasaccharide to form the low affinity AT-H intermediate with the
F122L/N135A mutant. This might be accomplished by the larger heparin
providing an electrostatic guiding effect that enhances the initial
rate of association.
Thrombin and Factor Xa Inhibition--
Table
IV lists the association rates
(kassoc) of F121A/N135A and F122L/N135A with
thrombin and factor Xa in the absence and presence of full-length
heparin or pentasaccharide. Association rates were obtained by
measuring the apparent inhibition rate (kapp)
and stoichiometry of inhibition (SI) for the different ATIII-target
enzyme-cofactor combinations at pH 7.4 and 0.15 I and
corrected for the content of inactive material and/or substrate partitioning by multiplying kapp × SI. Values
of kassoc for F121A/N135A and F122L/N135A inhibition
of thrombin and factor Xa in the absence and presence of
full-length heparin and pentasaccharide are similar to previously
published values for the control N135A molecule (19). Therefore,
F121A/N135A and F122L/N135A are properly folded and exhibit the same
profiles of full-length heparin and pentasaccharide activation of
proteinase inhibition observed for plasma antithrombin and the control
recombinant ATIII. Specifically, the reactivities (kassoc) of F121A/N135A and F122L/N135A with
thrombin and factor Xa were accelerated three orders of
magnitude by full-length heparin, whereas
pentasaccharide-mediated factor Xa inhibition rates increased by only
two orders of magnitude, and thrombin inhibition was minimally stimulated by the addition of pentasaccharide.
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Table IV
Association rate constants of N135A, F121A/N135A, and
F122L/N135A with thrombin and factor Xa in the absence
and presence of full-length heparin or pentasaccharide at pH 7.4, 0.15I, and 25 °C
Apparent rate constants (kapp) and stoichiometries
(SI) for the inhibition of thrombin and factor Xa in the absence
(uncat) of cofactor and the presence full-length heparin
(HAH) or pentasaccharide (H5) were measured at pH
7.4, 0.15I, and 25°C as described under "Experimental
Procedures." Association rate constants (kassoc)
were obtained by multiplying the kapp and SI values
to correct for the content of inactive material in the preparation and
for substrate partitioning. Tabulated values are average ± S.E.
for 3-9 measurements, except as noted.
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Although the initial rates for association of F121A/N135A and
F122L/N135A and their heparin complexes with thrombin and factor Xa are
normal, stoichiometry of proteinase inhibition data indicate that,
following acylation with the target enzyme, partitioning into the
substrate arm of the branched pathway of serpin inhibition is increased
compared with the N135A control. Stoichiometries for F121A/N135A
uncatalyzed inhibition of thrombin and factor Xa were in the 3.3-4.4
range. In the presence of pentasaccharide, inhibition stoichiometries
ranged from 3.6 to 4.2, and in the presence of full-length heparin,
values between 4.8 and 5.4 were obtained. In contrast, the heparin
binding stoichiometry for F121A/N135A was 0.8, which would correspond
to a proteinase inhibition SI of 1.25 if all heparin-binding molecules
in the sample were competent for proteinase inhibition and
no substrate partitioning occurred. The mismatch between the proteinase
inhibition stoichiometries and the heparin binding stoichiometry of
F121A/N135A suggests that Phe-121 plays a role in promoting ATIII
utilization of the complex formation arm, rather than the substrate
pathway arm, of the branched serpin inhibition pathway. Consistent with
this alternative, a band with the mobility of cleaved antithrombin was
readily observed on non-reducing SDS-polyacrylamide gels of F121A/N135A
complex formation reactions (data not shown). Stoichiometries for
F122L/N135A inhibition of thrombin and factor Xa in the absence of
heparin were in the 1.9-2.0 range. In the presence of pentasaccharide they ranged from 2.4 to 2.5, whereas in the presence of full-length heparin, values between 2.3 and 2.6 were obtained. The heparin binding
stoichiometry for F122L/N135A ranged from 0.8 to 1.0. Small amounts of
a band with the mobility of cleaved antithrombin were also observed on
non-reducing SDS-polyacrylamide gels of F122L/N135A complex formation
reactions (data not shown). The gel data and the mismatch between the
proteinase inhibition stoichiometries and the heparin binding
stoichiometry of F122L/N135A suggest that the wild type Phe-122 residue
of antithrombin III also contributes to blocking flux through the
substrate arm of the branched pathway, however, its importance in this
regard is less than that of the adjacent Phe-121 residue. The observed
substrate tendencies of the F121A/N135A and F122L/N135A variants imply
that the nature of the binding interaction between the
pentasaccharide-binding region of antithrombin and heparin influences
the efficiency of acylated strand 4A insertion and/or stable inhibitory
complex formation. This possibility is consistent with previous studies showing that antithrombin exhibits increased substrate behavior in the
presence of heparin (19, 32).
 |
DISCUSSION |
As illustrated in Fig. 3,
phenylalanines 121 and 122 are located below the pentasaccharide and in
the center of a cluster of positively charged residues (Arg-47,
Lys-114, Lys-125, and Arg-129) known to participate in heparin binding
(9-12, 33). To investigate the roles of Phe-121 and Phe-122 in heparin
binding and activation of ATIII, we made F121A and F122L substitution mutants on a
-antithrombin-like N135A background. F121A/N135A and
F122L/N135A association rates for inhibition of thrombin and factor Xa
in the absence and presence of full-length heparin and pentasaccharide
were similar to control, and SDS-stable inhibitory complexes with
target enzymes formed, indicating proper folding and function with
respect to proteinase inhibition. However, the mutants displayed
distinct heparin binding and activation defects. F121A/N135A exhibited
a 13-fold decrease and F122L/N135A exhibited a >2000-fold decrease in
affinity for full-length heparin. In both cases, reduced binding was
entirely due to the loss of non-ionic interactions (hydrogen bonds, van
der Waals, and hydrophobic interactions), whereas the number of ionic
interactions remained unchanged. For binding of full-length heparin to
F122L/N135A under physiological conditions of pH and ionic strength,
formation of the weak AT-H intermediate in the two-step-induced fit
mechanism for heparin activation of ATIII occurs normally, as indicated
by its normal K1. However, a moderate reduction
in the value of k+2 and a large increase in the
value of k
2 revealed that the >2000-fold loss
of binding affinity results from a ~20-fold reduction in the rate of
the AT-H to AT*H conformational transition coupled with a >200-fold
decrease in stability of the activated AT*H complex. The effects of the
Phe-122 mutation on pentasaccharide binding are comparable to its
effects on the binding of full-length heparin; however, in addition a
slight decrease in the affinity of the first binding step is apparent
for the pentasaccharide.

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Fig. 3.
Phe-121 and Phe-122 are located in the center
of a cluster of positively charged heparin binding residues under the
bound pentasaccharide. The pentasaccharide-binding region
from the I-chain of pdb.1E03 is shown. Phe-121 side chain is
yellow. Phe-122 side chain is green. Arg-47,
Lys-114, Lys-125, and Arg-129 side chains are blue. The carbon trace of antithrombin III is gray, except for
residues 113-119, which form the P-helix and are drawn in
red. All non-hydrogen atoms of the pentasaccharide are
shown, with carbon in green, oxygen in red, and
sulfur in yellow. Individual sugars of the pentasaccharide
are denoted in alphabetical order from the nonreducing
end.
|
|
The findings of our work on the functional contributions of
phenylalanines 121 and 122 to antithrombin heparin binding and activation were evaluated from a structural point-of-view using high
resolution x-ray structures for human plasma
antithrombin bound
(pdb.1E03) and not bound (pdb.1E05) to pentasaccharide. From this
analysis, we have identified a series of Phe-121- and Phe-122-mediated
non-ionic interactions that appear to make significant contributions to
the strength of ATIII-heparin binding. The structural analysis also
revealed several Phe-121- and Phe-122-helix P interactions that appear
to be critical for the conformational activation step and stabilization
of the high affinity AT*H complex, which were defective in the kinetic
studies of the F122L/N135A mutant.
Phe-121 and Phe-122 Contributions to the Non-ionic Component of
ATIII Heparin Binding--
Fig. 3 shows that the most direct
interaction between Phe-122 and the pentasaccharide involves the CZ of
its phenylalanine side chain and the 3-O-sulfate on sugar F. Considering that extensive direct, non-ionic interactions between
Phe-122 and the pentasaccharide are not observed in the
co-crystal structure, it is paradoxical that the large loss of heparin
binding affinity measured for the F122L mutant should be entirely
accounted for by the loss of non-ionic interactions between ATIII and
heparin (Table II). This disparity between the functional data and
first inspection of the crystal structure can be resolved by
hypothesizing that heparin makes non-ionic interactions with ATIII that
do not directly involve the Phe-122 residue but are
Phe-122-dependent.
Further examination of the co-crystal structure (Fig. 3) reveals that
the distal carbons of the Phe-122 phenyl ring form van der Waals and
hydrophobic interactions with the hydrocarbon stems of the Arg-47 and
Lys-114 side chains, positioning their respective terminal guanidinium
and amino groups for multiple ionic and hydrogen bond interactions with
the pentasaccharide. Measurements of the packing of the Phe-122
phenyl ring against the alkyl side chains of Arg-47 and Lys-114 in the
ATIII-pentasaccharide complex are presented in Table V. Table
VI lists measurements for specific ionic
and non-ionic interactions formed between the pentasaccharide and the Arg-47 guanidinium side chain or
Lys-114 epsilon amino group as a result of their positioning by the
hydrophobic interactions between Phe-122 and Arg-47 and between Phe-122
and Lys-114 described in Table V. The overall conclusion from
the information in Fig. 3 and Tables V and VI is that non-ionic
interactions between Phe-122 and the proximal stems of the Arg-47 and
Lys-114 side chains lead to extensive and specific contacts of their
distal charged groups and the pentasaccharide. In the case of Arg-47, the guanidinium position specified by the interaction of its stem and
the Phe-122 phenyl ring leads to four ionic and two non-ionic interactions involving the F, G, and H sugars of the pentasaccharide (see Fig. 3 and the right side of Table VI). In the case of
the Lys-114 side chain, three different ionic interactions and five different non-ionic interactions with the pentasaccharide form as the
result of the epsilon amino position specified by interactions of
Phe-122 with Lys-114 CD and CE. The ionic and non-ionic interactions of
Lys-114 with the cofactor are distributed over the pentasaccharide F,
G, and H sugars, as shown in Fig. 3 and on the left
side of Table VI.
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Table V
Packing of Phe-122 against Arg-47 and Lys-114
Van der Waals and hydrophobic contacts of Phe-122 atoms with Arg-47 and
Lys-114 atoms in the I-chain of antithrombin-pentasaccharide
complex (pdb.1E03).
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Table VI
Ionic and non-ionic contacts of antithrombin residues Arg-47 and
Lys-114 with bound pentasaccharide
Distances between the indicated atoms of antithrombin residues Arg-47
or Lys-114 and bound pentasaccharide were measured in the
I-chain of pdb.1E03.
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|
In the F122L variant the distal CZ, CE1, and CE2 carbons of the
normally occurring phenylalanine side chain are missing. As shown in
Table V, these are exactly the carbons that mediate van der Waals and
hydrophobic bonding of Phe-122 with Lys-114 and Arg-47. In the absence
of the intact Phe-122 phenyl ring, positioning of Arg-47 and
Lys-114 to develop the full range of non-ionic interactions described
in Table VI will occur less efficiently. Although the unchanged
Z value of F122L/N135A indicates that the normal number of
ionic interactions with the pentasaccharide form despite the
substitution, the substantial Kd' increase
suggests that the distal carbons of the phenylalanine ring are
necessary for participating in affinity-augmenting hydrophobic/van der
Waals interactions, and to precisely position the Lys-114 and Arg-47 epsilon amino and guanidinium groups for highly directional hydrogen bonding, which also contributes to the strength of the interaction. Thus, we propose that a substantial component of the >2000-fold loss
in binding energy results from the reduced ability of the substituting
leucine residue to specifically promote the ATIII-pentasaccharide van
der Waals, hydrophobic, and hydrogen bond interactions listed in Table
VI. With respect to Lys-114, affected interactions in the F122L/N135A
mutant would include the hydrophobic interaction between the stem of
the lysine and sugar H of the pentasaccharide, and a trifurcated
hydrogen bond between its epsilon amino group and three sites on sugar
G. For Arg-47, the bifurcated hydrogen bond between the Arg-47
guanidinium and two sulfates of the H sugar are disfavored in the
F122L/N135A mutant.
Van der Waals and hydrophobic interactions between the hydrocarbon stem
of lysine 125 and the phenyl rings of Phe-121 and Phe-122 may also
organize its epsilon amino group for favorable ionic and non-ionic
hydrogen bond interactions with the D, E, and F sugars of the
pentasaccharide. However, it is not possible to address this issue
directly by inspection of the pdb.1E03 co-crystal structure due to the
absence of density for the Lys-125 side chain distal to its
carbon
(see Fig. 3). The low electron density may be due to population of more
than one conformation in pentasaccharide-bound ATIII. This appears
possible based on the position of the Lys-125 C
and C
atoms, and
the potential of its epsilon amino group to form hydrogen bond or ionic
interactions with multiple polar atoms on the D, E, and F sugars and
different van der Waals/hydrophobic interactions with Phe-121 and
Phe-122. The 13-fold loss of binding affinity observed for F121A
relative to its N135A parent may result from the loss of hydrophobic
and van der Waals interactions between the ring of Phe-121 and proximal
carbons of the Lys-125 side chain and/or the failure of the epsilon
amino group to be optimally positioned to hydrogen bond with the pentasaccharide.
Proposed Roles of Phe-121, Phe-122, the P-helix and Lys-114 in the
Heparin-dependent Conformational Change and Stabilization
of AT*H--
Fig. 4A shows
that Phe-121 and Phe-122 are partially exposed to solvent in the native
conformation of antithrombin (1E05.i, left panel) but shift
to a more hydrophobic environment when heparin is bound (1E03.i,
right panel). Burial of Phe-121 and Phe-122 is associated
with movement of heparin binding site lysine and arginine residues
(Fig. 4B) to positions in which extensive and specific ionic
and non-ionic interactions with the pentasaccharide develop, as
discussed above and enumerated in Table VI. Fig.
5 shows that Phe-121 and Phe-122 are
located in the center of the helix D, helix A, helix P, and N-terminal
polypeptide structural elements that rearrange during the protein
conformational change associated with heparin binding and activation.
In this central location, Phe-121 and Phe-122 are ideally situated to
contribute to and to coordinate the conformational change leading to
high affinity binding of heparin and increased reactivity of the
reactive loop with target proteinases.

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Fig. 4.
A, Phe-121 and Phe-122 are partially
exposed to solvent in the native conformation of antithrombin but shift
to a more hydrophobic environment when heparin is bound.
Drawings show the surface of antithrombin III in the
Phe-121/Phe-122 region of the heparin binding site as viewed from the
solvent. The left panel is the van der Waals surface for the
I-chain of native human antithrombin III, not bound to
heparin (pdb.1E05). The right panel shows the van der Waals
surface from the structure of the I-chain of the human
ATIII-pentasaccharide complex (pdb.1E03), with the overlying
pentasaccharide removed to permit viewing. Phe-121 is
yellow. Phe-122 is green. B, heparin
binding and burial of Phe-121 and Phe-122 are accompanied by
rearrangement of heparin binding site lysine and arginine residues. The
structures of the I-chains of human antithrombin III unbound
(pdb.1E05) and bound to pentasaccharide (pdb.1E03) were superpositioned
using the entire sets of carbon data and the least-squares
algorithm in QUANTA software. The carbon trace of native
antithrombin III (not bound to pentasaccharide) is yellow.
The carbon trace of antithrombin III in complex with
pentasaccharide is gray. Phe-121 is yellow, and
Phe-122 is green. Arg-47, Lys-114, Lys-125, and Arg-129 are
blue. The arrows illustrate the movement of
individual residues from their positions in native antithrombin
(tails) to their positions in the ATIII-pentasaccharide
complex (arrowheads).
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Fig. 5.
Phe-121 and Phe-122 are located in the center
of the helix D, helix A, helix P, and N-terminal polypeptide structural
elements that rearrange during the protein conformational change
associated with heparin binding and activation. The carbon
backbones of antithrombin III I-chains not bound (pdb.1E05)
and bound to pentasaccharide (pdb.1E03) were superpositioned as above.
Native, unbound ATIII backbone is yellow. The backbone of
ATIII in complex with pentasaccharide is gray, except for
residues 113-119, which form the P-helix and are drawn in
red. Phe-121 is yellow, and Phe-122 is
green. hA indicates helix A, hD
indicates helix D, and hP indicates helix P.
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When heparin binds antithrombin III, a short, one-turn
helix is
formed from residues 113-119 at the amino-terminal end of helix D
(red C
backbone in Figs. 3 and 5). This
conformation-dependent helix is called the P-helix (13), and it
is not present in native antithrombin (see Fig. 5) (34). Two naturally
occurring mutants disrupt P-helix formation due to substitutions of
serine 116 or glutamine 118 with the imino acid proline (35, 36). S116P and Q118P variant antithrombins exhibit heparin binding defects and
cause thrombosis, suggesting that P-helix formation is important for
heparin binding and for the physiological expression of antithrombin activity. Structural and kinetic considerations further suggest that
P-helix formation is of central importance in the conformational change
generating the high affinity activated AT*H complex and that Phe-122
and the key pentasaccharide binding residue lysine-114 act
cooperatively in the formation of AT*H.
Fig. 4B and Table VII show
that P-helix formation is accompanied by the development of close
contacts between Phe-121/Phe-122 and P-helix residues, including
Lys-114. In native ATIII, Phe-122 and Lys-114 are well separated (see
arrow tails in Fig. 4B). However, in the complex
with pentasaccharide, these residues have moved near to each other (see
arrowheads in Fig. 4B), and the Phe-122 phenyl
ring is in van der Waals/hydrophobic contact with the side chains of
P-helix residues Lys-114 and Thr-115 (see Table VII). Similarly,
Phe-121 and Gln-118 move into close contact with each other (4.0 Å,
see Table VII) in the structure of the P-helix-containing pentasaccharide-bound complex. Thus, extensive contacts between Phe-122 and Lys-114 in the crystal structure of the
ATIII-pentasaccharide complex, but not in the structure of native
antithrombin, suggest that these residues act cooperatively to form the
P-helix and to stabilize the activated AT*H complex. This hypothesis is
supported by similar three order of magnitude increases in the values
of k
2 (= koff) for
F122L/N135A (Table III) and K114A/N135A (11). Therefore, based on the
structural and kinetic considerations reviewed above, we propose that
Phe-122 and Lys-114 act cooperatively through non-ionic interactions to
promote ATIII binding to the pentasaccharide via a P-helix-mediated
protein conformational change mechanism that promotes the formation and
stabilization of the activated, high affinity AT*H complex.
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Table VII
Phe-121 and Phe-122-P helix contacts
Distances between indicated atoms of Phe-121 and Phe-122 and P helix
atoms were measured in the I-chain of the
ATIII-pentasaccharide complex (pdb.1E03).
|
|
Conclusion--
This work establishes the importance of
hydrophobic residues Phe-121 and Phe-122 for heparin binding and shows
that, under physiological conditions of pH and ionic strength, Phe-122
is critical for inducing a normal rate of conformational change and stabilizing the activated heparin-ATIII complex, AT*H. The rich network
of contacts between Phe-122, Lys-114, and the pentasaccharide and
P-helix may explain why high affinity binding to heparin and the
protein conformational change leading to ATIII activation are
intimately linked processes. In addition to aligning Lys-114 and Arg-47
to make the extensive ionic, hydrogen bond, and hydrophobic interactions with the pentasaccharide, Phe-122 and Lys-114 act cooperatively in the formation and stabilization of the P-helix, which
promotes high affinity binding to heparin and may also be a key element
in propagating the protein conformational change that increases
reactive loop reactivity with target enzymes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. James Herron for the use of his
spectrofluorometer, Dr. Maurice Petitou for pentasaccharide, and Dr.
James Huntington for introducing us to the 1E03 and 1E05 structures.
 |
FOOTNOTES |
*
This work was supported by American Heart Association
Western States Affiliate Postdoctoral Fellowship 0020132Y (to
M. A. J.), National Institutes of Health (NIH) Grant HL39888 (to
S. T. O.), Swedish Medical Research Council Grant 4212 (to I. B.), and NIH Grant HL30712 (to S. C. B.).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: University of
Utah Health Sciences Center, Pulmonary Division, 50 N. Medical Dr., Salt Lake City, UT 84132. Tel.: 801-585-6521; Fax: 801-585-3355; E-mail: susan.bock@m.cc.utah.edu.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212319200
 |
ABBREVIATIONS |
The abbreviations used are:
ATIII, antithrombin
III;
HAH, full-length high affinity heparin;
H5, heparin
pentasaccharide;
SI, stoichiometry of inhibition;
I, ionic
strength;
PEG, polyethylene glycol.
 |
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