Antithrombin III Phenylalanines 122 and 121 Contribute to Its High Affinity for Heparin and Its Conformational Activation*

Mohamad Aman JairajpuriDagger , Aiqin LuDagger , Umesh Desai§, Steven T. Olson, Ingemar Bjork||, and Susan C. BockDagger **

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha -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.


<UP>AT + H</UP> <LIM><OP><ARROW>↔</ARROW></OP><UL>K<SUB><UP>1</UP></SUB></UL></LIM><UP> AT-H</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB><UP>+2</UP></SUB></UL></LIM> <UP>AT*H</UP>

<UP><SC>Scheme</SC> 1</UP>

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,
k<SUB><UP>obs</UP></SUB>=((k<SUB>+<UP>2</UP></SUB>[<UP>H</UP>]<SUB><UP>o</UP></SUB>)<UP>/</UP>([<UP>H</UP>]<SUB><UP>o</UP></SUB><UP> + </UP>K<SUB><UP>1</UP></SUB>))+k<SUB>−2</SUB> (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 alpha -antithrombin (Protein Data Bank, pdb.1E05) and a 2.90-Å resolution structure of human plasma alpha -ATIII bound to pentasaccharide (pdb.1E03) were viewed and analyzed using QUANTA software.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta  isoform of antithrombin III, which is not glycosylated on asparagine-135. The N135A beta  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 alpha  and beta  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 beta  antithrombin isoform, relative to the alpha  antithrombin isoform, is experimentally advantageous with respect to purification and increased heparin binding affinity of the control. The pentasaccharide binding site of ATIII beta  (pdb.1E04) resembles that of ATIII alpha  (pdb.1E05), and previous studies have shown that conclusions about the mechanism of heparin binding and activation obtained from investigation of beta  antithrombin mutants (N135A background) apply to the antithrombin alpha  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.

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 Psi  being the fraction of Na+ bound per heparin charge and released upon binding to antithrombin (estimated to be 0.8 (17)).


<UP>log</UP> K<SUB>d<UP>,obs</UP></SUB><UP> = log </UP>K<SUB>d</SUB><UP>′ + </UP>Z<UP>&psgr; log</UP>[<UP>Na<SUP>+</SUP></UP>] (Eq. 2)
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.

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 Delta 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.

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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha  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 alpha  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.

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 beta  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 Calpha and Cbeta 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 alpha  carbon data and the least-squares algorithm in QUANTA software. The alpha  carbon trace of native antithrombin III (not bound to pentasaccharide) is yellow. The alpha  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 alpha  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.

When heparin binds antithrombin III, a short, one-turn alpha  helix is formed from residues 113-119 at the amino-terminal end of helix D (red Calpha 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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