Heparin Enhances the Specificity of Antithrombin for Thrombin and Factor Xa Independent of the Reactive Center Loop Sequence

EVIDENCE FOR AN EXOSITE DETERMINANT OF FACTOR Xa SPECIFICITY IN HEPARIN-ACTIVATED ANTITHROMBIN*

Yung-Jen Chuang, Richard Swanson, Srikumar M. Raja, and Steven T. OlsonDagger

From the Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois, Chicago, Illinois 60612

Received for publication, December 21, 2000

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antithrombin is a member of the serpin family of serine proteinase inhibitors whose primary physiologic function is to inhibit and thereby regulate several serine proteinases in the blood coagulation pathway (1, 2). Inhibition of these proteinases results from the serpin trapping the enzymes as stable acyl-intermediate complexes of a regular substrate reaction through a major conformational change. A unique feature of antithrombin is that it requires activation by the polysaccharide, heparin, to inhibit its main target enzymes, thrombin and factor Xa, at a physiologically significant rate. Heparin activates antithrombin through two distinct mechanisms whose relative contribution depends on the proteinase inhibited (3, 4). In both mechanisms, antithrombin binds to a sequence-specific pentasaccharide in heparin (5) which induces an activating conformational change in the reactive center loop of the serpin (6-9). Such conformational changes are alone sufficient to accelerate antithrombin inactivation of factor Xa through an allosteric mechanism. By contrast, the conformational changes negligibly affect the rate of thrombin inhibition, heparin accelerating antithrombin inhibition of this enzyme instead through a bridging mechanism wherein thrombin binding to a bridging site on heparin next to bound antithrombin enhances thrombin recognition of the serpin (3, 4, 10).

Although it is well established that conformational activation of antithrombin by the heparin pentasaccharide specifically enhances the reactivity of the serpin with factor Xa, the basis for this enhanced reactivity has not been determined. The x-ray crystal structures of antithrombin (11, 12) and its complex with the heparin pentasaccharide (9) have shown that heparin induces an increased exposure of the reactive center loop which may allow unhindered access of the proteinase to the loop and also permit optimal proteinase interaction by increasing loop flexibility. The factor Xa specificity of the conformationally activated serpin suggests that the loop sequence must also be critical for discrimination between factor Xa and thrombin. Supporting this idea, the preferred P4 to P1 substrate sequence for factor Xa recognition in the natural substrate prothrombin, IEGR or IDGR, does resemble the P4-P1 IAGR sequence found in antithrombin, whereas the proposed FPRSFR P3-P3' optimal thrombin recognition sequence (13) deviates significantly from the AGRSLN sequence in antithrombin. The factor Xa specificity of the antithrombin reactive loop sequence is further supported by our finding that mutation of the factor Xa-preferred P2 Gly of the loop to the thrombin-preferred Pro enhances antithrombin specificity for thrombin at the expense of decreasing specificity for factor Xa in a manner dependent on allosteric activation of the serpin (14).

Together, these considerations have strongly suggested that the presence of a factor Xa recognition sequence in the reactive center loop and the need for conformational activation of the loop to make this sequence accessible to factor Xa is the basis for allosteric activation of antithrombin by heparin pentasaccharide. To rigorously test this hypothesis, we sought to determine whether changes in the antithrombin reactive center loop sequence to an optimal thrombin recognition sequence would increase the thrombin specificity of the serpin in a manner that was dependent on pentasaccharide activation and comparable to the activation-dependent increase in factor Xa specificity of the wild-type serpin. To accomplish this goal, we evaluated 12 antithrombin variants with single or multiple mutations in the putative P6-P3' proteinase binding region in the loop other than P1-P1' to a more favorable thrombin recognition sequence. The results of analyzing the effects of these mutations on antithrombin specificity for thrombin and factor Xa revealed that such reactive center loop changes significantly increased the specificity of antithrombin for thrombin, but the specificity enhancements were only modestly dependent on allosteric activation of the serpin and were greatly attenuated with a bridging heparin activator. Remarkably, the effects of the reactive center loop sequence changes on factor Xa specificity were quite modest, and all variants showed a large inhibition rate enhancement upon allosteric activation comparable to that of the wild-type serpin and independent of the loop sequence. Together, these results strongly argue against our initial hypothesis that exposure of a factor Xa-specific reactive center loop sequence in antithrombin by a pentasaccharide-induced conformational change in the loop is the basis for allosterically activating antithrombin for rapid factor Xa inhibition. Rather, our findings suggest that the specificity of antithrombin for factor Xa arises from the exposure of still unknown recognition determinants outside the P6-P3' reactive center loop sequence which are only made accessible upon heparin activation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

Construction, Expression, and Purification of Antithrombin Variants-- The cDNA for N135Q antithrombin was used as the template for mutating reactive center loop residues to eliminate glycosylation heterogeneity resulting from incomplete glycosylation at Asn-135 and to mimic the high heparin affinity beta -form of plasma antithrombin that lacks the Asn-135 carbohydrate chain (15, 16). Site-directed mutagenesis of human antithrombin cDNA was carried out in the single-stranded M13mp19 vector as described (7) using an antisense oligonucleotide encoding the desired mutation. This method was used for the construction of the G392P, A391H, and N396H mutations. Other mutants were made by annealing complementary oligonucleotides carrying the desired mutant codons to pAlter (Promega) or pMAStop (7) plasmids in which the antithrombin N135Q cDNA was preinserted and generating mutant plasmids by polymerase chain reaction. cDNAs for antithrombin-protease nexin-1 loop chimeras were generated in pAlter plasmids and then excised and ligated into the pMAStop expression plasmid. The chimeras were generated by first mutating the antithrombin P6-P2 sequence and then the P2'-P3' sequence to that of protease nexin-1. Variants containing G392P, A391F, and L395F mutations in all possible combinations were generated by stepwise mutation. Oligonucleotides used for generating mutations (antisense strand only) were as follows (mutant codons underlined): A391F, 5'-GTT TAG CGA ACG GCC GAA AAT CAC AAC AGC GG-3'; G392P, 5'-GTT TAG CGA ACG GGG AGC AAT CAC AAC-3'; L395F, 5'-CCT GTT GGG GTT AAA CGA ACG GCC AGC-3'; A391F/G392P, 5'-GG GTT TAG CGA ACG GGG GAA AAT CAC AAC AG-3'; A391F/L395F, 5'-GTT AAA CGA ACG GCC AAA AAT CAC AAC AGC-3'; G392P/L395F, 5'-GTT AAA CGA ACG CGG AGC AAT CAC AAC-3'; A391F/G392P/L395F, 5'-C CCT GTT GGG GTT AAA CGA ACG GGG AAA AAT CAC AAC AGC-3'; PN1 P6-P2, 5'-CAC CCT GTT GGG GTT TAG CGA ACG CGC GAT GAG GAT GGC AGC GGT ACT TGC AGC TGC TTC ACT-3'; PN1 P6-P3', 5'-GGC CTT GAA AGT CAC CCT GTT GGG CGG CGA GCT TCT GGC GAT GAG GAT GGC AGC GGT ACT TGC-3'; V388A/V389A/I390A, 5'-G CGA ACG GCC AGC AGC GGC AGC AGC GGT ACT TGC AG-3'; A391H, 5'-TAG CGA ACG GCC ATG AAT CAC AAC AGC-3'; N396H, 5'-CAC CCT GTT GGG ATG TAG CGA ACG GCC-3'. All mutations were confirmed by DNA sequencing.

Baby hamster kidney cells were cotransfected with the expression vector carrying the reference or mutant cDNA together with selection plasmids and stably transfected cell lines obtained as described previously (7, 15). Recombinant antithrombins were isolated from serum-free cycles of medium collected from the stably transfected baby hamster kidney cells grown to confluence in roller bottles, with expression levels routinely reaching 15-40 mg/liter (7, 15). Recombinant antithrombins were purified by heparin-agarose chromatography to resolve the high heparin affinity glycoform corresponding in affinity to plasma beta -antithrombin followed by DEAE-Sepharose and Sephacryl S-200 chromatography (15, 17). Concentrations of recombinant antithrombins were determined from the absorbance at 280 nm using a molar absorption coefficient of 37,700 M-1 cm-1 (18).

Proteinases-- Human alpha -thrombin was a gift of Dr. John Fenton (New York State Department of Health, Albany, NY). Human factor Xa (predominantly alpha ) was obtained by activation of purified factor X followed by purification on soybean trypsin inhibitor-agarose as described (19) or generously provided by Dr. Paul Bock (Vanderbilt University, Nashville, TN). Proteinase concentrations were based on active-site titrations that indicated >90 and >70% active enzyme for thrombin and factor Xa, respectively (20). Human neutrophil elastase was purchased from Athens Research and Technology (Athens, GA). Bovine beta -trypsin was isolated from commercial enzyme (Sigma) by soybean trypsin inhibitor-agarose chromatography as described (21).

Heparins-- The alpha -methyl glycoside of a synthetic heparin pentasaccharide corresponding to the antithrombin-binding sequence in heparin was generously provided by Dr. Maurice Petitou (Sanofi Recherche, Toulouse, France). A full-length heparin containing the pentasaccharide with an average molecular weight of ~8000 (~26 saccharides) was isolated from commercial heparin by size and antithrombin affinity fractionation (17). Concentrations of pentasaccharide and full-length heparins were determined by stoichiometric titrations of antithrombin with the saccharides monitored by changes in protein fluorescence (4, 17).

Experimental Conditions-- All experiments were done at 25 or 37 °C as noted in buffers consisting of 20 mM sodium phosphate, 0.1 mM EDTA, 0.1% (w/v) polyethylene glycol 8000 containing either 0.1 M NaCl (I 0.15) at pH 7.4, 0.25 M NaCl (I 0.3) at pH 7.4, or 0.125 M NaCl (I 0.15) at pH 6.0. An exception was experiments with trypsin that were done in 100 mM Hepes containing 0.1 M NaCl, 0.01 M CaCl2, and 0.1% polyethylene glycol 8000, pH 7.4.

Stoichiometry of Inhibition-- Thrombin (5-100 nM), factor Xa (10-100 nM), or trypsin (100 nM) were incubated with increasing molar ratios of recombinant antithrombin to enzyme in the absence or presence of heparin levels equimolar with the maximum inhibitor concentration (0.1-2 µM) for times sufficient to complete the reaction based on measured second-order rate constants (17). Residual enzyme activity was then measured by 50-100-fold dilution of an aliquot of the reaction mixtures into 100 µM S2238 (Chromogenix) for thrombin, 200 µM Spectrozyme FXa (American Diagnostica) for factor Xa, or 100 µM S-2222 (Chromogenix) for trypsin, followed by monitoring the initial rate of substrate hydrolysis from the linear absorbance increase at 405 nm. In some cases thrombin or trypsin activity was measured by dilution of reaction mixtures into 50 µM tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin (Sigma) and substrate hydrolysis monitored from the linear fluorescence increase at excitation and emission wavelengths of 380 and 440 nm, respectively. All substrates contained 50-100 µg/ml Polybrene (Aldrich) to neutralize any heparin present. The inhibition stoichiometry was obtained by extrapolating linear least squares fits of the decrease in enzyme activity with increasing molar ratio of inhibitor to enzyme to the ratio yielding complete enzyme inhibition.

Electrophoresis-- SDS-polyacrylamide gel electrophoresis analysis was performed using the Laemmli discontinuous buffer system and a 10% polyacrylamide gel under nonreducing conditions (22). The products of antithrombin-proteinase reactions were analyzed after reacting 2-5 µM proteinase with a 1-3-fold molar excess of antithrombin in the absence or presence of full-length heparin equimolar with the inhibitor for 5-30 min in I 0.15 buffer. Reactions were quenched with 250 µM Phe-Pro-Arg-chloromethyl ketone (Calbiochem) for thrombin or 500 µM Glu-Gly-Arg-chloromethyl ketone (Bachem) for factor Xa prior to addition of SDS sample buffer and boiling. Neutrophil elastase cleavage of wild-type and V388A/V389A/I390A variant antithrombins was analyzed by incubating 3 µM serpin with 0.3 nM enzyme and 10 µM unfractionated heparin (Sigma) for varying times up to 60 min in I 0.15 buffer at 37 °C followed by quenching with SDS sample buffer and boiling for 3 min. Protein bands were detected by Coomassie Blue R-250 staining.

Heparin Binding-- The KD for heparin binding to recombinant antithrombins was measured by titrating 50 nM antithrombin with at least a 10-fold molar excess of polysaccharide in I 0.3 buffer, and monitoring heparin binding from the intrinsic protein fluorescence enhancement at excitation and emission wavelengths of 280 and 340 nm, respectively (4, 17). Stoichiometries of heparin binding were determined by similar titrations in I 0.15 buffer where binding is considerably tighter (4). Titrations were fit by the quadratic equilibrium binding equation with KD, the stoichiometry and the maximal relative fluorescence change being the fitted parameters (17). Stoichiometries determined at I 0.15 were assumed in fitting titrations at I 0.3 where KD was best determined.

Kinetics of Antithrombin Inhibition of Proteinases-- Second-order rate constants for the association of variant antithrombins with proteinases were measured under pseudo first-order conditions by using a molar excess of inhibitor over enzyme of at least 10 times the inhibition stoichiometry (16). Reactions contained 10-300 nM antithrombin and 0.1-10 nM proteinase with or without catalytic levels of pentasaccharide or full-length heparins ranging from 0.25 to 20 nM. For pentasaccharide-accelerated antithrombin-thrombin reactions or for pentasaccharide or full-length heparin-accelerated antithrombin-trypsin reactions, a 1.5-4-fold molar excess of heparin over antithrombin was used to saturate the inhibitor (>= 92%). The time-dependent decrease in enzyme activity was measured by quenching samples at varying times into substrate and determining the initial rate of chromogenic or fluorogenic substrate hydrolysis as in determinations of reaction stoichiometry. Enzyme inhibition progress curves were computer-fitted by a single exponential function with a zero end point to obtain the observed pseudo first-order rate constant, kobs. Apparent second-order association rate constants for uncatalyzed and heparin-catalyzed reactions were determined from the least squares slope of the linear dependence of kobs on the antithrombin or the heparin concentration, respectively, in accordance with Equation 1 that applies when [AT]o [H]o,


k<SUB><UP>obs</UP></SUB>=k<SUB><UP>uncat</UP></SUB>×[<UP>AT</UP>]<SUB>o</SUB>+k<SUB>H</SUB>×[<UP>H</UP>]<SUB>o</SUB>×([<UP>AT</UP>]<SUB>o</SUB>/(K<SUB>AT,H</SUB>+[<UP>AT</UP>]<SUB>o</SUB>)) (Eq. 1)
where kuncat and kH are the second-order rate constants for uncatalyzed and heparin-catalyzed reactions, respectively, and [AT]o and [H]o represent the total antithrombin and heparin concentrations, and KAT, H is the dissociation constant for the antithrombin-heparin interaction (16). The expression multiplying kH represents the antithrombin-heparin complex concentration which under the conditions of the experiments ([AT]o KAT,H) was closely approximated by [H]o. Association rate constants for reactions of thrombin with antithrombin-pentasaccharide complex or of trypsin with antithrombin complexes with either pentasaccharide or full-length heparins were obtained by dividing the measured or fitted kobs at saturating heparin by the antithrombin concentration. Apparent second-order rate constants were corrected for the different fractions of wild-type and variant antithrombins reacting through the inhibitory pathway by multiplying by the measured stoichiometry of proteinase inhibition (1).

Rapid Kinetics of Antithrombin-Proteinase Reactions-- Resolution of the noncovalent and covalent reaction steps of the reaction of wild-type and variant antithrombin-heparin complexes with thrombin was done by monitoring the reaction kinetics with a reporter fluorogenic substrate in an SX-17MV Applied Photophysics stopped-flow instrument under pseudo first-order conditions in I 0.15 buffer as in previous studies (14). Reactions contained 0.06-75 nM thrombin and a 40-400-fold molar excess of antithrombin-heparin complex generated by saturating 0.025-2 µM full-length heparin with a 1.1-2-fold molar excess of antithrombin. Reactions were continuously monitored from the exponential decrease in the rate of hydrolysis of the fluorogenic substrate, tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin, present at 5 µM. kobs values were averaged for each inhibitor-heparin complex concentration and corrected for the uncatalyzed reaction due to excess free inhibitor. The dependence of kobs on the antithrombin-heparin complex concentration ([AT·H]) was fit by the hyperbolic Equation 2,
k<SUB><UP>obs</UP></SUB>=k[<UP>AT·H</UP>]/(1+[<UP>S</UP>]<SUB>o</SUB>/K<SUB>M</SUB>)/(K<SUB>T,ATH</SUB>+[<UP>AT·H</UP>]/(1+[<UP>S</UP>]<SUB>o</SUB>/K<SUB>M</SUB>)) (Eq. 2)
where k represents the limiting rate constant for conversion of the noncovalent heparin-antithrombin-thrombin ternary complex to a covalent serpin-proteinase complex with release of heparin, KT,ATH is the dissociation constant for formation of the ternary complex, [S]o is the concentration of fluorogenic substrate, and KM is the Michaelis constant for substrate hydrolysis by thrombin. KM was fixed at the measured value of 4.5 ± 0.4 µM, and KT,ATH and k were the fitted parameters.

Kinetics of Dissociation of Antithrombin-Proteinase Complexes-- Rate constants for dissociation of covalent antithrombin-proteinase complexes were measured as in previous studies by continuously monitoring the recovery of proteinase activity in the presence of a reporter chromogenic substrate (14, 23, 24). Briefly, complexes were formed by reacting 5-10 µM antithrombin with 0.5-1 µM proteinase and 1 µM full-length heparin for 10 min at 25 °C. In the case of the triple P3/P2/P2' AGL right-arrow FPF variant and protease nexin-1 loop swap variants, heparin was either omitted or the complex formed at higher ionic strength to minimize any substrate reaction (14). Complexes were diluted to 0.3-6 nM in 400 µM S2238 or 400 µM Spectrozyme FXa in I 0.15 buffer containing 100 µg/ml Polybrene at 37 °C, conditions which effectively block reassociation of residual inhibitor with dissociated enzyme (17), and the initial rate of complex dissociation was continuously monitored from the parabolic increase in the rate of substrate hydrolysis at 405 nm for ~80 min (<1% complex dissociation). The time-dependent absorbance changes were fit by a second-order polynomial equation to obtain the initial rate of complex dissociation using the independently measured turnover number for enzyme hydrolysis of the substrate under these conditions to relate absorbance changes to changes in enzyme concentration (14, 23, 24). Initial rates of complex dissociation were plotted against the concentration of complex and the first-order dissociation rate constant determined from the least squares slope of this linear plot.

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Design of Thrombin-specific Reactive Center Loop Variants of Antithrombin-- Table I summarizes the reactive center loop mutations that were made in antithrombin to improve its specificity for thrombin. Such mutations were confined to the P6-P3' residues since the proteinase recognition sequence is typically contained in this region (25). P3 and P3' residues were individually changed to basic His side chains due to the reported thrombin preference for basic residues in these positions (26, 27), and specificity changes were analyzed under pH conditions where the His side chain was expected to be either uncharged or charged. The critical P3, P2, and P2' specificity-determining residues were substituted with the proposed thrombin consensus residues, Phe, Pro, and Phe, respectively (26), by stepwise mutation. The entire P6-P3' sequence was replaced with the corresponding sequence of the serpin, protease nexin-1, since the latter serpin is an ~100-fold faster inhibitor of thrombin than antithrombin in the absence of a cofactor (28). The contribution of P6-P4 residues to specificity was evaluated by mutating these residues simultaneously to Ala. For this particular mutant, the mutation was verified at the protein level by showing that the rate of cleavage of the wild-type P4-P3 Ile-Ala bond by neutrophil elastase (29) was greatly curtailed in the mutant (data not shown). All variants were homogeneous following purification and were functional in forming SDS-stable complexes with thrombin and in having such complex formation accelerated by heparin.

                              
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Table I
Engineered reactive center loop variants of antithrombin
Reactive center loop residues are designated according to the nomenclature of Schechter and Berger (38) with mutated residues in bold and underlined.

Stoichiometries of Proteinase Inhibition by Variant Antithrombins-- In the absence of heparin, all antithrombin variants behaved like the wild-type serpin in showing an ~1:1 stoichiometry for inhibiting thrombin, factor Xa, and the prototype P1 Arg-specific proteinase, trypsin (Table II). Most variant antithrombins also resembled the wild-type inhibitor in showing similar increases in stoichiometry of inhibition of thrombin and factor Xa of greater than 1 mol of inhibitor/mol of proteinase in the presence of pentasaccharide or full-length heparins. In the case of the factor Xa reaction, both pentasaccharide and full-length heparins elevated the stoichiometry to similar extents, whereas in the case of the thrombin reaction, only the full-length heparin increased the stoichiometry, an effect that paralleled the ability of both types of heparin to catalyze factor Xa inhibition but only the full-length heparin to catalyze thrombin inhibition (4).

                              
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Table II
Association rate constants and stoichiometries for variant antithrombin-proteinase reactions
Second-order association rate constants (kassoc in units of M-1 · s-1) and stoichiometries of inhibition (SI given as mol of inhibitor/mol of proteinase) were determined in the absence or presence of pentasaccharide (H5) or full-length heparins (H26) at 25 °C, I 0.15, pH 7.4, as described under "Materials and Methods." Multiplication of kassoc by SI corrected kassoc for the different extents of substrate reaction. Reported values are averages of either two determinations ± range or three or more determinations ± S.E. except for trypsin reactions in which case results from a single inhibition progress curve are reported.

The heparin-dependent increases in inhibition stoichiometry result from heparin promoting an alternative reaction of antithrombin as a substrate of proteinases in competition with the inhibitory reaction when thrombin and factor Xa are the target enzymes (3, 4). This heparin-dependent substrate reaction was evident from the appearance of cleaved antithrombin on SDS-polyacrylamide gel electrophoresis in the presence but not in the absence of the polysaccharide. The substrate reaction was much more pronounced in the case of those variants containing a P2 Gly right-arrow Pro mutation or protease nexin-1 loop residues when thrombin was the proteinase and could be stimulated by both pentasaccharide and full-length heparins, although much more by the full-length heparin (Table II). Full-length heparin-catalyzed reactions of P2 Pro variants and of protease nexin-1 loop variants with thrombin thus showed stoichiometries in the range of 20-30 and 9-12, respectively, in contrast to the value of ~2 for the wild-type and all other variants under these conditions. Such variants thus reacted preferentially as substrates of thrombin rather than inhibitors of the enzyme in the presence of a full-length heparin activator. Inhibition stoichiometries were not significantly elevated by heparin when trypsin was the target enzyme.

Heparin Binding and Conformational Activation of the Variant Antithrombins-- To determine whether the mutations affected the ability of antithrombin to bind heparin and be conformationally activated, variant and wild-type antithrombins were titrated with a full-length high affinity heparin and heparin binding, and conformational activation of the serpins was followed by monitoring the progressive appearance of the ~40% tryptophan fluorescence enhancement that reports these events up to saturation of the inhibitor with the polysaccharide (4, 6). Similar KD values of ~10-30 nM were determined at I 0.3, and binding stoichiometries of ~1:1 were measured at I 0.15 for wild-type and variant antithrombin interactions by fitting titration curves by the quadratic equation for equilibrium binding (Table III). Similar fluorescence enhancements of 40-50% were also found to be induced by heparin at saturation in these titrations (Table III). These results indicated that none of the reactive center loop mutations affected heparin binding to the serpin or the ability of the serpin to be conformationally activated to any significant extent.

                              
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Table III
Interaction of heparin with antithrombin variants
Dissociation constants and relative maximal fluorescence enhancements for heparin binding to antithrombin variants were determined from titrations of antithrombin with full-length heparin at 25 °C, I 0.3, pH 7.4, whereas heparin binding stoichiometries were determined from separate titrations at 25 °C, I 0.15, pH 7.4, as described under "Materials and Methods." Reported values ± S.E. are from global fits of at least two titrations.

Association Rate Constants for Variant Antithrombin-Proteinase Reactions-- The changes in antithrombin specificity produced by the reactive center loop mutations were analyzed by measuring second-order rate constants for the association of mutant and wild-type antithrombins with the target enzymes, thrombin and factor Xa, and in some cases also with the less specific enzyme, trypsin. To resolve the effects of conformational activation of antithrombin from the effects of heparin bridging antithrombin and proteinase on specificity, second-order rate constants were measured in the absence of heparin, in the presence of the heparin pentasaccharide and in the presence of a full-length bridging heparin containing the pentasaccharide (Table II) (4). Apparent second-order rate constants were corrected for the different extents of substrate reaction of the variants to provide valid comparisons of the association rates along the inhibitory pathway and thereby changes in specificity (1). This correction involved multiplication of the apparent rate constant by the measured stoichiometry of inhibition (Table II). The relative changes in antithrombin specificity for thrombin and factor Xa produced by the mutations for both the native and heparin-activated inhibitor are summarized in Fig. 1.


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Fig. 1.   Effect of reactive center loop mutations on antithrombin specificity for thrombin and factor Xa. The relative specificity of mutant versus wild-type antithrombins for inhibiting thrombin (top panel) or factor Xa (bottom panel) is indicated by the log of the ratio of corrected second-order association rate constants for the inhibitor-proteinase reactions (Table II) in the case of unactivated antithrombin (gray bars), pentasaccharide-activated antithrombin (black bars), or full-length heparin-activated antithrombin (white bars).

Of the single reactive center loop mutations, the P2 Gly to Pro change produced the largest increase in thrombin specificity over wild-type antithrombin, and this specificity enhancement was greatest when the variant inhibitor was activated by pentasaccharide (15-fold) as compared to when the variant was not activated (3.6-fold) or activated by full-length heparin (2.1-fold). A more modest specificity enhancement was produced by the P2' Leu to Phe change that was again maximal for the pentasaccharide-activated serpin (5.5-fold) relative to the unactivated (2.5-fold) or full-length heparin-activated inhibitor (1.6-fold). Other single mutations of P3 Ala to Phe or to His, or of P3' Asn to His only marginally increased or slightly decreased thrombin specificity with or without heparin activation. Converting the largely neutral His side chain at pH 7.4 in the P3 and P3' His variants to a mostly positively charged side chain by lowering the pH to 6 modestly increased or did not affect thrombin specificity, respectively, independent of heparin activation (Table IV).

                              
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Table IV
Association rate constants and stoichiometries for reactions of P3H and P3'H antithrombin variants with proteinases at pH 6 
Rate constants (kassoc in units of M-1 s-1) and stoichiometries of inhibition (SI) ± S.E. were measured at 25 °C, I 0.15, pH 6.0, as detailed under "Materials and Methods." kassoc was corrected for flux along the substrate pathway by multiplying by SI.

Stepwise mutations of P3, P2, and P2' residues of antithrombin to the proposed consensus residues preferred by thrombin, i.e. P3 Phe, P2 Pro, and P2' Phe (26), resulted in the largest enhancement in antithrombin specificity for thrombin. The effects of these successive mutations on thrombin specificity depended on the sequence in which the P3, P2, and P2' residues were mutated, indicating that the specificity changes caused by each residue were not additive and thus depended on cooperative interactions between the substituted residues. An example of the cooperative effects of these substitutions is exemplified by the effects of the P2 Pro and P2' Phe mutations alone and in combination on the specificity of the pentasaccharide-activated inhibitor. The P2 Pro mutation alone enhanced thrombin specificity 15-fold, whereas when this mutation was made subsequent to the P2' Phe mutation, the specificity enhancement was reduced to 6.3-fold. Likewise, the P2' Phe change alone enhanced thrombin specificity 5.5-fold but when made subsequent to the P2 Pro change enhanced specificity a lesser 2.3-fold. Similar analysis of double mutant cycles leading to the triple mutant (30) showed that each mutation contributed to specificity enhancement in a manner that depended on the presence or absence of the other two residues. The variant antithrombin containing all three mutations showed the highest increase in thrombin specificity relative to the wild-type inhibitor of 10-fold for the unactivated inhibitor, 55-fold for the pentasaccharide-activated inhibitor, and 4.8-fold for the full-length heparin-activated inhibitor.

Replacement of the entire P6-P3' region with the corresponding sequence of the serpin, protease nexin-1, produced a maximal 4.2-fold increase in thrombin specificity for the pentasaccharide-activated variant serpin, slight decreases in specificity for the unactivated variant, and a smaller 1.7-fold enhanced specificity for the full-length heparin-activated variant. Most of this specificity enhancement was achieved by replacement of just the P2-P6 residues of antithrombin with those in protease nexin-1. Substitution of the P6 to P4 Val-Val-Ile sequence with Ala-Ala-Ala produced only small decreases in thrombin specificity with or without activation by either heparin.

A notable feature of the thrombin specificity enhancements of most variants was that a significant fraction of the enhancement (as much as 10-fold) was realized already with the unactivated inhibitor. Thus, although maximal increases in thrombin specificity were found when the variants were activated by heparin pentasaccharide, such activation increased thrombin specificity at most ~9-fold over that of the unactivated variant inhibitor. Whereas this increase exceeded the 1.6-fold enhancement in thrombin specificity of wild-type antithrombin due to allosteric activation, it did not come close to approaching the ~200-fold increase in factor Xa specificity resulting from allosteric activation of the wild-type inhibitor. Also of note were the smaller increments in thrombin specificity of the antithrombin variants over that of the wild-type serpin of at most ~5-fold observed when the inhibitors were activated by a full-length bridging heparin. Thus, heparin bridging of antithrombin and thrombin appeared to attenuate the recognition of the antithrombin reactive center loop by thrombin. Because variants containing the P2 Pro mutation or the protease nexin-1 P6-P2 or P6-P3' residues preferentially reacted as a substrate of thrombin when activated by the full-length heparin, the apparent rate constants for thrombin inhibition by such variants in the presence of the full-length heparin were actually decreased as much as 7-fold relative to the corresponding wild-type inhibitor reaction.

The effects of the reactive center loop mutations on factor Xa specificity were surprisingly very different. Instead of the progressive decreases in factor Xa specificity expected to accompany the stepwise increases in thrombin specificity of the variant antithrombins, the reactive center loop mutations produced relatively small changes in factor Xa specificity with or without heparin activation of the inhibitor. For the unactivated inhibitor, the mutations either had no significant effect (P3 Phe, P3 His, P3' His, P6-P2 PN1) or decreased factor Xa specificity up to 9-fold (all other variants) relative to wild-type antithrombin with the greatest decrease associated with the P2 Pro replacement. Activation by either pentasaccharide or full-length heparins resulted in smaller decreases in factor Xa specificity of maximally 2.5-fold, and in several cases specificity was increased as much as 3-fold (P3 His, P3 Phe, P2' Phe, and P3 Phe/P2' Phe) compared with the wild-type inhibitor. Evaluation of the specificity changes of the P3 and P3' His variants at pH 6 showed similar modest increases or decreases in specificity, respectively, with or without heparin activation (Table IV). The insensitivity of reactive center loop changes in antithrombin to its specificity for inhibiting factor Xa was particularly evident from the modest effects on specificity resulting from the swapping of the antithrombin P6-P3' region with that of protease nexin-1. Minimal decreases in factor Xa specificity no greater than 2-fold were thus observed for either the unactivated or heparin-activated variant despite the seven amino acid changes made in this variant. Most striking of all was the finding that for every one of the reactive center loop changes, the pentasaccharide rate enhancement observed for the wild-type serpin of ~200-fold was unchanged or modestly increased up to ~600-fold for the variant serpins. As with the wild-type inhibitor, the full-length heparin further increased factor Xa specificity ~2-fold in most cases due to a small bridging effect of the larger heparin, although for mutants containing either a P3 Phe or P2' Phe, the increase was somewhat greater (3-6-fold).

For the stepwise mutation of antithrombin P2, P3, and P2' residues that resulted in the largest increment in thrombin specificity, the effects of the mutations on trypsin inhibition with and without heparin activation were additionally studied. The mutations produced either no change or only small decreases (maximally 3-fold) in the rate constants for trypsin inhibition both for the unactivated as well as for the heparin-activated variant antithrombins. The ~7-fold maximal rate enhancement of the wild-type inhibitor reaction produced by the full-length heparin was consequently similar (3-7-fold) for the variants. These findings are in keeping with trypsin being relatively nonspecific for substrate amino acids flanking the primary P1 Arg residue (31).

Reaction Step Affected by the P3/P2/P2' AGN right-arrow FPF Mutation in Antithrombin-- For the P3/P2/P2' AGN right-arrow FPF triple mutant antithrombin that produced the largest increase in thrombin specificity, it was of interest to evaluate which step or steps of the multistep serpin inhibitory pathway were responsible for the specificity enhancement. To address this question, the kinetics of thrombin inhibition by triple mutant and wild-type antithrombins complexed with the full-length heparin were compared by stopped-flow fluorometry using a fluorigenic substrate to monitor proteinase inhibition. Comparison was also made with the reaction of the variant containing just the P2 Gly right-arrow Pro mutation using data obtained from a previous study (14). Fig. 2 compares the dependence of the pseudo first-order inhibition rate constant for wild-type and variant inhibitor reactions measured by the continuous assay as a function of the antithrombin-heparin complex concentration. Saturation of kobs was evident in all cases, but the limiting rate constants at saturation for the mutant inhibitors were decreased relative to the wild-type inhibitor, and saturation was achieved at much lower antithrombin-heparin complex concentrations for the triple mutant than for the wild-type or single mutant inhibitors. Fitting of the data by the hyperbolic equation given under "Materials and Methods" indicated dissociation constants of 300 ± 30, 220 ± 40, and 17 ± 7 nM for formation of the initial heparin-antithrombin-thrombin ternary complex and limiting rate constants of 3.1 ± 0.1, 0.43 ± 0.03, and 0.15 ± 0.01 s-1 for reaction of antithrombin and thrombin in the ternary complex to form a covalent complex for wild-type, single mutant, and triple mutant inhibitor reactions, respectively. The apparent decrease in limiting rate constant for the mutants as compared with the wild-type inhibitor could be ascribed to the greater fraction of the mutants that reacted as a substrate (Table II) (14). Correction of the limiting rate constant for the different extents of substrate reaction by multiplying by the stoichiometry of inhibition resulted in values of 6.8 ± 0.5, 11 ± 2, and 4.8 ± 1.1 s-1 for wild-type, single mutant, and triple mutant inhibitor reactions, respectively. These results indicate that the single P2 Gly right-arrow Pro mutation enhances the specificity of antithrombin for thrombin mostly by increasing the rate constant for the covalent reaction step without affecting the noncovalent serpin-proteinase interaction, whereas the triple mutation enhances serpin specificity for thrombin primarily by increasing the affinity of the noncovalent complex with minimal effect on the covalent step. Such findings, taken together with the cooperativity of antithrombin reactive center loop interactions with thrombin revealed by double mutant cycles, suggest that antithrombin has a flexible reactive center loop whose conformation can adapt to produce an optimal active-site interaction either in the Michaelis complex or in the transition state leading to the covalent acyl-enzyme complex.


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Fig. 2.   Rapid kinetics of full-length heparin-catalyzed reactions of wild-type antithrombin and P2 Gly right-arrow Pro and P3/P2/P2' AGL right-arrow FPF variant antithrombins with thrombin. Shown is kobs for reactions of thrombin with wild-type (), P2 P (), or P3/P2/P2' FPF (black-triangle) variant antithrombins complexed with full-length heparin as a function of the antithrombin-heparin complex concentration corrected for the competitive effect of a reporter fluorogenic substrate used to monitor inhibition. Rate constants were measured by stopped-flow fluorometry as described under "Materials and Methods." Solid lines are fits to the hyperbolic equation in the text. The top and bottom panels show the data on two different scales.

Effect of Reactive Center Loop Mutations on Serpin-Proteinase Complex Stability-- To determine whether the mutations made in the reactive center loop of antithrombin affected the stability of the covalent antithrombin-proteinase complex, the rate constants for dissociation of complexes of P3/P2/P2' AGN right-arrow FPF, P6-P4 VVI right-arrow AAA, and protease nexin-1 P6-P3' loop swap variants as well as wild-type antithrombin with thrombin and factor Xa were measured over a range of complex concentrations as in previous studies (14, 17, 23, 24) (Table V). Whereas P3/P2/P2' AGN right-arrow FPF and protease nexin-1 loop variant inhibitor complexes were dissociated with rate constants up to ~2-fold slower than those of the wild-type inhibitor complexes with thrombin and factor Xa as proteinase, similar to the effect of the single P2 Gly right-arrow Pro mutation reported previously (14), the P6-P4 VVI right-arrow AAA mutant inhibitor complexes were dissociated with rate constants up to ~2-fold greater than wild-type complexes. These results suggest that changes in P6 to P3' reactive center loop residues of antithrombin other than P1 and P1' have only minimal effects on complex stability.

                              
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Table V
Dissociation rate constants for variant antithrombin-proteinase complexes
Dissociation rate constants (kdiss) were measured at 37 °C, I 0.15, pH 7.4, from the slopes ± S.E. of linear plots of the initial rates of complex dissociation as a function of complex concentration as described under "Materials and Methods."


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reactive Center Loop Contribution to Antithrombin Specificity-- Our studies were designed to test the hypothesis that the enhanced factor Xa specificity of allosterically activated antithrombin arises from a factor Xa-specific sequence in the antithrombin reactive center loop that becomes accessible to the proteinase only after activation. Such an hypothesis predicts that changes in the putative factor Xa recognition sequence in the reactive center loop to a sequence preferred by thrombin would convert antithrombin to an allosterically activated inhibitor of thrombin and a poor inhibitor of factor Xa. The results of testing this prediction have shown that the thrombin specificity of antithrombin can be significantly enhanced in a manner dependent on allosteric activation by introducing reactive center loop changes more favorably recognized by thrombin. In particular, introducing the proposed optimal P3-P2' sequence for thrombin (26) into the antithrombin reactive center loop resulted in the largest increase in specificity (55-fold), whereas substitution with the protease nexin-1 P6-P3' loop sequence or the P3 and P3' residues with a positively charged histidine at pH 6 produced more modest enhancements in thrombin specificity. Substituting the protease nexin-1 P6-P3' sequence into the reactive center loop of alpha 1-antichymotrypsin similarly produced only modest increases in thrombin specificity of this serpin (32), suggesting that the high reactivity of protease nexin-1 with thrombin may involve recognition by elements in the serpin outside the P6-P3' region. Changes in the Val-Val-Ile P6-P4 sequence did not significantly affect thrombin recognition in native or activated states of the serpin, suggesting that these residues are not important for binding thrombin. The maximal increase in thrombin specificity achieved with the triple P3/P2/P2' AGN right-arrow FPF mutation exceeds that produced in the serpin, alpha 1-proteinase inhibitor, by similar changes in the reactive center loop to a thrombin-preferred sequence (30) and is comparable to the thrombin specificity of protease nexin-1 in the absence of heparin (28). The reactive center loop sequence of this variant antithrombin may thus be near optimal for interacting with the active-site of thrombin, in keeping with recent studies of thrombin specificity assessed by screening a combinatorial library of synthetic substrates (33).

Surprisingly, a significant fraction of the thrombin specificity enhancements achieved in the variant antithrombins were realized without allosteric activation of the serpin. The reactive center loop sequence thus plays a role in antithrombin recognition of thrombin, but this recognition appears to be only marginally dependent on allosteric activation of the inhibitor. Trypsin inhibition by antithrombin was similarly only modestly affected by allosteric activation with either wild-type or mutant serpins, suggesting that the reactive center loop of native antithrombin is largely accessible to thrombin and trypsin and does not depend on allosteric activation for its rapid association with these enzymes. The structural basis for allosteric activation of antithrombin is known to involve the expulsion of the reactive center loop from beta -sheet A of the protein core in which the loop is partially buried at the P15-P14 hinge in native antithrombin (8, 9, 34). The expulsion results in the exposure of the loop in a manner similar to that seen in the structures of other native serpins (35, 36), and it has been presumed that this expulsion is important for increasing accessibility of the loop to target proteinases. However, our findings suggest that the loop is already accessible to thrombin and trypsin when the hinge region is buried and that the modest increases in association of the loop with these enzymes upon exposure of the hinge can be ascribed to the increased flexibility of the loop when it is not constrained by burial of the hinge region. Such an increased flexibility could improve a substrate-like interaction between the loop and the enzyme which is characteristic of serpin-proteinase interactions (1) by enabling the loop to adopt a conformation most optimal for proteinase interaction.

It was also surprising to find that the reactive center loop changes produced relatively small effects on the specificity of antithrombin for factor Xa whether or not the serpin was allosterically activated by heparin pentasaccharide, the largest specificity changes being associated with the P2 Pro substitution. These findings are consistent with the broad specificity of the S4 and S3 subsites of factor Xa and more restricted specificity of the S2 subsite of the enzyme for Gly indicated by x-ray crystallography (37) and combinatorial substrate screening (33). Most interesting was the finding that allosteric activation of any of the variant antithrombins still enhanced factor Xa specificity to the same or a greater extent than the wild-type serpin. These findings imply that the reactive center loop residues flanking the P1-P1' scissile bond including the P4 and P2 mimics of the prothrombin sequence are not strong determinants of antithrombin specificity for factor Xa in either native or activated states of the serpin and do not appear to mediate the enhanced specificity of allosterically activated antithrombin for the enzyme. Previous studies suggest also that the P1' residue of the serpin is not important for factor Xa specificity and that replacement of P2' and P3' residues with those in prothrombin do not significantly enhance reactivity (27), implying that the P1 Arg is the most critical specificity-determining residue in this region, a conclusion supported by our recent P1 Arg mutagenesis studies (51).

Our initial hypothesis that the reactive center loop sequence and its differential accessibility in native and activated states is responsible for the factor Xa specificity of activated antithrombin is therefore not supported by our findings. It would instead appear that the enhanced factor Xa specificity of activated antithrombin is minimally dependent on the reactive loop sequence and arises from the unique ability of factor Xa, unlike thrombin and trypsin, to discriminate between the constrained and flexible loop conformations of native and activated antithrombin. In support of factor Xa having such an unusual ability, a similar level of discrimination between the two antithrombin loop conformations could be engineered in thrombin by changing the active-site to be more factor Xa-like (39). However, the finding that swapping the reactive center loop of antithrombin into antichymotrypsin resulted in a slow rate of factor Xa inactivation comparable to that of native, unactivated antithrombin (32) argues that the reactive center loop sequence of antithrombin has an intrinsically low reactivity with factor Xa even in a serpin in which the loop is normally fully exposed (35).

We believe that a more plausible explanation for the enhanced factor Xa specificity of activated antithrombin is that it does not involve enhanced factor Xa recognition of the P6-P3' region at all, but instead involves the recognition of an antithrombin exosite outside of this region that is made accessible through conformational activation. According to this idea, factor Xa resembles thrombin and trypsin in accessing and recognizing the native and activated loop conformations of antithrombin similarly, with only modest enhancements in recognition of the activated loop due to its increased conformational flexibility. Other serpins appear to utilize such nonreactive center loop determinants to recognize their target proteinases (32, 40, 41). Examination of the x-ray crystal structures of two specific protein inhibitors of factor Xa, leech antistasin (42) and tick anticoagulant peptide (43) and the modeled or elucidated structures of complexes of these inhibitors with factor Xa support the idea that the enhanced factor Xa specificity of allosterically activated antithrombin may result from an interaction between an antithrombin exosite and a complementary exosite on factor Xa. Such structures thus reveal the importance of a basic exosite sequence in factor Xa, Lys-222---Lys-223---Arg-224, located adjacent to the active site and constituting part of the sodium-binding site of the enzyme (44), in mediating the interaction of the enzyme with both antistasin and tick anticoagulant peptide. Whether this enzyme exosite is involved in recognizing a complementary exosite in antithrombin that becomes accessible through heparin activation remains to be determined.

Contribution of Heparin Bridging to Antithrombin Specificity-- All antithrombin variants showed large increases in thrombin specificity when complexed with a full-length heparin due to the longer heparin providing a bridging site for thrombin to bind adjacent to the bound inhibitor (3, 4). However, the thrombin specificities of the mutant antithrombins were not much greater than that of the wild-type inhibitor when bound to the full-length heparin (maximally 5-fold) as compared to when they were bound to the pentasaccharide (maximally 55-fold). Thrombin recognition of the reactive center loop of the variant antithrombins thus appears to be poorer when thrombin is constrained to also bind heparin in the ternary bridging complex. Reactive center loop interactions and heparin bridging interactions with the proteinase are thus not additive and hence cooperative. Consistent with this view, heparin binding to the thrombin exosite that mediates heparin bridging of thrombin with antithrombin has been shown to affect allosterically active-site interactions of thrombin with its inhibitors and substrates (45). Factor Xa reactivity with the antithrombin variants complexed with the full-length heparin was only slightly greater than when the variants were activated by pentasaccharide, varying from 2- to 3-fold to as much as 4- to 6-fold higher. The small enhancement in specificity results from heparin bridging (4), with the variability in this enhancement again indicating cooperativity between heparin bridging and reactive center loop interactions with factor Xa. The relatively modest bridging effect observable with factor Xa results from the heparin-binding exosite in this enzyme being inaccessible to heparin due to an intramolecular interaction between the exosite and the acidic gamma -carboxyglutamic acid domain of the enzyme (46).

Heparin bridging thus appears primarily responsible for achieving large enhancements in the thrombin specificity of wild-type antithrombin with changes in the reactive center loop sequence only modestly affecting specificity. The overriding effect of heparin-bridging interactions in determining thrombin specificity suggests that the reactive center loop sequence of antithrombin need not be optimal for interaction with thrombin and may have evolved to allow the inhibitor to be selective for procoagulant proteinases and limit its reaction with the anticoagulant proteinase, activated protein C (30). A poor thrombin recognition sequence in wild-type antithrombin may also have been favored based on our finding that sequence changes that enhance thrombin specificity also resulted in variants that preferentially reacted as substrates rather than inhibitors of the enzyme when complexed with a physiologic bridging heparin (14).

Reactive Center Loop Contribution to Complex Stability-- The finding that mutations in the P6-P3' reactive center loop sequence had small or no effects on complex stability despite the marked effects of many of the mutations on the rate of formation of the stable complex with thrombin and to a lesser extent with factor Xa supports a unique mechanism for stabilization of these complexes that is different from that of lock-and-key inhibitors. A hallmark of the lock-and-key mode of complex stabilization is the destabilizing effect of amino acid substitutions in reactive center loop residues of such inhibitors on complex formation, as reflected by increases in the rate constants for complex dissociation (47). Our finding that reactive center loop mutations in antithrombin minimally affect the rate constants for dissociation of antithrombin-proteinase complexes thus supports the idea that reactive center loop interactions with the proteinase active site do not significantly contribute to stabilizing the complexes. These findings agree with the recent x-ray structure of a serpin-proteinase complex (48) that supports a model for serpin-proteinase complex stabilization in which reactive center loop interactions with the enzyme are disrupted as a consequence of loop cleavage at the acyl-intermediate stage allowing the loop to fully insert into beta -sheet A of the serpin and translocate the tethered proteinase to the distal end of sheet A. Complex stabilization arises according to this model from the reactive center loop burial in sheet A eliminating reactive center loop interactions with the acyl-linked proteinase and inducing a distortion of the proteinase catalytic apparatus (49, 50).

Summary-- In summary, our studies have provided new insights into the origin of antithrombin specificity for thrombin and factor Xa and how heparin enhances specificity for these enzymes. We have shown that antithrombin specificity for thrombin depends on thrombin recognition of the reactive center loop sequence but that this sequence is not optimal for thrombin recognition in either the native or heparin-activated serpin. Heparin enhances antithrombin recognition by the enzyme instead by providing a secondary interaction site on heparin adjacent to the bound serpin for the enzyme to bind. By contrast, our findings suggest that antithrombin specificity for factor Xa is minimally dependent on the reactive loop sequence other than the P1 residue in either native or heparin-activated states due to a broader sequence specificity of this enzyme. Heparin enhances antithrombin recognition by the enzyme in this case through exposure of an antithrombin exosite outside the P6-P3' loop region capable of interacting with a complementary factor Xa exosite. Heparin additionally provides a bridging site for factor Xa, but optimal utilization of this site requires the presence of physiologic levels of calcium to expose a complementary heparin-binding exosite in factor Xa (46). Importantly, our findings rule out alterations in loop accessibility as an important mechanism for heparin activation of the serpin, a conclusion that is supported by the antithrombin P1 residue mutagenesis studies we have recently reported (51).

    ACKNOWLEDGEMENTS

We thank Peter Gettins for critical reading of the manuscript and both Peter Gettins and Ingemar Björk for helpful discussions of this work.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL 39888 (to S. T. O.).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.

Dagger To whom correspondence should be addressed: Center for Molecular Biology of Oral Diseases, University of Illinois, Rm. 530E Dentistry (M/C 860), 801 S. Paulina St., Chicago, IL 60612. Tel.: 312-996-1043, Fax: 312-413-1604.

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M011550200

    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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