Deletion of P1 Arginine in a Novel Antithrombin Variant (Antithrombin London) Abolishes Inhibitory Activity but Enhances Heparin Affinity and Is Associated with Early Onset Thrombosis*

Srikumar M. RajaDagger , Neetu ChhablaniDagger , Richard SwansonDagger , Elizabeth Thompson§, Mike Laffan§, David A. Lane§, and Steven T. OlsonDagger

From the Dagger  Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612 and § Department of Haematology, Hammersmith Hospital Campus, Faculty of Medicine, Imperial College, London W12 0NN, United Kingdom

Received for publication, January 3, 2003, and in revised form, February 12, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a KD reflecting an ~10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antithrombin, a member of the serpin family of protein proteinase inhibitors, is the principal regulator of blood coagulation proteinases in plasma (1, 2). The essential role of this protein in hemostasis and in sustaining life is clear from the association of inherited or acquired deficiencies of the serpin in humans with an increased risk of thrombosis (2) and from the finding that complete deficiency in mice results in embryonic lethality due to a consumptive coagulopathy (3). Efficient regulation of clotting proteinases by antithrombin requires the polysaccharide cofactors heparin and heparan sulfate, which act to increase the moderate rates of inhibition of clotting proteinases by the serpin several thousand-fold (1). Only a fraction of heparin molecules are functional in this regard due to their possessing a specific pentasaccharide sequence capable of binding antithrombin with high affinity and inducing activating conformational changes in the serpin (4-7). Endothelial cell surface heparan sulfate molecules are thought to be to be the natural activator of antithrombin, but only a limited fraction (~1%) of such molecules contain the pentasaccharide sequence and are anticoagulantly active (8). The crystal structures of antithrombin free and complexed with the heparin pentasaccharide reveal that pentasaccharide binding to an allosteric site on the inhibitor transmits conformational changes to a reactive proteinase binding loop on the inhibitor surface that enhances the loop accessibility to proteinases (9, 10). However, mutagenesis studies suggest that not only the proteinase binding loop common to serpins but also a unique exosite region adjacent to the loop are made accessible for proteinase interaction as a result of heparin activation (11).

Natural variants of antithrombin have provided valuable insights into the anticoagulant function of the serpin. Such variants initially led to the identification of the pentasaccharide binding site as well as verification of the proteinase binding site of the inhibitor before the crystal structures became available (1, 2). Antithrombin variants have additionally provided significant insights into the relative physiologic importance of antithrombin interactions with clotting proteinases and heparin (2). Individuals expressing variant antithrombins with abnormal proteinase binding are thus typically heterozygous for the genetic defect and are usually at risk for thrombosis. However, those individuals expressing a variant antithrombin with a defect in heparin binding may be heterozygous or homozygous, and only homozygotes are typically more susceptible to thrombosis.

In the present report we describe a novel dysfunctional variant of antithrombin associated with early onset thrombosis in the proband and a clear family history of thrombosis in affected individuals. Sequencing of the gene for the variant revealed an in-frame deletion of the P1 Arg of the serpin. Purification of the variant antithrombin confirmed the complete absence of inhibitory activity but also revealed an abnormally high affinity for heparin. The latter property was shown to result in the variant acting as an effective antagonist of the heparin-catalyzed inhibition of clotting proteinases by normal antithrombin, providing an explanation for the unusually severe thrombosis associated with this mutation. The abnormally high heparin affinity of the variant suggests that the deletion of the reactive loop residue causes activation of the variant without heparin and provides new insights into the structural basis of heparin activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Coagulation Tests-- Patient antithrombin activity was measured as heparin cofactor activity using a commercially available kit (Dade-Behring, Deerfield, IL). Antithrombin antigen was measured using antibody-coated microlatex particles in a commercially available kit (Diagnostica Stago, Parsippany, NJ). The reference plasmas used were 100% reference plasma from Technoclone for antithrombin activity and the 7th British standard supplied by the National Institute for Biological Standards and Control for antithrombin antigen. Routine testing did not identify any other thrombophilic trait in the proband (protein C and protein S were normal, and the proband tested negative for the genetic lesions, Factor V Leiden, and prothrombin 20210A). Crossed immunoelectrophoresis of antithrombin with heparin included in the gel was performed according to Sas et al. (12) as described (13).

Sequencing of the Variant Antithrombin Gene-- Genomic DNA was extracted from peripheral blood leukocytes by standard methods. The 7 exons (and their flanking regions) were amplified by the polymerase chain reaction using primers described in full elsewhere (14). Amplified products were sequenced using an ABI 377 sequencer.

Purification of Normal and Variant Antithrombins-- Normal antithrombin was purified from outdated human plasma by chromatography on heparin-Sepharose, DEAE-Sepharose, and Sephacryl S-200 columns as previously described (15). The variant antithrombin was isolated from ~200 ml of plasma of the proband by adsorption to 20 ml of heparin-Sepharose gel equilibrated in 0.1 M Tris-Cl, 0.15 M NaCl, pH 7.4, washing the gel with equilibrating buffer containing 0.40 M NaCl to remove unbound protein, and then step-eluting the bound antithrombin with buffer containing 2.5 M NaCl. SDS-PAGE of the purified protein revealed a major antithrombin band in addition to a minor protein contaminant. After dialysis into 0.1 M NaHCO3, 0.15 M NaCl, pH 8.3, the protein was repurified by binding to heparin-Sepharose and eluting with a NaCl gradient according to the procedure used to purify normal antithrombin from plasma. Protein fractions eluting at the end of the gradient were pooled, concentrated, and dialyzed into 20 mM sodium phosphate, 0.1 M NaCl, 0.1 mM EDTA, pH 7.4. The concentration of normal functional antithrombin in the purified inhibitor was assessed by titrating 100 nM thrombin with 0-2 molar eq of purified inhibitor in I 0.15 buffer. After overnight incubation at 25 °C, the residual thrombin activity was determined from the initial rate of hydrolysis of the substrate, S-2238, monitored from the change in absorbance at 405 nm. The amount of active antithrombin was determined from the abscissa intercept of a plot of the linear decrease in activity versus the molar ratio of inhibitor to enzyme added. A 10% molar excess of thrombin over the normal antithrombin found in the preparation (5.8 µM) was then added, and the mixture was incubated for 20 min at 25 °C, a time sufficient to fully complex the functional inhibitor based on the measured inhibition rate constant (>99.9%). Any remaining thrombin was inactivated by the addition of 30 µM D-Phe-Pro-Arg-chloromethyl ketone (Calbiochem), and the mixture was chromatographed in ~5-mg batches on a 5-ml Hi-Trap Heparin-Sepharose column (Amersham Biosciences) equilibrated in dialysis buffer. After loading the sample at 0.5 ml/min, the column was washed with equilibrating buffer for 10 min, a gradient from 0.1 to 3 M NaCl in the same buffer was applied over the next 35 min, and elution was then continued with 3 M NaCl limit buffer for 15 min, all at a flow rate of 1 ml/min. Two incompletely resolved peaks detected by protein fluorescence eluted between 18 and 28 min followed by a well resolved peak eluting between 35 and 50 min. Fractions containing the high heparin affinity variant antithrombin peak were pooled, concentrated, and dialyzed into equilibration buffer. Concentrations of normal and variant antithrombins were determined from the absorbance at 280 nm using an extinction coefficient of 37,700 M-1cm-1 (16).

Heparins-- The pentasaccharide corresponding to the antithrombin binding sequence in heparin and a variant of this sequence having higher affinity for antithrombin (compound 83 in van Boeckel and Petitou (17)) were synthesized by Sanofi Recherche and generously provided by Dr. Maurice Petitou. A full-length heparin of ~26 saccharides containing the pentasaccharide binding sequence for antithrombin was isolated from commercial heparin by size and antithrombin affinity fractionation as described (15). Concentrations of all heparins were determined by stoichiometric fluorescence titrations of plasma antithrombin with the saccharides as described (7, 15).

Proteinases-- Human alpha -thrombin was prepared by purifying prothrombin from plasma followed by activation of the zymogen and purification of the enzyme as described (18, 19). Human factor Xa (predominantly alpha -form) was purchased from Enzyme Research Laboratories (South Bend, IN). Concentrations of active proteinases were determined by comparison of the activities in standard enzyme assays with those of active-site titrated preparations of the enzyme as in previous studies (20).

SDS and Native PAGE-- SDS-PAGE and nondenaturing electrophoresis of normal and variant antithrombins and of their interactions with thrombin, heparin, and neutrophil elastase (Athens Research and Technology) were done with the Laemmli discontinuous buffer system (21). Samples containing thrombin or neutrophil elastase were treated with a >100-fold molar excess of D-Phe-Pro-Arg-chloromethyl ketone or methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (Bachem), respectively, before denaturation and electrophoresis. For reactions of antithrombin with neutrophil elastase, 5 µM normal or variant antithrombin was mixed with 0.1 µM elastase with or without 5 µM heparin pentasaccharide at 25 °C, and samples were taken after 5-80 min and quenched with chloromethyl ketone inhibitor before electrophoresis.

Experimental Conditions-- All experiments were performed at 25 °C in 20 mM sodium phosphate buffers containing 0.1 mM EDTA and 0.1% polyethylene glycol 8000 adjusted to pH 7.4. NaCl was either absent or added to 0.10 or 0.25 M to give ionic strengths of 0.05, 0.15, and 0.30.

Heparin Binding to Antithrombin-- Normal or variant antithrombins were titrated with heparin, and binding of the saccharide was monitored from the increase in tryptophan fluorescence that accompanies the binding interaction with an SLM 8000C spectrofluorometer (SLM Instruments, Rochester, NY) as in previous studies (15, 22). Titrations to assess heparin binding stoichiometry were done at antithrombin concentrations (200-800 nM) much higher than KD (>10-fold) and in I 0.05 (pentasaccharide) or 0.15 (full-length heparin) buffer, whereas titrations to determine the equilibrium dissociation constant were done at inhibitor concentrations closer to KD in I 0.3 buffer (50-200 nM). Fluorescence data were computer-fit by nonlinear regression analysis to the equilibrium binding equation for a 1:1 binding interaction (7, 15). Stoichiometries measured in titrations at lower ionic strength were assumed in titrations at higher ionic strength.

Fast Protein Liquid Chromatography Analysis of Heparin Binding-- Normal or variant antithrombins (10 µg) were applied to a 5-ml Hi-Trap heparin-Sepharose column equilibrated in I 0.15 buffer, washed, and eluted with the program used to purify the variant inhibitor given above.

Heparin Binding Kinetics-- Normal or variant antithrombins were reacted with at least a 5-fold molar excess of heparin to achieve pseudo-first-order conditions, and the kinetics of heparin binding were continuously monitored from the increase in protein fluorescence in an SX-17MV stopped-flow instrument (Applied Biophysics, Leatherhead, UK) as in previous studies (7, 22). The rise in fluorescence was well fit by a single exponential function in all cases, which yielded the observed pseudo-first-order binding rate constant (kobs). kobs from 10-20 reaction traces were averaged for each heparin concentration. Heparin concentrations ranged from 0.1 to 1 µM for determinations of overall association and dissociation rate constants at ionic strengths of 0.15 and 0.3. Polysaccharide concentrations were extended up to 32 µM for resolution of the two steps of the binding process for the full-length heparin in I 0.15 buffer.

Proteinase Inactivation Kinetics-- The proteinase inhibitor activity of the variant antithrombin was assessed by reacting 100 nM variant with 10 nM thrombin or factor Xa in the absence or presence of 300-500 nM heparin in I 0.15 buffer in a total volume of 0.1 ml. Control reactions were done in the absence of inhibitor to establish the activity of the uninhibited enzyme and in the presence of normal antithrombin to determine the normal rate of enzyme inhibition. After varying intervals of time up to 60 min, reactions were quenched by the addition of 0.9 ml of chromogenic substrate, either 100 µM S-2238 (Chromogenix) for thrombin reactions or 100 µM Spectrozyme FXa (American Diagnostica) for factor Xa reactions, containing 50-100 µg/ml Polybrene to neutralize heparin. Residual enzyme activity was then determined by monitoring the initial linear rate of absorbance increase at 405 nM for several minutes. The effect of the variant antithrombin on the kinetics of normal antithrombin inhibition of thrombin was determined under pseudo-first-order conditions by reacting 100 nM normal antithrombin with 10 nM thrombin with or without 1 nM full-length heparin in the presence of 0-500 nM variant antithrombin. Reactions in the absence of heparin additionally contained 50 µg/ml Polybrene. Reactions were quenched after varying times with 0.9 ml of 100 µM S-2238 containing 50 µg/ml Polybrene, and the residual thrombin activity was measured from the initial rate of substrate hydrolysis. Progress curves of the loss in thrombin activity were fit by a single exponential function with a zero activity end point to obtain kobs. In some cases, inactivation was allowed to proceed for a fixed time of 3 or 5 min, and kobs was calculated from the expression for a first-order reaction, (ln(vo/vt))/t, where vt and vo represent the enzyme activities after reaction for time t and for unreacted enzyme, respectively. The dependence of kobs on the variant antithrombin concentration was fit by the following equation for competitive binding of heparin to normal and variant antithrombins,


k<SUB><UP>obs</UP></SUB>=k<SUB><UP>uncat</UP></SUB><UP>×</UP>[<UP>AT</UP>]<SUB><UP>0</UP></SUB><UP>+</UP>k<SUB>H</SUB>×[<UP>H</UP>]<SUB>0</SUB><UP>×</UP>[<UP>AT</UP>]<SUB>0</SUB><UP>/</UP>([<UP>AT</UP>]<SUB>0</SUB><UP>+</UP>K<SUB><UP>AT,</UP>H</SUB>×(<UP>1+</UP>[<UP>AT*</UP>]<SUB>0</SUB>/K<SUB><UP>AT*,H</UP></SUB>)) (Eq. 1)
where kuncat and kH are the second order rate constants for reactions of free and heparin-complexed normal antithrombin with thrombin, [AT]o and [AT*]o are the total concentrations of normal and variant antithrombins, respectively, [H]o is the total heparin concentration, and KAT,H and KAT*,H are the dissociation constants for heparin binding to normal and variant antithrombins. The expression multiplying kH represents the concentration of normal antithrombin-heparin complex in the presence of the competitor variant antithrombin. The expression derives from the cubic equation defining the concentration of antithrombin-heparin complex in the presence of a competitor antithrombin under the conditions, [AT]o, [AT*]o [H]o, in which the concentrations of the free antithrombins can be equated with their total concentrations (23). Values for kobs measured in the absence of the variant antithrombin, denoted kobs,o, and also in the absence of heparin, denoted k'obs,o, enabled simplification of this equation to the form
(k<SUB><UP>obs</UP></SUB>−k′<SUB><UP>obs,</UP>o</SUB>)/(k<SUB><UP>obs,</UP>o</SUB>−k′<SUB><UP>obs,</UP>o</SUB>)=(<UP>1+</UP>[<UP>AT</UP>]<SUB>0</SUB>/K<SUB><UP>AT,</UP>H</SUB>)/(1+[<UP>AT</UP>]<SUB>0</SUB>/K<SUB><UP>AT,</UP>H</SUB>+[<UP>AT*</UP>]<SUB>0</SUB>/K<SUB><UP>AT*,</UP>H</SUB>) (Eq. 2)
Data were fit by this equation by fixing KAT,H at the measured value of 19 nM with KAT*,H a fitted parameter.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a Novel Variant Antithrombin in a Patient with Early Onset Thrombosis-- The 16-year-old proband presented in the clinic with deep vein thrombosis. Coagulation test screening identified an abnormality in the antithrombin of the patient, as indicated from the 100% antigen levels but ~50% activity (Fig. 1 and Table I ), suggesting the presence of an inactive antithrombin variant. No other functional abnormality could be detected and the genetic risk factors, factor V Leiden and prothrombin 20210A, were not present. Further analysis of the patient antithrombin by crossed immunoelectrophoresis in the presence of heparin indicated a normal profile, consistent with the variant antithrombin binding heparin with an affinity at least as high as that of the normal protein. Sequencing of the coding region of the two antithrombin alleles confirmed that one of the alleles contained an in-frame deletion of 3 base pairs corresponding to the codon for the P1 Arg-393 residue (results not shown but available on request to the authors). Such a mutation has not been previously described. Investigation of the family medical history revealed several cases of thrombosis, including pulmonary embolism, mesenteric thrombosis, and still births. Thrombosis was correlated with antithrombin deficiency in several cases that were tested (Fig. 1).


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Fig. 1.   Pedigree of proband. The proband is indicated by the arrow, family member affected with thrombosis is indicated by , and those documented with antithrombin deficiency are indicated by . Individuals affected with thrombosis and antithrombin deficiency are indicated by . Table I provides further relevant clinical information on family members.


                              
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Table I
Family history of propositus

Purification of the Variant Antithrombin-- Antithrombin was purified from the plasma of the proband by heparin-agarose chromatography as in previous studies (15, 24). Comparison of the specific activity of the isolated proband antithrombin with that purified from normal pooled plasma showed that about twice as much of the former protein than of the latter was required to neutralize a fixed amount of thrombin (Fig. 2). This observation was consistent with the presence of an inactive antithrombin variant in the purified antithrombin. To separate the anticipated variant and normal antithrombins present in the patient antithrombin, thrombin was added in slight molar excess over the amount of normal antithrombin measured in the preparation. After allowing sufficient time to fully complex the normal inhibitor, the protein mixture was subjected to heparin-agarose affinity chromatography (see "Experimental Procedures" for details). A peak with high heparin affinity anticipated to be the unreacted variant antithrombin was well separated from two low heparin affinity peaks whose elution positions corresponded to those expected for normal antithrombin-thrombin complex and residual free thrombin. SDS-PAGE showed that the isolated antithrombin variant was pure and comigrated with normal antithrombin but was unable to form an SDS-stable complex with thrombin (Fig. 3A) or with factor Xa (not shown) either in the absence or presence of heparin. The variant, termed antithrombin London or Delta P1 antithrombin to indicate that the P1 Arg residue is deleted, also showed no detectable ability to inhibit thrombin or factor Xa enzymatic activity with or without added heparin (<0.001% normal antithrombin activity, data not shown). Nondenaturing PAGE showed an increased mobility of Delta P1 antithrombin as compared with normal inhibitor (Fig. 3B), consistent with deletion of the positively charged P1 Arg residue in the variant. Both normal and variant bands also showed a similar large mobility shift upon the addition of a molar excess of a super high affinity pentasaccharide (17), demonstrating that the variant retained high heparin affinity. Neutrophil elastase cleaved the variant inhibitor at a rate comparable with that of the normal inhibitor in the absence or presence of heparin pentasaccharide (not shown), indicating a similar accessibility of the loop to the enzyme in both inhibitors whether they were in the native or activated conformation.


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Fig. 2.   Reduced functional activity of antithrombin purified from the proband. 0-2 molar eq of purified normal or patient antithrombin was mixed with 100 nM thrombin, and residual thrombin activity was measured after ~16 h as described under "Experimental Procedures." The residual activity expressed as the % of the uninhibited activity is plotted versus the molar ratio of antithrombin (AT) to thrombin and fit by linear regression (solid lines).


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Fig. 3.   SDS and nondenaturing PAGE of purified normal and variant antithrombins. A, SDS-PAGE of purified normal (lanes 1-3) and variant (lanes 5-7) antithrombins (5 µM) alone (lanes 1 and 5) or reacted with 0.5 molar eq of thrombin in the absence of heparin for 5 min (lanes 2 and 6) or in the presence of 0.1 µM full-length heparin (lanes 3 and 7) for 1 min. Unreacted thrombin is shown in lane 4. B, purified normal (lanes 1, 3, and 5) and variant (lanes 2 and 4) antithrombins (3 µg) were electrophoresed in a nondenaturing gel in the absence (lanes 1, 2, and 5) or presence of a 2-fold molar excess of the super high affinity pentasaccharide (lanes 3 and 4).

Heparin Binding to Delta P1 Antithrombin-- The heparin affinity of the P1 Arg deletion variant of antithrombin was initially compared with that of normal antithrombin by analyzing the NaCl concentration required to elute the protein from a heparin-agarose matrix. Interestingly, the variant protein eluted at a much higher salt concentration of 1.9 M than that required to elute the normal inhibitor (1.3 M), indicating that the variant bound heparin with a significantly higher affinity than normal. Direct equilibrium binding studies were conducted by titrating variant and normal antithrombins with both pentasaccharide and full-length high affinity heparins and monitoring polysaccharide binding from the 40% increase in tryptophan fluorescence that signals this binding (15). The stoichiometry of heparin binding was first analyzed by titrating normal and mutant inhibitors with heparin at inhibitor concentrations much higher than the anticipated KD and at low ionic strengths (I 0.05-0.15) where binding was tight. Although the purified variant antithrombin bound 0.62 ± 0.03 mol of heparin/mol of protein (n = 3) when initially tested, the heparin binding stoichiometry increased to 0.92-0.95 (n = 3) after rechromatography of the inhibitor on heparin-agarose, values experimentally indistinguishable from those observed with normal plasma antithrombin. The variant inhibitor thus appeared to be fully functional in binding heparin after removal of some nonfunctional, presumably latent, inhibitor formed during the initial three-step purification (25). The intrinsic protein fluorescence of uncomplexed normal and variant antithrombins was the same within experimental error and both inhibitors underwent similar maximal fluorescence enhancements of ~40% upon binding heparin.

Because of the high heparin affinity of the normal inhibitor and anticipated higher affinity of the variant under these conditions, measurements of the dissociation equilibrium constants were done at an ionic strength higher than physiologic to reduce the affinity and thereby allow accurate determination of KD (Fig. 4). The variant antithrombin bound pentasaccharide and full-length heparins with dissociation constants of 43 ± 4 and 23 ± 4 nM, whereas the corresponding values for binding of these heparins to normal antithrombin were 400 ± 30 and 150 ± 30 nM. A similar difference in KD values of 45 ± 7 and 290 ± 10 nM was measured for the binding of variant and normal antithrombins to a second preparation of the full-length heparin. The variant antithrombin thus bound the two heparins with 7-9-fold higher affinities than normal antithrombin. The lower affinity of the pentasaccharide than of the full-length heparin for both normal and variant antithrombins indicates that the additional ionic interaction, which is made by the full-length heparin just outside the pentasaccharide binding site in normal antithrombin, is similarly made in the variant inhibitor (7, 10).


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Fig. 4.   Higher affinity binding of heparin to Delta P1 antithrombin than to normal antithrombin. Normal (circles) or variant antithrombins (AT) (triangles) (60-200 nM) were titrated with pentasaccharide (panel A) or full-length heparins (panel B) in I 0.3 buffer and heparin binding to the protein was monitored from increases in tryptophan fluorescence as described under "Experimental Procedures." Data were fit by the quadratic equilibrium binding equation assuming functional antithrombin concentrations determined in stoichiometric heparin binding titrations at lower ionic strength.

Rapid Kinetics of Heparin Binding-- Rapid kinetic studies of heparin binding to normal and variant antithrombins were performed to characterize the basis for the abnormally high heparin affinity of the Delta P1 Arg antithrombin variant. The observed pseudo-first-order rate constant for binding of the full-length heparin, measured at physiologic ionic strength (I 0.15) by continuously monitoring the protein fluorescence change that signals binding, increased as a function of heparin concentration in a saturable manner for both antithrombins (Fig. 5). This behavior reflects the two-step nature of the binding interaction established in previous studies (6, 7), which is depicted in the following reaction scheme.


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Fig. 5.   Delta P1 antithrombin binds heparin with a lower dissociation rate constant than normal antithrombin. Observed pseudo-first-order rate constants (kobs) for the binding of heparins to normal (circles) or variant (triangles) antithrombins (AT) were measured as a function of the heparin concentration by continuously monitoring the rate of protein fluorescence changes in a stopped-flow spectrofluorometer as described under "Experimental Procedures." A, experiments done in I 0.15 buffer over a wide range of full-length heparin concentrations where saturation of kobs occurred. B, experiments conducted at low heparin concentrations with both pentasaccharide (open symbols) and full-length heparins (filled symbols) in I 0.3 buffer to better determine overall association and dissociation rate constants. Solid lines indicate nonlinear regression fits of data in panel A by the hyperbolic equation in the text or linear regression fits of data in panel B.


(Eq. 3)
According to the scheme, heparin initially forms a low affinity interaction with antithrombin with dissociation constant, K1, in a rapid equilibrium binding step, which then induces the inhibitor to undergo an activating conformational change with a forward rate constant, k+2, and reverse rate constant, k-2, which results in a high affinity interaction. The expected dependence of kobs on heparin concentration ([H]o) for this scheme is given by the following equation.
k<SUB><UP>obs</UP></SUB>=k<SUB>−2</SUB>+k<SUB>+2</SUB>×[<UP>H</UP>]<SUB>0</SUB>/(K<SUB>1</SUB>+[<UP>H</UP>]<SUB>0</SUB>) (Eq. 4)
Notably, the kinetic data for heparin binding to normal and mutant inhibitors displayed an indistinguishable dependence of kobs on heparin concentration that was well fit by this equation (Fig. 5). Values for K1 of 21 ± 2 µM and for k+2 of 290 ± 10 s-1 obtained from fitting the normal antithrombin data were thus the same within experimental error as the values obtained by fitting the variant antithrombin data. However, the value of k-2 given by the intercept on the ordinate axis was indistinguishable from zero for both inhibitor interactions, in keeping with the small value previously measured for this parameter at this ionic strength (7). These results suggested that the greater heparin affinity of the variant than of normal antithrombin was due to an effect of the mutation on the rate constant for reversal of the conformational activation step, k-2. To verify this possibility, the kinetics of heparin binding were measured at low heparin concentrations and at a higher ionic strength to increase the value of k-2 and better determine its value. In the range of low heparin concentrations well below the value of K1 ([H]o K1), the equation for kobs simplifies to the linear function,
k<SUB><UP>obs</UP></SUB>=k<SUB>−2</SUB>+(k<SUB>+2</SUB>/K<SUB>1</SUB>)×[<UP>H</UP>]<SUB>0</SUB>=k<SUB><UP>off</UP></SUB>+k<SUB><UP>on</UP></SUB><UP>×</UP>[<UP>H</UP>]<SUB>0</SUB> (Eq. 5)
The intercept and slope of the limiting linear variation of kobs at low heparin concentrations thus yields k-2 and k+2/K1, respectively, which represent the overall dissociation and association rate constants, koff and kon, for the two-step binding process. kobs showed the expected linear dependence on heparin concentration for the binding of pentasaccharide and full-length heparins to the two antithrombins at low heparin concentrations (Fig. 5). Whereas the plots were virtually indistinguishable for full-length heparin binding to normal and variant inhibitors, they were clearly distinguishable for pentasaccharide binding. Similar slopes corresponding to kon were found for the binding of either heparin to normal and variant antithrombins, but the intercept representing koff was significantly lower for the Delta P1 variant than for normal antithrombin, this difference being most marked for the pentasaccharide interaction. These results confirmed that the higher heparin affinity of the variant antithrombin was due to a marked reduction in the dissociation rate constant with no significant effect on the association rate constant. Table II summarizes kon and koff values obtained from these experiments and other experiments at I 0.15. 


                              
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Table II
Association and dissociation rate constants for heparin binding to normal and variant antithrombins
Values for kon and koff were measured from the linear dependence of the pseudo-first-order binding rate constant, kobs, on heparin concentration from 0.1 to 1.5 µM heparin as described under "Experimental Procedures."

Delta P1 Antithrombin Effects on the Heparin-catalyzed Normal Antithrombin-Thrombin Reaction-- The substantially higher heparin affinity of Delta P1 antithrombin relative to normal antithrombin suggested that the variant could have a deleterious effect in individuals heterozygous for the mutation because of its ability to block activation of normal antithrombin by heparin. To test this possibility, we examined the ability of the mutant antithrombin to antagonize the heparin-catalyzed reaction of normal antithrombin with thrombin. Delta P1 Arg antithrombin was found to progressively reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin to that of the uncatalyzed rate as the concentration of the mutant inhibitor was increased in the reaction (Fig. 6). Moreover, substoichiometric levels of the mutant inhibitor were sufficient to cause the bulk of the rate decrease. The mutant antithrombin showed no ability to inhibit thrombin itself in the presence of heparin and had no effect on the uncatalyzed antithrombin-thrombin reaction, consistent with the effect of the mutant inhibitor being solely to compete with the normal inhibitor for binding the limiting heparin component. Analysis of the decrease in kobs for the heparin-catalyzed antithrombin-thrombin reaction as a function of the added Delta P1 Arg antithrombin concentration according to a simple competitive heparin binding model (Fig. 6, inset) indicated that the variant inhibitor bound heparin with a dissociation constant of 1.6 ± 0.1 nM, representing a ~12-fold higher affinity than the normal inhibitor at the lower physiologic ionic strength of these experiments.


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Fig. 6.   Antagonism of the heparin-catalyzed antithrombin-thrombin reaction by Delta P1 antithrombin. Reactions of 100 nM normal antithrombin with 10 nM thrombin were done in I 0.15 buffer in the absence (, open circle ) or presence (black-triangle, diamond , down-triangle, , triangle ) of 1 nM full-length heparin without variant antithrombin (, black-triangle) or with 50 nM (diamond ), 100 nM (down-triangle, open circle ), 200 nM (), or 500 nM (triangle ) variant antithrombin as described under "Experimental Procedures." Control reactions of 370 nM variant antithrombin and 10 nM heparin with 10 nM thrombin are shown by ×'s. Inset, kobs for the heparin-catalyzed antithrombin-thrombin reaction in the presence of Delta P1 antithrombin, corrected for the uncatalyzed reaction and expressed relative to the corrected kobs for the reaction in the absence of variant antithrombin, is plotted as a function of the ratio of variant to normal antithrombins. Data are shown for fixed-time assays with variant antithrombin concentrations ranging from 5-300 nM (black-square). The solid line shows a nonlinear regression fit by the competitive binding equation given under "Experimental Procedures."


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified a novel antithrombin variant in a patient with early onset thrombosis and a family history of thrombosis that segregates with antithrombin deficiency in affected individuals. The mutation in the proband was identified by sequencing of the two antithrombin alleles and showing an in-frame deletion of the codon for the P1 Arg residue of antithrombin in one of the two alleles. Such a natural mutation has not been previously reported and was expected to produce a variant antithrombin that lacked inhibitor function. Natural and engineered mutations of the P1 Arg thus cause dramatic losses in inhibitor activity (20, 26, 27), in keeping with earlier studies showing that the P1 Arg residue is the principal reactive site for recognition of the serpin by the trypsin-like proteinases of the blood clotting cascade (28). The presence of an abnormal antithrombin in the proband was indicated from the normal antigen levels but 50% functional levels of the serpin in the plasma and confirmed from the 50% reduced specific activity of the antithrombin purified from plasma by heparin-agarose affinity chromatography. The inactive antithrombin variant was separated from the normal inhibitor by complexing the active antithrombin fraction with thrombin and rechromatographing the mixture on a heparin affinity matrix, taking advantage of the large reduction in heparin affinity that accompanies the binding of the serpin to proteinases (24, 29). The electrophoretic properties of the mutant inhibitor and the lack of inhibitor function were consistent with deletion of the charged P1 Arg residue.

Although lacking in inhibitor activity toward either thrombin or factor Xa, the mutant antithrombin was surprisingly found to bind either full-length or pentasaccharide heparins with an affinity substantially higher than that of the normal inhibitor. This was shown to be due to a decreased rate constant for reversal of the conformational activation step of the two-step heparin binding mechanism that shifted the conformational equilibrium more in favor of the activated high heparin affinity state. Such findings imply that the variant antithrombin is already partially activated without heparin. Thus, partial activation would explain the higher heparin affinity of the variant because the small equilibrium fraction of the inhibitor normally in the activated high heparin affinity state would be increased (6, 7, 30, 31). Moreover, the observation that the heparin-induced conformational activation of the serpin is more favorable for the variant than for the normal inhibitor implies that the energy cost for conformational activation of the variant is lower, and therefore, the conformational activation equilibrium in the absence of heparin will also favor the activated state more for the variant than for the normal inhibitor.

Because activation is associated with a ~40% increase in tryptophan fluorescence, it would be expected that the variant inhibitor might show a larger basal fluorescence and smaller fluorescence enhancement upon binding heparin than the normal inhibitor (32). However, no significant differences were noted in either the basal or the heparin-enhanced fluorescence of the two antithrombins. Nevertheless, these observations are still consistent with a partial activation of the variant. Thus, assuming that in the normal inhibitor there is less than 1% activated antithrombin in equilibrium with unactivated antithrombin, a reasonable assumption based on the 300-fold increased reactivity of the fully activated inhibitor toward factor Xa1 and the inability to detect direct binding of heparin to a preequilibrium fraction of activated antithrombin (6, 27), full activation of the inhibitor is predicted to enhance the inhibitor affinity greater than 100-fold.2 The observed 7-9-fold increase in affinity would thus represent less than 10% inhibitor activation and, thus, is unlikely to be detectable by the anticipated <4% increase in basal fluorescence given the experimental error of our measurements. Such a small extent of inhibitor activation would be consistent with the inability to also detect any preequilibrium fraction of activated antithrombin in kinetic studies of heparin binding to the variant antithrombin (27, 30). The native inhibitor conformation must, therefore, still dominate over the activated conformation in the variant antithrombin despite some partial activation.

The structural basis for partial activation of the Delta P1 variant can be understood from the established mechanism by which heparin activates antithrombin as has been deduced from the structures of native and activated inhibitors (9, 10) and from structure-function analyses of mutant inhibitors (32, 33) (Scheme 1). In this mechanism, the serpin is maintained in a low heparin affinity, low activity state by partial burial of the N-terminal P15 and P14 residues of the reactive proteinase binding loop in beta -sheet A of the protein core. Binding of the heparin pentasaccharide to helix D and surrounding regions comprising the heparin binding site of the serpin on one side of the beta -sheet induces the inhibitor into the high heparin affinity activated state by extending the D helix and expelling the portion of the proteinase binding loop buried in sheet A to increase the exposure of the loop and its central P1 Arg residue (10, 20). The activated state is accessible in the absence of heparin through a conformational equilibrium, but the equilibrium highly favors the low activity state. Heparin shifts the equilibrium to the high activity state only because of the preferential binding of heparin to the activated state (30). The deletion of the P1 Arg residue in Delta P1 antithrombin could be expected to influence the conformational equilibrium of the native inhibitor because this deletion would shorten the length of the loop. The shortened loop would thus induce strain in the native buried loop conformation that could be relieved in the activated exposed loop conformation.


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Scheme 1.   Proposed mechanism of partial activation of the Delta P1 antithrombin variant. Antithrombin exists predominantly in a native low heparin affinity state, in which the reactive loop (solid black curve) is partially inserted in beta -sheet A (arrows) and the P1 Arg (the side chain depicted in stick representation) interacts with the serpin body when heparin is not bound. However, the native state is in equilibrium with a minor fraction of an activated high heparin affinity state in which the reactive loop is expelled from sheet A and the P1 Arg is exposed. Preferential binding of heparin to helix D (depicted by a coil) in the activated inhibitor state shifts the equilibrium in favor of this state and causes helix D to extend and to thereby contract sheet A and result in expulsion of the reactive loop from the sheet. Deletion of the P1 Arg in the loop is proposed to induce strain in the partially inserted loop of the native state that can be relieved in the activated state by expelling the loop from sheet A. The conformational activation equilibrium would, thus, be shifted in favor of the activated conformation in the variant antithrombin.

Support for this proposal has recently been provided by a study which noted that antithrombin has a three-residue insertion on the primed side of the reactive loop when aligned with other serpins (34). Progressive deletion of these residues corresponding to the P6'-P8' positions resulted in partial activation of the inhibitor as judged from an enhanced basal rate of factor Xa inhibition, which is a sensitive indicator of activation. However, only small effects of the mutations on heparin affinity of at most 2-fold were noted. Moreover, single residue deletions resulted in modest 2-fold increases in factor Xa inhibition, and a double-residue deletion increased the factor Xa inhibition rate constant ~10-fold, i.e. <5% of the ~300-fold maximal inhibition rate enhancement produced by activation of the wild-type inhibitor. Deletion of the P1 residue, thus, appears to have a more marked activating effect on the serpin than the deletion of any single residue in the unique loop insertion. This finding can be rationalized from the x-ray structure of antithrombin, which reveals that two of the three inserted residues in the antithrombin loop reside in strand 1 of beta -sheet C, which anchors the reactive loop to the serpin body on the primed side (9, 10), rather than being a part of the exposed, flexible loop. Deletion of the P1 residue may thus more accurately reflect the effect of loop shortening on the conformational activation equilibrium than deletion of the P6'-P8' residues, since the latter deletions may shorten the loop less effectively and cause perturbations of C-beta -sheet structure. Interestingly, the expected strain in the reactive loop produced by deletion of the P1 residue did not noticeably affect accessibility of the loop to proteinase in either the native or activated inhibitor states, as judged from the comparable rate of cleavage by neutrophil elastase of the P4-P3 Ile-Ala bond of the loop in normal and mutant inhibitors. The exposure and flexibility of the loop, thus, appear to be less affected by the single residue deletion.

It is thought that individuals with heterozygous type II antithrombin deficiency resulting from the presence of an abnormal antithrombin with defective proteinase binding function show evidence of thrombotic disease later in life than that seen in family members carrying the Delta P1 Arg variant unless there is coinheritance of additional genetic risk factors such as factor V Leiden or the presence of strong environmental risk factors (2, 35). Presumably this reflects the fact that 50% levels of the inhibitor are just sufficient to maintain the balance of hemostasis. However, in the case of the Delta P1 Arg variant, the higher than normal heparin affinity provides a reasonable explanation for the unusually severe thrombotic phenotype observed in this family, characterized by mesenteric thrombosis, still births, pulmonary embolism, and early onset spontaneous venous thrombosis, all associated with inhibitor deficiency. This is because the inactive mutant inhibitor can interfere with the function of the normal inhibitor by blocking activation of the latter by endogenous heparan sulfate molecules on the endothelium. The mutant inhibitor is, thus, predicted to preferentially bind to the limited heparan sulfate activators estimated to comprise ~1% of cell surface heparan sulfate molecules (8). As a consequence the effective levels of functional inhibitor in vivo would be less than the 50% measured in vitro and would thereby be expected to compromise the regulation of coagulation proteinase activity.

    ACKNOWLEDGEMENT

We thank Peter Gettins for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01-HL-39888 and grants from the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M300062200

1 If the reactivity of native antithrombin with proteinases is all due to a preequilibrium fraction of activated antithrombin, then at most 0.03% of the inhibitor would be activated since inhibitor fully activated with heparin is 300-fold more reactive toward factor Xa than the native inhibitor. If native antithrombin reactivity partly reflects an intrinsic reactivity of the unactivated inhibitor, then the preequilibrium fraction of activated antithrombin would be <0.03%.

2 Heparin pentasaccharide binding to native antithrombin causes the conformational equilibrium to favor the activated over the native conformation by 700-fold (7,19). If one assumes that the conformational equilibrium in the absence of heparin favors the native over the activated conformation >100-fold, then binding of pentasaccharide to the activated conformation is predicted to be >70,000-fold higher affinity than to the native conformation because of the thermodynamic linkage between binding and conformational activation (30). Because conformational activation enhances the measured heparin pentasaccharide affinity for native antithrombin by 700-fold, full activation of the inhibitor would be expected to increase the measured affinity by >70,000/700 = 100-fold.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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