Inactivation of Human Antithrombin by Neutrophil Elastase

Vol. 264, No. 18, Issue of June 25, pp. 10693-10500.1989 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, h C .

Inactivation of Human Antithrombin by Neutrophil Elastase KINETICS OF THE HEPARIN-DEPENDENT REACTION* (Received for publication, November 10, 1988)

Robert E. Jordan$, RichardM. Nelsong, Jaleh Kilpatrickll, James 0.Newgren, Pamela C. Esmon, and Michael A. Fournel From the Departmentsof Physiology and Biochemistry Research and Development, Cutter Biological Group, Miks Laboratories, Berkeky, California94710

Human neutrophil elastase catalyzes the inactivation One of the major natural regulatory mechanisms of blood of antithrombin by a specific and limited proteinolytic clotting involves the inhibition of coagulation proteinases by cleavage. This inactivation reaction is greatly accel- plasma antithrombin (1-3). This inhibitor, a 58,000-dalton erated by an active anticoagulant heparin subfraction glycoprotein, is a member of a large superfamily of related with high binding affinityfor antithrombin. A poten- proteins (4) to which the acronym serpin (serine protease tially complex reaction mechanism is suggested by the inhibitor) has recently been applied (5, 6). binding of both neutrophil elastase and antithrombin The inhibition of coagulation enzymes by antithrombin is in vitro kinetic behavior of this system greatly potentiated by heparin (7). A highly specific interacto heparin. The was examined under two different conditions: 1) at a tion between antithrombin and achemically and functionally constant antithrombin concentration in which the ac- distinct subfraction of heparin molecules is responsible for tive anticoagulant heparin was varied from catalytic the acceleratory effect (8-10). The binding interaction with to saturating levels; and 2) at a fixed, saturating hep- heparin induces a conformational change inantithrombin arin concentration and variable antithrombin levels. (11)with two apparent consequences. First, the enhanced rate Under conditions of excess heparin, the inactivation of inhibition of coagulation enzymes which donot themselves could be continuously monitored by a decrease in the strongly interact with heparin (e.g. Factor Xa) can be attribultraviolet fluorescence emission of the inhibitor.A K,,, uted to an increased intrinsic reactivity of heparin-bound of approximately 1 PM for the heparin-antithrombin antithrombin (12). Secondarily, the conformational change complex and a turnovernumber of approximately 200/ itself strengthens the interaction of antithrombin with hepamin was estimated fromthese analyses. Maximum acceleratory effects of heparin on the inactivation of rin (13, 14)therebycontributing to the approximation of antithrombin occur at heparin concentrations signifi- heparin binding enzymes (e.g. thrombin) to theinhibitor (1519).It is generally accepted that themechanisms derived from cantlylowerthan those requiredtosaturateantithese in vitroobservations relate directly to thein vivo functhrombin. The divergence in acceleratory effect and tioning of the antithrombin-heparin system. antithrombin binding contrasts with the anticoagulant Although heparin does not normally circulate in plasma, functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely the inner luminal surface of the blood vessel possesses the is not active heparin species as an integral surface component (20). to derive from the fact that neutrophil elastase consumed in the inactivation reaction. A size depend- The normally nonthrombogenic properties of the vascular ence was observed for the heparineffect since an an- endothelium have been attributed, in part,to the presence of ticoagulantly active octasaccharide fragmentof hepa- this bound heparin (21). Since endogenous vascular heparin levels are low in comparison to the amounts of circulating rin, with avid antithrombin binding activity, was without effect on theinactivation of antithrombin by antithrombin (22), it is likely that endothelial bound heparin neutrophil elastase. Despite the completely nonfunc- parallels the observed catalytic behavior of soluble heparin in tional nature of elastase-cleaved antithrombin and the vitro in promoting the inhibition of coagulation enzymes. In altered physical properties of the inhibitoras indicated certain coagulopathic conditions, however, the ability of the by fluorescence and sodium dodecyl sulfate-polyacryl- heparinjantithrombin system to modulate coagulation events amide gel electrophoresis, the inactivated inhibitor exmaybe diminished. Inflammatory states characteristically hibited a circulating half-life in rabbits that indiswas involve a localized activation of neutrophils. This can lead to tinguishable from native antithrombin. These results a release of a proteolytic enzyme, neutrophil elastase, capable point to an unexpected and apparently contradictory of inactivating human antithrombin (23). It has been profunction for heparinwhich may relate to the properties posed that thepathological decreases of plasma antithrombin of the vascularendothelium in pathological situations. observed in acute sepsis may derive from this proteolytic inactivation by the neutrophil enzyme (24). This inactivation * The costs of publication of this article were defrayed in part by phenomenon may resemble a similar inactivation of antithe payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 thrombin catalyzed by certain snake venom proteases (25, solely to indicate this fact. 26). Carrel1 and Owen (27) have further suggested that inac$ To whom correspondence should be addressed Dept. of Biophar- tivation by proteolytic cleavage may be a common charactermaceutical Research and Development, Centocor, 244 Great Valley istic of serpin family members and may, in fact, constitute a Pkwy., Malvern, PA 19355. Tel.: 215-889-4537. Present address: Dept. of Biochemistry, University of Vermont, physiological switch mechanism. We have recently reported that the inactivation of human College of Medicine, Burlington, VT 05405. ll Present address: Dept. of Process Development, Chiron Corp., antithrombin by neutrophil elastase is a heparin-dependent 4560 Horton St., Emeryville, CA 94608. reaction (28). Several hundred-fold rate enhancements led us

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Heparin-dependent Inactivation

of

Antithrombin

to speculatethat under certain conditions, thespecific interactionbetweenantithrombinandanticoagulantlyactive endothelial heparin might provide a mechanism for the enzymatic destruction of the inhibitor in uiuo. This apparently contradictory role for heparin and its potential implications for physiological clotting events has prompted the present more detailed examinationof the heparin/antithrombin/elastase system.

served shift toward a higher molecular weight polydispersity following affinity fractionation (34). Preparations of low affinity, anticoagulantly inactive heparin were prepared by a modification of the above procedure in which limiting amounts of heparin were adsorbed on immobilized antithrombin in 0.01 M Tris-HC1, 0.15 M NaCl. The unbound fraction was depleted repetitively under similar conditions until heparin preparations possessing less than 5 activity units/mg were obtained. Heparin functional activity was determined using a modification of a two-stage chromogenic substrate assay (36) employing Factor Xa and S-2222. All activities were related to the anticoagulant unitage EXPERIMENTAL PROCEDURES of a USP heparin standard. Znnctioation of Antithrombin by Neutrophil Elastase-Inactivation Materials reactions were carried out at antithrombin concentrations between Human neutrophil elastase derived from purulent sputum was 0.5 and 2 P M and employed catalytic amounts of neutrophil elastase (generally 5 nM). Antithrombin and heparin were preincubated briefly obtained from Elastin Products Co. (Pacific, MO). Functional human neutrophil elastase concentrations were established by comparison of at 37 "C in a buffer consisting of 0.02 M Tris-HC1, 0.15 M NaCl, pH amidolytic activity to an enzyme standard supplied by Dr. James 7.5. The inactivation reaction was initiated by the addition of enzyme. Travis (University of Georgia) which was greater than 95% active as Aliquots were removed from the incubation mixture at the desired time intervals and diluted (1:20) into a buffer containing the elastase determined by active site titration. Human a1 proteinase inhibitor was isolated as described by Glaser inhibitor a1 proteinase inhibitor (1-5 pglml) in0.05 M Tris-HC1,0.15 M NaCI, pH 8.4. This level of a1 proteinase inhibitor was in excess et al. (29). Heparin-Sepharose was prepared by covalent attachment of porcine mucosal heparin (Scientific Protein Laboratories, Wau- of the amount of elastase present and resulted in the immediate nakee, WI) to cyanogen bromide-activated Sepharose CL-4B. QAE- inactivation of the proteinase. The inclusion of a1 proteinase inhibSepharose (Fast-flow) was obtained from Pharmacia LKB Biotech- itor prevented the further inactivation of antithrombin in the follownology Inc. The KABI chromogenic substrates S-2222 and S-2238 ing assay step in which relatively high heparin concentrations are were obtained from Helena Laboratories (Beaumont, TX). Theelas- present. Residual functional antithrombin was determined as heparin methoxysuccinyl-Ala-Ala-Pro-Val-para-nitroanilidecofactor activity by a modification of the chromogenic assay system tase substrate of Abildgaard et al. (37) employing bovine thrombin and s-2238. was obtained from Sigma. An octasaccharide fragment of porcine mucosal heparin (Mr, ap- Determinations of antithrombin concentrations involved quantificaproximately 2050) possessing potent anti-Factor Xaactivity and high tion of functional activity versus a standard curve employing pooled binding affinity for human antithrombin was kindly provided by Dr. human plasma. The calculation of rates of antithrombin (AT) inactivation by Robert Rosenberg (Massachusetts Institute of Technology). elastase in the presence of heparin fractions assumed first-order kinetics for this process. Initial velocities ( V,) for individual inactiMethods vation curves were established as V, = k*[AT,] after determining the Purification of Antithrombin for Kinetic Studies-Human anti- first-order constant, k, from the slope of the plot: ln([AT,]/[AT]) thrombin was repurified from a commercial inhibitor concentrate versus t. derived from pooled normal plasma and isolated by affinity chromaFluorescence Methods-Fluorescence measurements were carried tography on immobilized heparin. Repurification was undertaken to out at 37 "C using a Perkin-Elmer MPF-44A instrument equipped minimize the normal size heterogeneity of antithrombin and toinsure with a temperature-controlled sample compartment. Excitation of the absence of certainpotentialcontaminants. The predominant antithrombin solutions was at 285 nm. The interaction of heparin circulating species of human antithrombin (58,000 daltons) was sep- with antithrombin was monitored by the heparin-induced enhancearated from the tighter binding, lower molecular weight form of the ment of the ultraviolet fluorescence emission of the inhibitor(38-40). inhibitor (30) by sodium chloride gradient chromatography on hepa- Binding curves were established from the magnitude of fluorescence rin-Sepharose (31). Isolated under these conditions, antithrombin emission at 330 nm as a function of heparin concentration. Fluoreswas free of contamination by platelet factor four, an avid heparin- cence readings were corrected, if necessary, for any contribution due binding and heparin-neutralizingcomponent derived from platelet a to the heparin itself. granules. The absence of platelet factor four was confirmed by a The elastase-induced reversal of the heparin-dependent enhanceradioimmunoassay for this component (Abbott Laboratories, Chi- ment of antithrombin fluorescence was monitored as a time-dependcago, IL). Finally, the antithrombin was subjected to two cycles of ent decay of fluorescence emission at 330 nm. The initial rate of QAE-Sepharose chromatography to adsorb residual heparin contam- fluorescence decay was determined by establishing the slope ( S ) , in inants deriving from the affinity matrix used in earlier steps. Anti- arbitrary fluorescence units per min, of the tangent to the initial thrombin was eluted as an unbound pool when applied in 0.02 M portion of the fluorescence curve. Thetotal observed change in fluorescence (AFbt) was assumed to reflect the complete conversion Tris-HC1, 0.15 M NaCl, pH 7.5. Fractionation of Heparin-Porcine mucosal heparin was separated of functional antithrombin to theelastase-cleaved species. The reacinto species with differing affinities for human antithrombin by tion velocity ( V,) was calculated as: V, = (S/AFbJx AT,. Analyses chromatography on columns of immobilized antithrombin as previ- were carried out at varying antithrombin concentrations (AT,) in the ously described (9). Affinity matrices were prepared by covalent presence of a constant, saturating level of high affinity heparin (0.15 mg/ml = 10 p ~ ) . linkage of human antithrombin to Affi-Gel 15 (Bio-Rad) at 5-10 mg SDS'-Polvacrvlamide ElectroDhoresis-SDS-Dolvacrvlamide eel of protein/ml of gel using the conditions specified by the manufac- " turer. Coupling was carried out in the presence of an excess of N- electrophoresis analysis was carried out on 8-15% gradient gels using acetylated heparin (32). This latter step minimizes coupling through the Pharmacia PHAST system. Samples were reduced with 5% antithrombin lysine residues required for the interactionwith heparin mercaptoethanol. Gels were stained with Coomassie Blue. The molecular weight standards were obtained from Bio-Rad. (7,33). Protein Concentrations-Antithrombin concentrations were deterThe active heparin species was separated by application of crude heparin to the affinity column in 0.02 M Tris, pH 7.5, containing 0.5 mined based on absorbance at 280 nm using an extinction coefficient of 6.5 (1%solution at 280 nm) and a mass of 58,000 daltons (41). The M NaCl. The amount of heparin (milligrams) applied was approximately equivalent to the amount of immobilized antithrombin (mil- concentration of the neutrophil elastase standard was estimated from of ligrams) calculated to be present on the gel matrix. After washing the its absorbance at 280 nm employing an extinction coefficient (1%) column in application buffer, bound heparins were desorbed in a step 9.85 and a mass of 30,000 (42). Heparin Concentrations-Quantitative determinations of heparin elution with 3 M NaCl in Tris buffer. The high salt eluates from multiple chromatography cycles were pooled, dialyzed, and lyophilized concentrations were made by the carbazole method (43) and assumed a uronic acid content of 33% for all porcine heparin fractions. before further use. The anticoagulant activities of theseaffinityElastase Amidolytic Activity-Neutrophil elastaseactivity was fractionated heparin preparations were 450-550 units/mg in contrast to the 150 units/mg for unfractionated heparin. A higher average monitored in column fractions and incubation mixtures using the molecular mass (15,000 versus 12,000 daltons) was also assumed for The abbreviation used is: SDS, sodium dodecyl sulfate. the affinity-fractionated heparin in keeping with the previously ob"

.

.

1

I

Heparin-dependent Inactivation chromogenic substrate methoxysuccinyl-Ala-Ala-Pro-Val-paranitroanilide (44). Elastase activity was detected after addition of sample to 1 mM substrate in a total volume of 0.8 ml. Amidolysis was quenched at appropriate time points by the addition of 0.2 ml of 50% acetic acid. Activity was expressed as the change in absorbance at 410 nm/min/ml of the test solution. Animal Studies-Purified human antithrombin was iodinated with ['251]NaI using the solid-state lactoperoxidase method employing Enzymobeads (Bio-Rad) as described by Marcum et al. (45) to a specific radioactivity of 8.65 pCi/sg protein. A portion of the labeled antithrombin (59 pg) was incubated with 0.2 pg of heparin and 0.5 pg of neutrophil elastase at 37'C for 1 h. Followingremoval of the heparin by batch adsorption on QAE-Sepharose, the incubate was chromatographed over heparin-Sepharose to remove both unreacted antithrombin and neutrophil elastase and theunbound peak collected into phosphate-buffered saline containing 2%bovine serum albumin (Sigma). Groups of three New Zealand White rabbits (male, 2-3 kg) were injected with 10-25 pCi/kg of either native or elastase-inactivated antithrombin. Sequential blood samples were collected over a 72-h time course. Radioactivity present in each sample was determined using an LKB Model 1282 y counter, and the resulting values were subjected to pharmacokinetic curve-fitting employing the RSTRIP program (Micromath, Salt Lake City, UT).

of Antithrombin I

10495 1

I

b

RESULTS

A previous report from this laboratory demonstrated that heparin is required for the inactivation of human antithrombin by neutrophil elastase (28). This effect was specific for the active anticoagulant subfraction of heparin. In order to further evaluate this unexpected cofactor behavior, the present study of the kinetics of inactivation was undertaken. Several investigations have demonstrated aconformational rearrangement in antithrombin structure resulting from the binding interaction with heparin (13,38,40,61).A significant increase in the ultraviolet fluorescence emission of the inhibitor is associated with this binding interaction (11). In the present study, we observed the heparin-enhanced fluorescence emission profile of antithrombin to be largely reversed by proteolytic inactivation by neutrophil elastase. Representative fluorescence emission spectra at an antithrombin concentration of 1 pM are shown in Fig. 1. Spectra in the absence of heparin (a)and in the presence of a saturating level (10 pg/ml) of the active anticoagulant heparin species ( b ) are shown. In addition, the fluorescence profile resulting from a 20-min incubation with neutrophil elastase is represented by the spectrum of intermediate intensity (c). The ratio of neutrophil elastase to antithrombin was 1:200 in this experiment. Incubation for longer periods of time did not result in furtherdecreases of fluorescence emission intensity. The rateof fluorescence decay was studied by measurement of the fixed wavelength emission at 330 nm as a function of time. For this purpose, elastase was added directly to the cuvette containing antithrombin and 10 pg/ml of the active heparin species. In the fluorescence decay curve shown in Fig. 2, antithrombin was present at 1 ~ L and M neutrophil elastase was added to a final concentration of 5 nM. The decrease in fluorescence was essentially complete by 8 min. In agreement with the spectral data of Fig. 1, the final emission intensity remained significantly higher than the base line established for antithrombin before addition of heparin. In order to establish a correspondence between the change in fluorescence emission and antithrombin function, the residual inhibitory activity of antithrombin was measured at timed intervals. An assay procedure wasdeveloped (see "Methods") which included as anecessary step the inhibition of the neutrophil elastase enzyme. This was required to prevent further inactivation of antithrombin in the subsequent assay step for antithrombin function which contains heparin as a necessary component (37). A parallel incubation was

300

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Wavelength (nrn) FIG. 1. Fluorescence emission spectra. A cuvette containing antithrombin at 1 p~ was excited at 285 nm and the emission spectrum recorded over the indicated wavelength range (a).The active heparin fraction was then added to the cuvette to a final concentration of10 PM (0.15 mg/ml), and the emission profile was re-recorded ( b ) .Finally, neutrophil elastase was added to the cuvette to a final concentration of 5 nM and incubated for 20 min after which the emission spectrum was again recorded (c). The sample compartment was maintained at 37 "C for all spectral determinations. The buffer was 0.02 M Tris, pH 7.5, containing 0.15 M NaC1.

carried out under condkions identical to those employed in the fluorescence experiment and aliquots were withdrawn for the determination of inhibitory activity. The comparison of fluorescence decay to the loss of inhibitory activity is shown in the inset to Fig. 2. The results of these two different techniques are in close correspondence with a complete loss of inhibitory function occurring at the point at which the minimum fluorescence was reached. The extentof proteolytic cleavage of antithrombin by neutrophil elastase under the conditions of the incubation described in Fig. 2 was examined by SDS-polyacrylamide gel electrophoresis (Fig. 3). Aliquots at thedesignated time points were immediately quenched by dilution in denaturing buffer under reducing conditions. The results indicate a progressive conversion of the intact 58-kDa antithrombin to a major polypeptide of lower molecular mass over the 8-min period. The observed change in molecular mass is consistent with the reported elastase cleavage site (27) at a position 5 kDa from the carboxyl-terminal end of the inhibitor. The time-dependent decrease of fluorescence emission provided a direct and convenient method for estimating the rates of antithrombin inactivation by neutrophil elastase. For reasons of consistency, a single, saturating heparin concentration was employed for all determinations. This level, 150 pg/ml , selected in order to be saturating (approximately 10 p ~ )was at even the highest antithrombin levels tested ( 5 pM). Fluorescence decay curves were recorded, as shown in the example in Fig. 2, for a wide range of inhibitor concentrations (62.5


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FIG. 4. Relationship between the rate of elastase digestion of antithrombin determined from fluorescence decay and inhibitor concentration. Initial reaction rates were determined as described ("Methods"). Theactive heparin fraction was present in all incubations a t 10 PM. A constant level of neutrophil elastase of 5 nM was employed in all incubations. Symbols represent the average of duplicate determinations a t each antithrombin concentration. Inset, Lineweaver-Burk transformation of rate data.

FIG. 2. Time-dependent decrease of antithrombin fluorescence emission following addition of neutrophil elastase. Antithrombin a t 1 P M was exciteda t 285 nm and thefluorescence emission turnover number of approximately 200/min. In these initial a t 330 nm measured as indicated a t point a. Active heparin was added studies only the single, saturating heparin concentration of to a concentration of 10 PM and the fluorescenceemissionagain 150 pg/ml was employed. Dependenciesbetweenreaction measured ( b ) .Neutrophil elastasewas added to the cuvette with rapid velocities and heparin concentrations within the saturating mixing (c) toa final concentration of 5 nM and the measurementof fluorescence a t 330 nm resumed as a function of time. The conditions range were not examined. However, it is likely that changes were identical to Fig. 1. In the inset to the Figure, the fluorescence in mucopolysaccharide concentration may greatly influence reaction rates considering the complex nature of this cofactor curve from the main panel is re-plotted (dotted line) in percentage terms using theemission intensity before addition of enzyme and the and its ability to interact with both neutrophil elastase and final measuredemission as 100 and 076, respectively. For comparison, antithrombin. the functional inhibitory activity of antithrombin, determinedby the Despite the potential complexity of the mechanism underdiscontinuous activity assay under identical conditions, is also shown a linearrelationship was lying thesekineticexperiments, (solid square symbols).

obtained in the Lineweaver-Burk transformation of the velocity data (see inset to Fig. 4). Thisanalysis yielded a calculated K,,, of approximately 1.1p M and a V,,, of 1.5 pM/ - 92.5kD min. For an examinationof inactivation rates in thepresence of catalyticheparin levels, the fluorescence method was not - 45.0 useful due to thelow fluorescence signals generated at these heparin concentrations. To measure antithrombin inactiva- 31 .O tionunderthese conditions, thediscontinuousfunctional - 21.5 activityassay described above was employed. Inactivation - 14.4 curves,generated at various concentrations of the active anticoagulant heparinspecies, have previously been published 2 4 6 8 0 0 . 5 1 (28). An example of an inhibition curve, generated in the Minutes presence of 100 ng/ml of heparin, can be found in Fig. 7. For FIG. 3. SDS-polyacrylamide gel electrophoresis analysis of the estimation of reaction velocities, the individual progress antithrombin cleavage by neutrophil elastase. The digestion curves were treated as first-order reactions. The first-order reaction employed conditions and reactant concentrations identical analysisas rateconstant, k, was calculatedbyregression tothose described in Fig. 2. Aliquots were withdrawnfromthe described ("Methods") and rates of inactivation were calcuincubation at theindicated time points andimmediately quenched in lated as K*[AT,] for the individual inactivation curves. SDS denaturing buffer containing 5% mercaptoethanol and heated at 100 "C for 3 min. Electrophoresis was carried out on the Pharmacia Fig. 5 describes the relationship between heparin concenof antithrombin inactivation. Results are PHAST system on 10-15% acrylamide gradient gels and was moni- tration and the rate tored with Coomassie Blue. plotted on a logarithmic scale to encompass the wide range of heparin concentrations employed (0.002-100 pg/ml; 0.13 nMnM-5 p ~ ) Initial . reaction velocities were estimated directly 6.7 p ~ )Antithrombin . was present at 2 p~ and elastase at5 as theslope of the tangent to the initial region of the fluores- nM in these determinations. For comparison, Fig. 5 alsoshows cence curve. the experimentally determined bindingof the active heparin A plot of initial reaction rates, asderived from fluorescence species to 2 p M antithrombin. This binding curve was estabdata, versus antithrombin concentration is presented in Fig. lished from the heparin-induced enhancementof antithrom4. The reaction kinetics clearlyshowed saturation a t high bin fluorescence as previously described (39) employing idenconcentrations of antithrombin under the conditions of these tical conditions as used for the inactivation experiments. A experiments. The maximal velocity of approximately 1 p M / significant displacement of the two phenomena is evident, min at an enzyme concentration of 5 nM corresponds to a with promotionof inactivation occurring a t significantly lower


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T 1.200

0.080, 0

2

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0.01 0.1 1 Hmporin conc. (pg/ml)

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FIG.5. The heparin concentration dependence for promotion of the rate of inactivation of antithrombin compared to the binding of heparin to antithrombin. The active heparin fraction was employed in all determinations. Inactivation rates(solid circles) were determined from the loss of inhibitory activity of antithrombin after addition of neutrophil elastase to 5 nM. An antithrombin concentration of 2 p~ was employed in all analyses. The binding of the active heparin species to 2 p~ antithrombin (open triangks) was monitored by fluorescence titration as previously described (39). i

i

concentrations of heparin than requhed for binding to antithrombin. The lack of accord between the binding of the active anticoagulant heparin species to antithrombin and itsacceleration of the inactivation of antithrombin by neutrophil elastase could reflect a kinetic contribution resulting from an interaction between heparin and the enzyme itself. Attempts to directly measure the solution-phase elastase-heparin interaction by spectral techniques such as fluorescence polarization failed due to a visible precipitation of the complex. A similar precipitation phenomenon has previously been observed to occur with thrombin and heparin (19). Therefore, an estimation of the affinity of these two components was attempted by affinity chromatography on heparin-Sepharose. In Fig. 6, representative elution profiles of human antithrombin (panel A ) and human neutrophil elastase (panel B) are compared under identical sodium chloride gradient conditions. In this experiment, antithrombin eluted at approximately 0.8 M sodium chloride, while elastase eluted from the heparin matrix at 0.6 M sodium chloride. The results suggest a lower affinity interaction between heparin and elastase than between heparin and antithrombin. The strength of the heparin-elastase interaction isof a similar magnitude as observed previously for coagulation Factor IX and thrombin (46) and suggests that thebinding between heparin and the neutrophil enzyme may be an important component in the inactivation mechanism. The heparin employed in the above studies was purified from crude porcine mucosal heparin and, with an average molecular mass of approximately 15,000 daltons,corresponded to a chain length of approximately 50 saccharide residues. To determine the potential chain length requirements for heparin-induced promotion of the elastase reaction, we studied the effect of a low molecular weight heparin fragment (octasaccharide) with full ability to bind to antithrombin and to stimulate its inhibitory activity against coagulation Factor Xa (35). The results of this experiment are shown inFig. 7. Incontrast tothe rapid inactivation of antithrombin in the presence of 100 ng/ml of anticoagulantly active heparin ( M , = 15,000),there was no apparent stimulation by 10 pg/ml of the active octasaccharide fragment. This suggests at least a 100-fold difference in the two heparin species on a weight basis and amore than 500-fold difference in potency on a molar basis.

0.040“

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FIG.6. Heparin-Sepharose chromatography of human antithrombin (panel A ) and human neutrophil elastase (panel B ) . Affinity chromatography experiments were carried out under identical conditions employing either 5 mgof antithrombin or 100 pgof neutrophil elastase. The chromatographic behavior of the respective proteins was monitored by absorbance in the case of antithrombin and amidolytic activity for neutrophil elastase. The column (1.1 X 8.8 cm) was pre-equilibrated in the application buffer (0.02 M Tris, 0.25 M NaCl, pH 7.5). Elution was accomplished by a linear gradient of NaCl from 0.25 to 1.2 M in the Tris buffer. Chromatograms were run at ambient temperature.

,,i a4

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FIG.7. Potentiation of the rate of inactivation of antithrombin (2 &M) by neutrophil elastase (5 nM) by high and low molecular weight heparins. The functional inhibitory activity of antithrombin was determined at each time point (see “Methods”). The inactivation curve carried out in the presence of 100 ng/ml of active heparin of M, = 15,000 is represented by the solid circles. Similar incubations, employing the anticoagulantly active octasaccharide fraction at 100 ng/ml (A),1 pg/ml (O),and 10 pg/ml (0)are also shown. Antithrombin incubated with neutrophil elastase in the absence of heparin is represented by the open triangles.

A comparison of the in vivo clearance of purified human antithrombin and elastase-inactivated antithrombinwas carried out in rabbits. The elastase-inactivated antithrombinwas prepared as described under “Methods” and was essentially free of contaminating heparin, neutrophil elastase or residual functional antithrombin. The ‘261-labeledproteins were in-

10498


jected into rabbits and blood samples collected for pharmacokinetic analysis. The catabolic ( B phase) half-life for each protein species was computed and found to be equivalent: 23.1 2.1 (S.E.) h for the native antithrombin and 23.7 k 1.1 (S.E.) h for the elastase-inactivated species.

of Antithrombin

talus adamanteus (25). The latterproteolysis, however, differs from that with neutrophil elastase inthat inactivation occurs slowly over a several-hour period and is associated with multiple cleavage sites within the inhibitor. Additionally, proteolytically inactivated antithrombin can be formed under certain circumstances as aconsequence of the inhibition reaction DISCUSSION with coagulation enzymes (48,49). The resulting inactivated Antithrombin binds with high affinity and specificity to a antithrombin species, which can also be detected as a dissominor subfraction of heparin molecules. As a result of com- ciation product of enzyme-inhibitor complexes in vitro (50plexing with this heparin fraction, antithrombin becomes a 52), exhibits bond cleavage at the reactive center Arg-Ser more potent inhibitor of coagulation enzymes. The binding bond. The physical properties of reactive site-cleaved antiinteraction is reversible and allows heparin to function as a thrombin molecule closely resemble those of elastase-inacticatalytic cofactor. Theanticoagulant activity of clinically vated antithrombin (62) as would be expected from the close administered heparin and thenormally nonthrombogenic na- proximity of the twocleavage sites (27). This cleavage of ture of the heparin-containing vascular lining is presumed to antithrombin by its targetenzyme (as in the cases of thrombin derive from this mechanism of action. However, the interac- and Factor Xa) would seem antithetical to its functioning as tion between heparin and antithrombin may lead to a quite a regulatory inhibitor. This latterreaction, both in extent and different outcome as suggested by the recent observation that rate, was found to be promoted by the active anticoagulant heparin promotes the inactivation of antithrombin by neutro- heparin fraction in an ionic strength-dependent fashion (53). phil elastase (28). This latter reaction differs significantly The physiological significance of this inactivation of antifrom the heparin-catalyzed covalent complex formation be- thrombin by its target enzymes is uncertain. This is also true tween antithrombin and coagulation enzymes in that a stable for the present elastase-mediated inactivation of antithromcomplex is not formed and elastase is not consumed. Despite bin. However, in bothcases, antithrombin inhibitory function this fundamental difference, a shared requirement for the is lost due to limited proteolytic cleavage promoted by its own active anticoagulant heparin species suggests that the hepa- cofactor, the specific antithrombin-binding subfraction of rin-antithrombin complex is a common component of each heparin. Rates of neutrophil elastase-mediated proteolysis of antimechanism. Two techniques were employed to assess the heparin-stim- thrombin were conveniently determined from the time-deulated inactivation of antithrombin by neutrophil elastase. A pendent reversal of heparin-enhanced fluorescence emission. discontinuous, chromogenic substrate-based assay was used Only active heparin fractions, and not other sulfated glycoto measure residual levels of inhibitory activity of antithrom- saminoglycans, promote the inactivation of antithrombin by bin when inactivation kinetics were studied at different hep- neutrophil elastase (28). This requirement for the antithromarin concentrations. Alternatively, for kinetic analyses at a bin-binding heparin species strongly suggests that the hepafor elastase attack. constant, saturating level of heparin and varying antithrom- rin-antithrombin complex is the substrate bin concentrations, a continuous fluorescence technique was At all concentrations of antithrombin examined (62.5 nM-5 pM), the inhibitor was essentially fully complexed with hepdeveloped. The fluorescence method derives from the increase in anti- arin. This could be shown directly by the inability to enhance thrombin fluorescence emission at 330 nm which occurs when further the fluorescence by additional heparin and was also the protein is fully complexed with heparin (11). We have consistent with a dissociation constant of approximately 100 found that thisenhancement islargely reversed after cleavage nM for the interaction between the active heparin fraction by neutrophil elastase. The affinity of elastase-inactivated and human antithrombin. The applicability of the fluorescence method was born out antithrombin for heparin is markedly diminished at 37 "C. Although some residual binding affinity has been detected by the close correlation of the decrease in fluorescence emisusing 'H NMR difference spectroscopy (62), addition of even sion to the loss of inhibitory function as well as the limited very highheparin concentrations to inactivated antithrombin proteolytic cleavage evidenced by SDS-polyacrylamide elecdid not result in an enhanced fluorescence emission (data not trophoresis. The apparent change in mass under reducing shown). Thus, the altered fluorescence profile is likely to be conditions, corresponding to a loss of approximately 5 kDa, related to a novel conformation of the proteolytically cleaved is consistent with the reported cleavage at isoleucine 390 (27), inhibitor which differs from that of native antithrombin. An a site which is 42 amino acids from the carboxyl terminus. Rates of inactivation of antithrombin were clearly saturable analogous situation has been reported for a1 proteinase inhibitor, a proteinclosely related to antithrombin (4), for which with respect to the concentration of the inhibitor. In the a radically altered conformation was observed following lim- present study, the observed maximum velocity of approxiabsence ited cleavage bypapain at thereactive center of the inhibitor mately 1pM/min is at least 100-fold faster than in the (47). Ovalbumin, another member of the serpin family, also of heparin. Because of the inherent difficulty in guaranteeing demonstrates a major conformational shift following prote- the absence of small amounts of contaminating heparin in olysis by subtilisin. Carrel1 and Owen (27) have proposed that even the most highly purified antithrombin preparations, and selective proteolytic cleavage within the peptide loop contain- the highly catalytic nature in which heparin promotes this ing the reactive center may represent a physiological switch inactivation phenomenon, it is likely that base-line rates are always overestimated. Small amounts of contaminating hepmechanism for this group of proteins. A limited proteolytic cleavage of antithrombin by neutro- arin in purified antithrombin preparations may have contribphil elastase had previously been observed to cause a complete uted to the inability to detect furtherheparin potentiation of inactivation of the inhibitor (23). However, astimulatory this reaction by previous investigators (23). The present syseffect by heparin in this phenomenon was not detected. On tem has the obvious potential forsignificant kinetic complexthe otherhand,a clear stimulatory effect of heparin was ity due to the presence of a cofactor, heparin, and for comdemonstrated for the proteolytic inactivation of antithrombin plexes of undefined stoichiometry between both elastase and by the proteinase I1 enzyme from the venomous snake Cro- antithrombin with heparin. Nevertheless, the velocity deter-

*

Heparin-dependent Inactivation minations derived from the fluorescence decay data appeared to conform to the behavior of more standard enzyme/substrate systems and exhibited a linear Lineweaver-Burk relationship. From this analysis, a K,,, of approximately 1 p M was derived. Although the relevance of this value for the physiological setting is uncertain due to the in vitro conditions and specific heparin fractions employed, the results are at least consistent with a potential in viuo functioning of this system since the normal circulating concentration of antithrombin in plasma is approximately 2 p M (55). Although an examination of reaction kinetics at saturating heparin was useful for establishing certain basic characteristics of the elastase/antithrombin system, in actuality such a situation would never occur in vivo. Heparin is present only as a minor component on vascular endothelial surfaces (20). For this reason, the acceleratory effects of catalytic concentrations of the active heparin fractiona t fixed concentrations of antithrombin (2 p ~ and ) neutrophil elastase (5 nM) were examined. Rates of antithrombin inactivation increased as the concentration of active heparin increased through the range of0.01-1 pg/ml. At concentrations above 1 pg/ml, reaction rates achieved a constant, plateau value. Interestingly, the heparin concentration at which maximum rates of inactivation were obtained (1-2 pg/ml; 66-133nM) was at least 10-fold lower than the concentration of antithrombin . disparity is particularly employed (116 pg/ml; 2 p ~ ) This evident when the concentration dependence for heparin stimulation of inactivation rates is compared directly to thebinding of the active heparin fractionto antithrombin. The reason for this lack of correspondence is unclear. Whatever the precise mechanism involved, the present results suggest an unexpected catalyticpotential of heparin to promote the inactivation of antithrombin. Neutrophil elastase bindswith considerable apparent affinity to heparin. Although difficult to measure in solution because of co-precipitation, the binding is readily demonstrated by chromatography of the enzyme on columns of immobilized heparin (28). Although a high affinity interaction between neutrophil elastaseand a limited subfraction of heparin cannot be ruled out, the evidence does not support such specificity. Rather, neutrophilelastase was found to bind with similar affinity to columns of either immobilized heparin or dextransulfate2and was shown to bind to several other sulfated glycosaminoglycans (56). In addition, the present results indicate that theaffinity of the neutrophil enzyme for heparin is weaker than the strong affinity exhibited by antithrombin for heparin. Nevertheless, the heparin-elastase interaction could contribute significantly to the rate of inactivation of antithrombin if diffusion of the enzyme along the heparin molecule leads to increased encounters with bound antithrombin. Such a situation hasbeen discussed previously in relation to the exceptionally high second-order rate constants observed for heparin-catalyzed inhibition of thrombin by antithrombin (18). In contrast to the latter example in which the enzyme acts asa bimolecular reactant, thepresent phenomenon is complicated further by the catalytic action of neutrophil elastase. This isa fundamentaldistinction between the present reaction and the better understood inhibition of coagulation enzymes by antithrombin (12-19). The kinetic models and equations which have been previously developed are not immediately applicable to thepresent phenomenon. TOascertain a possible heparin chain length requirement for promotion of antithrombin inactivation, an octasaccharide fragment with full antithrombin binding potential was examined. This heparin fragment has been shown to be inca-

* R. E. Jordan, unpublished results.

of Antithrombin

10499

pable of promoting the inhibition of thrombin (an enzyme with considerable affinity for heparin) but readily accelerates the inhibition of the weakly heparin-binding Factor Xa (35). These studies suggest that neutrophil elastase is ”thrombinlike” in its ability to react with antithrombin in a manner which is dependent on the chain length of heparin. Indeed, relatively high concentrations of the octasaccharide fragment were completely ineffective in promoting the inactivation of antithrombin. The physiological relevance of the present in vitro observations is currently unknown. In theory, significant amounts of elastase-inactivated antithrombin might be generated in viuo in circumstances associated with the release of neutrophil enzymes in inflammatory reactions. The recent development of an assay for detection of neutrophil elastase cleavage of fibrinogen in humanplasma gives evidence for the expression of elastase activity in vivo and suggests that a delicate balance exists in the regulation of this potentproteolytic enzyme (57). Further supporting evidence for a role for neutrophil elastase in coagulopathic events is suggested by the report of Sie et ai. (58) in which both antithrombin and heparin cofactor I1 (a second plasma inhibitor of thrombin), were shown to be inactivated in a heparin-dependent manner duringincubation with intact polymorphonuclear leukocytes in uitro. As a preliminary examination into the potential physiological relevance of the present system, the plasma clearance behavior of inactivated antithrombinwas examined for potential utility as a marker of inflammatory or prethrombotic events. Our results in rabbits indicate thatthe elastaseinactivated antithrombin demonstrates a circulating half-life comparable to the fully functional inhibitor. This is in sharp distinction to the very short, receptor-mediated clearance of the thrombin-antithrombin complex (59). This latter result, initially observed in animal systems and later confirmed in humans(60),prevents the accumulation of significant amounts of coagulation enzyme-antithrombin complexes in plasma. The present results suggest that the proteolytically inactivated form of antithrombin may not be subject to this rapid clearance from circulation. A role for heparin asa cofactor in the inactivation of antithrombin would seem paradoxical for this otherwise anticoagulant molecule. The present study was prompted, however, by the recent speculation that proteolytic inactivation may represent a physiological mechanism for regulation of proteolytic enzyme inhibitors of the serpin family (27). Interestingly, the same interaction with heparin which confers upon antithrombin its unique regulatory activity in inhibiting coagulation enzymes, may also be employed for its inactivation by neutrophil elastase. A similar functioning of this system in viuo could presumably lead to a localized loss of the normally antithrombotic character of the blood vessel wall. At present, we cannot deduce whether such an occurrence might be a normal and beneficial event or only a consequence of pathological circumstances. The present studies suggest that the regulation of hemostasis by heparin may be associated with additional complexity and subtlety. REFERENCES 1. Rosenberg, R. D., and Rosenberg, J. S. (1984) J. Clin. Znuest. 74,

1-6 2. Rosenberg, R. D. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice (Colman, R. W., Hirsh, J., Marder, V. J., and Salzman, E. W., eds) 2nd Ed., pp. 13731392, Lippincott/Harper, Philadelphia 3. Bjork, I., and Lindahl, U. (1982) Mol. Cell. Biochem. 48, 161-182 4. Hunt, L. T.,and Dayhoff, M. 0.(1980) Biochem. Biophys. Res. Commun. 95, 864-971

10500


5. Carrell, R.W., and Travis, J. (1985) Trends Biochem. Sci. 10, 20-24 6. Carrell, R. W., and Boswell, D. R. (1986) in Proteinase Inhibitors (Barrett, A. J., and Salvesen, G., eds) pp. 403-420, Elsevier Science Publishers, Amsterdam 7. Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505 8. Lam, L. H., Silbert, J. E., and Rosenberg, R. D. (1976) Biochem. Biophys. Res. Commun. 69,570-577 9. Hook, M., Bjork, I., Hopwood, J., and Lindahl, U. (1976) FEBS Lett. 66, 90-93 10. Andersson, L.-O., Barrowcliffe, T. W., Holmer, E., Johnson, E. A., and Sims, G. E.C. (1976) Thromb. Res. 9,575-583 11. Einarsson, R., and Andersson, L.-0. (1977) Biochim. Biophys. Acta 490,104-111 12. Jordan, R. E., Oosta, G. M., Gardner, W. T., and Rosenberg, R. D. (1980) J. Biol. Chem. 265,10081-10090 13. Olson, S. T., Srinivasan, K. R., Bjork, I., and Shore, J. D. (1981) J . Biol. Chem. 266, 11073-11079 14. Peterson, C. B., and Blackburn, M. N. (1986)J. Biol. Chem. 262, 7559-7566 15. Pomerantz, M. W., and Owen, W. G. (1978) Biochim. Biophys. Acta 535,66-77 16. Griffith, M. J. (1982) J. Biot. Chem. 257, 13899-13902 17. Nesheim, M. E. (1983) J. Biol. Chem. 258, 14708-14717 18. Hoylaerts, M., Owen, W. G., and Collen, D. (1984) J. Bwl. Chem. 269,5670-5677 19. Nesheim, M., Blackburn, M. N., Lawler, C. M., and Mann,K. G. (1986) J. Biol. Chem. 261, 3214-3221 20. Marcum, J. A., and Rosenberg, R. D. (1984) Biochemistry 23, 1730-1737 21. Damus, P. S., Hicks, M., and Rosenberg, R.D. (1973) Nature 246,355-357 22. Marcum, J. A., Reilly, C. F., and Rosenberg, R. D. (1986) Prog. Hemostasis Thromb. 8, 185-215 23. Jochum, M., Lander, S., Heimburger, N., and Fritz, H. (1981) Hoppe-Seyler’s 2.Physiol. Chem. 362,103-112 24. Duswald, K.-H., Jochum, M., Schramm, W., and Fritz, H. (1985) Surgery 98, 892-899 25. Kress, L. F., and Catanese, J. (1981) Biochemistry 20,7432-7438 26. Kress, L. F., and Catanese, J. (1980) Biochim. Biophys. Acta 615, 178-186 27. Carrell, R. W., and Owen, M. C. (1985) Nature 317, 730-732 28. Jordan, R. E., Kilpatrick, J., and Nelson, R. M. (1987) Science 237,777-779 29. Glaser, C. B., Chamorro, M., Crowley, R., Karic, L., Childs, A., and Calderon, M. (1982) Anal. Biochem. 124,364-371 30. Peterson, C. B., and Blackburn, M. N. (1985)J. Biol. Chem. 2 6 0 , 610-615

of Antithrombin 31. Brennan, S. O., George, P. M., and Jordan, R. E. (1987) FEBS Lett. 219,431-436 32. Pixley, R., and Danishefsky, I. (1982) Thromb. Res. 26, 129-133 33. Peterson, C. B., Noyes, C. M., Pecon, J. M., Church, F. C., and Blackburn, M. N. (1987) J. Biol. Chem. 262,8061-8065 34. Laurent, T. C., Tengblad, A., Thunberg, L., Hook, M., and Lindahl, U. (1978) Biochem. J. 175,691-701 35. Atha, D. H., Stephens, A. W., Rimon, A,, and Rosenberg, R. D. (1984) Biochemistry 23, 5801-5812 36. Teien, A. N., and Lie, M. (1977) Thromb. Res. 10, 399-410 37. Abildgaard, U.,Lie, M., and Odegard, 0. R. (1977) Thromb. Res. 11,549-553 38. Gettins, P. (1987) Biochemistry 26, 1391-1398 39. Jordan, R., Beeler, D., and Rosenberg, R. D. (1979) J. Biol. Chem. 254,2902-2913 40. Olson, S. T., and Shore, J. D. (1981) J. Biol. Chem. 256, 1106511072 41. Nordenman, B., Nystrom, C., and Bjork, I. (1977) Eur. J. Biochem. 7 8 , 195-203 42. Baugh, R. J., and Travis, J. (1976) Biochemistry 16,836-841 43. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330-334 44. Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M. (1979) J . Biol. Chem. 254,4027-4032 45. Marcum, J. A., Atha, D. H., Fritze, L. M. S., Nawroth, P., Stern, D., and Rosenberg, R. D. (1986) J. Biol. Chem. 261,7507-7517 46. Jordan, R. E., Oosta, G. M., Gardner, W. T., and Rosenberg, R. D. (1980) J. Biol. Chem. 255, 10073-10080 47. Loeberman, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 1 7 7 , 531-556 48. Fish, W. W., Orre, K., and Bjork, I. (1979) FEBS Lett. 98, 103106 49. Marciniak, E.(1981) Br. J . Haemutol. 48, 325-336 50. Jesty, J. (1979) J.Biol. Chem. 2 5 4 , 1044-1049 51. Bjork, I., Jackson, C. M., Jornvall, H., Lavine, K. K., Nordling, K., and Salsgiver, W. J. (1982) J. Biol. Chem. 267, 2406-2411 52. Danielsson, A., and Bjork, 1. (1980) FEBS Lett. 119.241-244 53. Olson, S. T. (1985) J. Biol. Chem. 260, 10153-10160 54. Marciniak, E. (1982) Blood 59, 576-581 55. Murano, G., Williams, L., Miller-Andersson, M., Aronson, D. L., and King, C. (1980) Thromb. Res. 18,259-262 56. Marossy, K. (1981) Biochim. Biophys. Acta 659, 351-361 57. Weitz, J. I., Landman, S. L., Crowley,K. A., Birken, S., and Morgan, F. J. (1986) J. Clin. Inuest. 78, 155-162 58. Sie, P., Dupouy, D., Dol, F., and Boneu, B. (1987) Thromb. Res. 47,657-664 59. Shifman. M. A,. and Pizzo. S. V. (1982) J . Biol. Chem. 2 5 7 , 3243-3248 ’ 60. Bauer, K. A,, Goodman, T. L., and Rosenberg, R. D. (1983) Clin. Res. 31, 534A 61. Stone. A. L., Beeler, D., Oosta, G . , and Rosenberg, R. D. (1982) Proc. Natll Acad. Sci. .U. S. A. 7 9 , 7190-7194 62. Gettins, P., and Harten, B. (1988) Biochemistry 27,3634-3639