©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Acceleration of Antithrombin-Proteinase Reactions by Low Affinity Heparin
ROLE OF THE ANTITHROMBIN BINDING PENTASACCHARIDE IN HEPARIN RATE ENHANCEMENT (*)

Virginia J. Streusand (3)(§), Ingemar Björk (4), Peter G. W. Gettins (2), Maurice Petitou (5), Steven T. Olson (1)(¶)

From the (1) From the Center for Molecular Biology of Oral Diseases and (2) Department of Biochemistry, University of Illinois-Chicago, Chicago, Illinois 60612, (3) Henry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202, (4) Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden, and (5) Sanofi Recherche, Gentilly Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (<0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity ( K19 ± 6 µ M) and 5-6-fold smaller fluorescence enhancement (8 ± 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to 100-fold higher polysaccharide concentrations and with a 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.


INTRODUCTION

Heparin is a naturally occurring glycosaminoglycan which acts as a potent antagonist of blood coagulation. This anticoagulant effect of heparin is primarily due to the polysaccharide accelerating the inactivation of blood clotting proteinases by their principal protein inhibitor, antithrombin, several thousandfold (for reviews, see Refs. 1 and 2) The accelerating activity results from a sequence-specific pentasaccharide region, present in about one-third of heparin polysaccharide chains (3, 4, 5) , which binds antithrombin with high affinity and induces an activating conformational change in the inhibitor (6, 7, 8, 9, 10, 11) . This conformational change appears to be primarily responsible for heparin acceleration when factor Xa is the target proteinase since the synthesized heparin pentasaccharide region is nearly as active as a full-length heparin containing this sequence in accelerating the inactivation of factor Xa by antithrombin (11, 12, 13) . However, the conformational change does not appear to be critical for heparin acceleration when thrombin is the target proteinase, since the pentasaccharide has negligible ability to accelerate the inactivation of this enzyme by antithrombin (11) . Instead, heparin rate enhancement of thrombin inhibition appears to be mostly due to the polysaccharide bridging antithrombin and thrombin in a ternary complex in which both the inhibitor and proteinase are bound to the same polysaccharide chain (12, 14, 15, 16, 17, 18, 19, 20, 21) .

Whereas heparin molecules containing the pentasaccharide binding sequence for antithrombin (high affinity heparin) account for the bulk of the anticoagulant activity of heparin preparations, heparin molecules lacking this sequence (low affinity heparin) nevertheless exhibit an anticoagulant effect of 5-10% that of high affinity heparin molecules. Low affinity heparin has been shown to bind antithrombin with about a 1000-fold weaker affinity than the high affinity polysaccharide and appears to induce a conformational change in the inhibitor, although one that is different in kind or extent (6, 7, 22, 23) . Low affinity heparin chains also can accelerate antithrombin-proteinase reactions, but the magnitude of these accelerations are relatively small and proteinase-dependent (24) . It has been suggested that the observed binding of low affinity heparin to antithrombin and accelerating effect of the polysaccharide on antithrombin-proteinase reactions may be accounted for by contaminating high affinity heparin in the low affinity heparin preparations studied (22) .

The purpose of the present study was to determine whether low affinity heparin possesses an intrinsic ability to accelerate antithrombin-proteinase reactions, to establish the mechanism of such an accelerating effect, and thereby to elucidate the role of the antithrombin-binding pentasaccharide in the heparin accelerating mechanism. A further goal was to evaluate whether pentasaccharide activation of antithrombin can potentiate the low affinity heparin acceleration of the antithrombin-thrombin reaction to an extent that duplicates the high affinity heparin acceleration, as has been proposed (25) . The results of our studies provide convincing evidence for an intrinsic accelerating activity of low affinity heparin on antithrombin-proteinase reactions and demonstrate a mechanism for this accelerating effect which resembles that of high affinity heparin. The reduced accelerating activity of the low affinity polysaccharide is further shown to be fully explained by the reduced affinity of the polysaccharide for antithrombin and its inability to induce a full activating conformational change in the inhibitor. Finally, our studies show that pentasaccharide binding to antithrombin diminishes rather than enhances the low affinity heparin acceleration of the antithrombin-thrombin reaction, in keeping with a predominant bridging mechanism of heparin rate enhancement of this reaction and a minimal role of the antithrombin conformational change in the rate enhancement. These findings suggest that nonspecific antithrombin binding sequences may contribute to the anticoagulant effects of clinically administered heparin or of endogenous heparin or heparan sulfate glycosaminoglycans involved in maintaining normal hemostasis.


MATERIALS AND METHODS

Proteins

Antithrombin was purified from outdated human plasma by heparin-agarose, DEAE-Sepharose and Sephacryl S-200 chromatography as described previously (19, 26) . Human -thrombin was a generous gift of Dr. John Fenton, New York State Department of Health, Albany, NY. Human -factor Xa was prepared by activation of purified factor X followed by chromatography on soybean trypsin inhibitor-agarose as described elsewhere (27) and, in part, generously provided by Dr. Paul Bock of Vanderbilt University, Nashville, TN. All proteins were judged pure by SDS-gel electrophoresis. Antithrombin concentrations were determined from the absorbance at 280 nm using a molar extinction coefficient of 37,700 Mcm(28) . Proteinase concentrations were determined by active-site titration as described previously (11) which indicated >70% active enzyme preparations.

Polysaccharides

Low affinity heparin was isolated from purified, size-fractionated heparin with a molecular weight of 7900 ± 10% by repeated antithrombin-agarose affinity chromatography as previously described (19) . The anti-factor Xa activity was measured after each affinity-purification step from the increase produced by 0.8 µ M polysaccharide on the pseudo first-order rate constant for the inactivation of 20 n M factor Xa by 200 n M antithrombin. After three such steps, the anti-factor Xa activity was reduced to 0.08 ± 0.01% of the activity of high affinity heparin with no further reduction observed after a fourth step. The resulting anticoagulant activity of the low affinity heparin measured from the prolongation of the plasma clotting time (29) was 1 ± 0.2% that of high affinity heparin.() High affinity heparin was a preparation previously described, having the same molecular weight as the low affinity polysaccharide (11) . For NMR difference spectra, low affinity and high affinity heparins with a molecular weight of 10,000 and with anti-factor Xa activities comparable to the 7900 Mheparins were employed. The -methyl glycoside of the pentasaccharide corresponding to the antithrombin binding sequence in heparin was synthesized as described elsewhere (30) . Dextran sulfate with an average molecular weight of 8000 was from Sigma. Fluorescein-labeled high affinity heparin was prepared by reacting intrinsic free amino groups in the 7900 Mhigh affinity polysaccharide with fluorescein 5-isothiocyanate (Molecular Probes) and purifying the labeled from the unlabeled polysaccharide by hydrophobic chromatography on octyl-Sepharose, as previously described (31, 32) . The labeled polysaccharide was further purified by antithrombin-agarose affinity chromatography. A labeling ratio of 0.90 ± 0.02 mol of fluorescein/mol of heparin was determined by analysis for heparin by an Azure A dye binding assay (33) and for fluorescein from the absorbance at 493 nm at pH 8.3 using an extinction coefficient of 68,000 Mcm. The latter value was determined for fluorescein isothiocyanate reacted with a 200-fold molar excess of ethanolamine under the same conditions. Titrations of antithrombin with the labeled heparin monitored by protein fluorescence changes (see below) indicated a binding stoichiometry and affinity similar to that of unlabeled high affinity heparin.

Exchange of Fluorescein-labeled High Affinity Heparin into Low Affinity Heparin

A mixture of 3 mg of low affinity heparin and 0.03 mg of fluorescein-labeled high affinity heparin or 0.03 mg of labeled high affinity heparin alone in 3.3 ml of equilibration buffer was applied to an antithrombin-agarose affinity column (30 mg of antithrombin coupled to 10 ml of Affi-gel 15 from Bio-Rad; capacity, 2 mg of high affinity heparin) equilibrated in 20 m M sodium phosphate, 0.25 M NaCl, 0.1 m M EDTA, pH 7.4. Following elution of low affinity heparin with the equilibration buffer, high affinity heparin was eluted by increasing the NaCl concentration in the buffer to 3 M. Fractions (1.8 ml) were assayed for total heparin by the Azure A dye binding method and for labeled heparin by fluorescence detection at 495 nm (excitation), 515 nm (emission).

Experimental Conditions

All experiments were conducted at 25 °C in an ionic strength ( I) 0.15, pH 7.4 buffer consisting of 20 m M sodium phosphate, 0.1 M NaCl, 0.1 m M EDTA, 0.1% polyethylene glycol 8000 except where otherwise noted.

Binding Studies

Binding of low affinity and high affinity heparins and heparin pentasaccharide to antithrombin was analyzed by fluorescence titrations in which the enhancement in protein tryptophan fluorescence accompanying polysaccharide binding was used to monitor the interaction (6, 9, 22) . High affinity heparin titrations were done with 10 n M antithrombin, whereas low affinity heparin titrations employed either 1 or 10 µ M inhibitor. Measurements were made on an SLM 8000 spectrofluorometer with fluorescence detection at 280 and 340 nm except for low affinity heparin titrations of 10 µ M antithrombin, in which was set at 295 nm to avoid inner filter corrections. Because the stoichiometry for low affinity heparin binding to antithrombin was indeterminate under the conditions of measurement, the 1:1 binding stoichiometry determined for high affinity heparin binding was assumed for low affinity heparin binding. Corrections for background fluorescence of the titrant were required for low affinity heparin (maximally 10% for titrations of 1 µ M antithrombin and <1% for titrations of 10 µ M inhibitor) and made by subtracting the signal observed in parallel titrations of polysaccharide into buffer. Titrations were analyzed by computer fitting to the quadratic binding equation (26) .

NMR Spectroscopy

H NMR spectra of antithrombin and antithrombin-heparin complexes were recorded at 500 MHz on a Bruker AMX500 spectrometer. Difference spectra were generated by computer subtraction as described previously (10) .

Kinetic Studies

The kinetics of low affinity heparin acceleration of antithrombin-proteinase reactions were measured under pseudo first-order conditions. In one set of experiments, 20 n M antithrombin was reacted with 1 n M thrombin or with 2 n M factor Xa at increasing concentrations of heparin or dextran sulfate. In some experiments, 0.5 or 1 µ M heparin pentasaccharide was also present. Reaction mixtures of 50 µl were incubated for increasing times in polyethylene glycol-coated polystyrene cuvettes (34) and then quenched with 1 ml of 50 µ M tosyl-Gly-Pro-Arg-7-amido-4-methyl coumarin (Sigma) in the case of the thrombin reaction or with 1 ml 100 or 200 µ M Spectrozyme FXa (American Diagnostica) in the case of the factor Xa reaction. Both substrates were in a high salt buffer consisting of 20 m M sodium phosphate, 1.0 M NaCl, 1 m M EDTA, 0.1% polyethylene glycol 8000, pH 7.4, plus 0.1 mg/ml Polybrene (Aldrich) (except for dextran sulfate reactions where Polybrene was absent) to quench the reaction. The residual enzyme activity was then determined from the initial linear rate of substrate hydrolysis monitored by the increase in fluorescence of 7-amido-4-methyl coumarin at 380 nm and 440 nm or by the increase in p-nitroaniline absorbance at 405 nm. p-Aminobenzamidine (2 m M) was included in the high affinity heparin-catalyzed antithrombin-thrombin reaction to reduce the reaction rate sufficiently for analysis by the discontinuous sampling method. Observed pseudo first-order rate constants ( k) were obtained from the slope of semilog plots of the decrease in enzymatic activity with time or by computer fitting the decay of enzyme activity by an exponential function (26) .

A second set of rapid kinetic experiments were monitored from the decrease in fluorescence accompanying the displacement of the probe, p-aminobenzamidine, from the proteinase by the inhibitor, as in previous studies (15, 35) . Antithrombin-thrombin reactions employed 0.25 µ M thrombin, 70 µ M p-aminobenzamidine, and variable antithrombin and heparin concentrations. Antithrombin-factor Xa reactions were done with 0.5 µ M factor Xa, 85 µ M p-aminobenzamidine, and either with 10 µ M inhibitor and variable heparin or 10 µ M heparin and variable inhibitor concentrations. Fluorescence changes were followed in an SLM 8000 spectrofluorometer equipped with a Milli-flow stopped-flow attachment with wavelengths set at 340 nm and 370 nm and with excitation and emission slits of 2 nm and 32 nm, respectively. Observed rate constants were obtained by fitting fluorescence decay curves from four to five averaged reaction traces by a single exponential function.

The dependence of kon polysaccharide concentration determined from studies at low antithrombin concentrations (20 n M) was computer fit by equations previously shown to describe the two different mechanisms of polysaccharide acceleration of antithrombin-proteinase reactions, i.e. either by a mechanism requiring antithrombin and proteinase to bind to the same heparin chain or one requiring just antithrombin to bind heparin (11, 19, 20) . For the former mechanism, which describes the heparin-catalyzed antithrombin-thrombin reaction, the equation for kis given by

On-line formulae not verified for accuracy

 

On-line formulae not verified for accuracy

For the heparin-catalyzed antithrombin-factor Xa reaction, the dependence of kon heparin concentration was fit by a mechanism requiring heparin binding to just antithrombin. This dependence is given by the equation (11) ,

  

On-line formulae not verified for accuracy

In fitting kinetic data by the above equations, second-order rate constants for uncatalyzed antithrombin-proteinase reactions were fixed at previously measured values for kand kgiven in . Similarly, kwas fixed at the value determined for the uncatalyzed reaction, since this parameter was poorly determined and indistinguishable from the uncatalyzed rate constant. Reported errors represent ± 2 S.E.


RESULTS

Purity of Low Affinity Heparin

Heparin with low antithrombin affinity was purified by repeated antithrombin-agarose affinity chromatography to a constant specific anti-factor Xa activity (see ``Materials and Methods'') equal to 0.1% that of high affinity heparin. To determine whether this activity was an intrinsic property of low affinity heparin or was due to small amounts of residual high affinity heparin, 30 µg of fluorescein-labeled high affinity heparin were added to 3 mg of low affinity heparin, and the exchange of labeled polysaccharide into unlabeled polysaccharide was measured after separating the polysaccharide mixture by antithrombin affinity chromatography. Fluorescein high affinity heparin was quantitatively separated from the low affinity polysaccharide by this procedure, as indicated by the appearance of 99% of the applied fluorescence in the high affinity polysaccharide fraction (Fig. 1). The residual 1% fluorescence, corresponding to 0.3 µg of labeled heparin which coeluted with low affinity heparin was also found in control experiments in which just the labeled high affinity polysaccharide was chromatographed (Fig. 1). Subsequent G-25 chromatography of this nonbinding fluorescent fraction revealed it to be free fluorescein (>90%) that had hydrolyzed from the labeled polysaccharide during the experiment. This indicated the presence of <0.03 µg or <0.001% high affinity heparin in the low affinity polysaccharide.() The levels of high affinity heparin predicted to be present in low affinity heparin in the above experiment assumed that the anti-factor Xa activity of high affinity heparin would not be affected by the low affinity polysaccharide. This assumption was confirmed by the finding that addition of 0.2% high affinity heparin to low affinity heparin increased the anti-factor Xa activity of the latter to an extent equal to that of the high affinity polysaccharide alone. Together, these observations suggested that the activity of low affinity heparin was not due to contaminating high affinity heparin.


Figure 1: Exchange of fluorescein-high affinity heparin into low affinity heparin after antithrombin-agarose chromatography of a mixture of the polysaccharides. Experimental details are given under ``Materials and Methods.'' Chromatography of a mixture of 3 mg of low affinity heparin and 0.03 mg of high affinity heparin ( circles) or of 0.03 mg of high affinity heparin alone ( triangles) was monitored by assays for total heparin ( open circles) and for fluorescent-labeled heparin ( closed circles and open triangles). The arrow depicts the change from low to high salt elution buffer.



Binding of Low Affinity Heparin to Antithrombin

To characterize the mechanism by which low affinity heparin accelerates antithrombin-proteinase reactions, binding of the polysaccharide to antithrombin was quantified by monitoring the protein fluorescence enhancement previously shown to accompany such binding. Titrations of antithrombin with low affinity heparin resulted in a saturable 8 ± 3% enhancement in protein fluorescence and an average dissociation constant (from four titrations) of 19 ± 6 µ M (Fig. 2), in agreement with previous studies (6, 7, 22) . Similar titrations of antithrombin with high affinity heparin gave a 47 ± 7% fluorescence enhancement and a Kof 19 ± 3 n M (average from four titrations); i.e. 1000-fold lower than the Kfor low affinity heparin binding to antithrombin (Fig. 2). Contrasting this large differential affinity of the two heparins for antithrombin, the two polysaccharides bound thrombin with indistinguishable Kvalues of 0.7 ± 0.1 µ M and 0.9 ± 0.1 µ M, respectively, as shown by previous equilibrium binding studies (19) (Fig. 2). These results demonstrated marked differences in the relative binding affinities of antithrombin and thrombin for the two heparins; i.e. a 100-fold higher affinity of antithrombin than of thrombin for high affinity heparin, but a 20-fold greater affinity of thrombin than of antithrombin for low affinity heparin.


Figure 2: Equilibrium binding of low affinity heparin and high affinity heparin to antithrombin and thrombin. Titrations of 10 n M antithrombin with high affinity heparin () or of 10 µ M antithrombin with low affinity heparin () were monitored by changes in intrinsic protein fluorescence as described under ``Materials and Methods.'' Titrations of thrombin with high affinity heparin () or with low affinity heparin () are taken from Olson (19) and were monitored from the 17% quenching of the fluorescence of active site-bound p-aminobenzamidine. Solid curves are computer fits of data by the quadratic binding equation.



NMR Difference Spectroscopy of Antithrombin-Heparin Complexes

H NMR difference spectroscopy was also used to compare the binding of low and high affinity heparins to antithrombin. The aromatic/amide region is particularly suited to making such a comparison, since several of the relatively few resonances in this region that are perturbed by heparin binding have been assigned to specific residues that lie in or close to the heparin binding site. Thus, prominent signals in the difference spectrum arise from the side-chains of tryptophan 49 (36) , histidines 1, 65, and 120 (37) , and tyrosine 131.() The perturbation of all of these side chains in a similar, though not identical, manner by both low and high affinity heparins (Fig. 3) indicates that both heparins bind at the same site and perturb the same contact residues. The aliphatic region of the difference spectrum (not shown) is less straightforward to interpret since there are many more perturbed and overlapping resonances, and no residue-specific resonance assignments. Perturbations arise not only from surface residues in the heparin binding site, but also from buried residues, that are only indirectly perturbed by heparin, through the heparin-induced conformational change (6, 7, 8, 9, 10, 11) . In this region the two heparins produce significantly different difference spectra, consistent with the conformational changes induced by the two species differing in kind or extent.


Figure 3: Comparison of the H NMR difference spectra between antithrombin and antithrombin-heparin complex for ( A) low affinity heparin and ( B) high affinity heparin. Spectra representing the average of 4000 scans were collected at 300 K for antithrombin alone (110 µ M) or of antithrombin-heparin complex (110 µ M, 1:1 mole ratio of antithrombin:heparin). The samples were in 20 m M sodium phosphate buffer, pH 7.15, containing 0.10 M NaCl. Negative peaks arise from residues in antithrombin that are perturbed by heparin binding. Positive peaks are from the same residues, but at the chemical shifts that obtain in the antithrombin-heparin complex. Assignments (for negative peaks) are: 8.14 ppm, His-65 C(2); 7.87 ppm, His-1 C(2); 7.74 ppm, His-120 C(2); 7.45 ppm, Trp-49; 7.06 ppm, His-1 C(4); 6.64 ppm, Tyr-131.



Kinetics of Low Affinity Heparin Acceleration of Antithrombin-Proteinase Reactions

Increasing concentrations of low affinity heparin progressively enhanced pseudo first-order rate constants ( k) for the inactivation of thrombin and factor Xa by antithrombin. However, the maximum rate enhancements were lower and the polysaccharide concentrations required to reach these maxima were considerably higher than those obtained with high affinity heparin (Fig. 4). Thus, low affinity heparin maximally enhanced kfor the antithrombin-thrombin reaction 60 ± 10-fold, as compared to 4400 ± 400-fold for high affinity heparin, and this enhancement required 100-fold higher concentrations of polysaccharide. Similarly, kfor the antithrombin-factor Xa reaction was maximally enhanced 140 ± 30-fold by low affinity heparin, as compared to the 600 ± 50-fold rate enhancement produced by high affinity heparin, and this reduced rate enhancement required 1000-fold higher levels of polysaccharide. Dextran sulfate with a chain-length similar to that of the two heparins also accelerated both antithrombin-proteinase reactions, although to an extent which was lower than that found for low affinity heparin; i.e. 10-fold for the antithrombin-thrombin reaction and 40-fold for the antithrombin-factor Xa reaction.


Figure 4: Polysaccharide concentration dependence of the acceleration of antithrombin-proteinase reactions by high affinity heparin, low affinity heparin, and dextran sulfate. Pseudo first-order rate constants ( k) for polysaccharide-accelerated reactions of 20 n M antithrombin with 1 n M thrombin ( circles) or with 2 n M factor Xa ( triangles) were measured as a function of polysaccharide concentration as described under ``Materials and Methods.'' Solid lines are computer fits of data by Equations 1 and 2 of the text, with K, K, k(for high affinity heparin-accelerated reactions), k (for low affinity heparin and dextran sulfate-accelerated reactions), and kbeing the fitted parameters. kvalues given on the left- and right-hand axes for the high affinity heparin-accelerated reactions refer to thrombin and factor Xa reactions, respectively. The former kvalues are 32.2-fold greater than measured values due to their correction for the competitive effect of p-aminobenzamidine.



Notably, the dependence of kon polysaccharide concentration was similar when the effects of the two heparin species or dextran sulfate on the same antithrombin-proteinase reaction were compared. However, this dependence differed for the two antithrombin-proteinase reactions, consistent with the different mechanisms of heparin acceleration of these two reactions demonstrated previously (11) . The polysaccharide concentration dependence of kwas thus bell-shaped for the antithrombin-thrombin reactions, suggesting that low affinity heparin and dextran sulfate act by bridging antithrombin and thrombin in a ternary complex, as previously shown for the high affinity heparin-accelerated reaction (14, 15, 16, 17, 18, 19, 20, 21) . Fitting of the low affinity heparin data by this mechanism suggested that the ascending portion of the bell-shaped dependence reflected the assembly of a ternary complex from a binary protein-heparin complex with a K(2 ± 1 µ M) similar to that measured for the thrombin-heparin interaction. The fit further indicated that the descending portion of the bell-shaped curve corresponded to antagonism of ternary complex formation by a protein-heparin binary complex with a K(70 ± 40 µ M) approaching that determined for the antithrombin-heparin interaction, given the error associated with the fitted parameters. The reverse situation was apparent for the high affinity heparin-accelerated reaction; i.e. the ascending portion of the bell-shaped curve corresponded to ternary complex assembly via an intermediate antithrombin-heparin binary complex ( K6 ± 4 n M), while the descending portion reflected the antagonism of this assembly by a thrombin-heparin binary complex ( K1.1 ± 0.4 µ M).

In the case of the heparin-enhanced antithrombin-factor Xa reaction, a sigmoidal heparin concentration dependence of k, reflecting a simple saturation curve, was evident for the reactions accelerated by both low- and high affinity heparin, as well as dextran sulfate. For the two heparin-enhanced reactions, the Kfor this saturation curve (8 ± 3 n M for high affinity heparin and 30 ± 10 µ M for low affinity heparin) corresponded closely to that directly measured for the antithrombin-heparin binary complex interaction. The data are thus consistent with prior studies which have indicated that heparin accelerates factor Xa inhibition primarily by conformational activation of antithrombin, with no significant role for a proteinase-heparin interaction (11, 13, 23, 25, 38, 39) . summarizes the second-order rate constants obtained from computer fits of the data of Fig. 4for the polysaccharide-accelerated antithrombin-proteinase reactions at saturation of the appropriate binary protein-polysaccharide complexes (see``Materials and Methods'').

Rapid Kinetic Evidence for Low Affinity Heparin Bridging of Antithrombin and Thrombin in a Ternary Complex

Rapid kinetic studies were performed to provide direct evidence for the assembly of the putative ternary complex in the low affinity heparin-accelerated antithrombin-thrombin reaction and to determine the rate at which thrombin and antithrombin react in this complex. The kinetics were analyzed as a function of low affinity heparin concentration in a range where thrombin-heparin but not antithrombin-heparin binary complexes would be saturated (10 µ M polysaccharide) and at increasing, fixed concentrations of antithrombin, over which ternary complex saturation was anticipated (15, 19) . In these studies, antithrombin binding to proteinase was monitored by the decrease in fluorescence accompanying the displacement of the probe, p-aminobenzamidine, from the proteinase active-site as the inhibitor is bound. A saturable dependence of kon heparin concentration was observed for each antithrombin concentration, and this dependence appeared to parallel the saturation of thrombin by the low affinity polysaccharide in each case, similar to the data of Fig. 4(Fig. 5). However, the limiting rate constant reached for each saturation curve increased in a nonproportional manner with increasing inhibitor concentration, suggesting progressive saturation of an intermediate thrombin-heparin-antithrombin ternary complex. A global fit of the data by equations describing the ternary complex mechanism (see ``Materials and Methods'') indicated a dissociation constant for ternary complex formation of 10 ± 4 µ M and first-order rate constant for reaction of antithrombin and thrombin in the ternary complex of 5 ± 1 s. Comparison with the kinetic parameters previously measured for the high affinity heparin-accelerated reaction (Fig. S1) indicate a 100-fold increased ternary complex Kbut a similar rate constant for antithrombin-thrombin reaction within the ternary complex for the low affinity heparin accelerated reaction.


Figure 5: Heparin and antithrombin concentration dependence of the acceleration of the antithrombin-thrombin reaction by low affinity heparin. kvalues for reactions of antithrombin with 0.25 µ M thrombin were measured as a function of the low affinity heparin concentration at different fixed antithrombin concentrations of 10 µ M (), 20 µ M (), 50 µ M (), and 100 µ M () by stopped-flow fluorometry as detailed under ``Materials and Methods.'' Solid lines are fits to equations describing the ternary complex bridging model for heparin action with k, K, and Kthe fitted parameters. Kwas fixed at the value of 19 µ M determined by equilibrium binding.




Figure S1:



Rapid Kinetic Evidence for Acceleration of Factor Xa Inactivation by Antithrombin-Low Affinity Heparin Binary Complex

Rapid kinetic studies were also used to verify that the acceleration of the antithrombin-factor Xa reaction by low affinity heparin is mediated by an antithrombin-heparin binary complex. The analyses were done as a function of low affinity heparin concentration at a fixed inhibitor concentration of 10 µ M and also as a function of antithrombin concentration at a fixed heparin concentration of 10 µ M. Reactions were monitored as above from the decrease in fluorescence accompanying the displacement of p-aminobenzamidine from the factor Xa active site. In each case, a similar saturable dependence of kwas found, reflecting a K(38 ± 17 µ M) in reasonable agreement with the value directly measured for the antithrombin-low affinity heparin binary complex interaction (Fig. 6). The 70 ± 20-fold rate enhancement obtained from these data was also in fair agreement with the kinetic data of Fig. 4 , considering the smaller data set analyzed in this case. These data confirmed that the reaction proceeded as a second-order association between antithrombin-heparin binary complex and free factor Xa, with no evidence for factor Xa binding to heparin being involved in the rate enhancement.

Effect of the Antithrombin-binding Heparin Pentasaccharide Fragment on Low Affinity Heparin Rate Enhancements

To confirm that the acceleration of antithrombin-proteinase reactions by low affinity heparin involved binding of the polysaccharide to the same site on antithrombin that mediates high affinity heparin binding, we examined the effect on these accelerations of the sequence-specific pentasaccharide responsible for high affinity heparin binding. Equilibrium binding titrations of antithrombin with pentasaccharide in the absence and presence of low affinity heparin (140-160 µ M), monitored by changes in protein fluorescence, demonstrated that low affinity heparin increased the apparent Kfor pentasaccharide binding to antithrombin to an extent compatible with low affinity heparin competing with the pentasaccharide for binding to the inhibitor (). Saturation of antithrombin with pentasaccharide levels sufficient to block low affinity heparin binding (0.5 µ M) nevertheless did not prevent low affinity heparin from accelerating the antithrombin-thrombin reaction in a concentration-dependent manner above the 1.7-fold acceleration produced by the pentasaccharide alone (Fig. 7). This rate enhancement exhibited a bell-shaped curve similar to that observed when low affinity heparin was varied in the absence of pentasaccharide. However, the rate enhancement was considerably reduced from that of low affinity heparin alone, i.e. from 60-fold to 16-fold (). Doubling of the pentasaccharide concentration minimally affected measured kvalues, suggesting that the maximum pentasaccharide effect on the low affinity heparin acceleration was being observed. Fitting of the data by the ternary complex mechanism indicated a similar Kfor a thrombin-heparin binary complex intermediate mediating ternary complex assembly (0.7 ± 0.3 µ M), but higher Kfor antagonism of ternary complex formation by antithrombin-heparin complexes (200 ± 100 µ M), although there was considerable uncertainty in the latter value. These data are consistent with pentasaccharide binding to antithrombin reducing its affinity for the full-length heparin and thereby the ability of the full-length polysaccharide to bridge antithrombin and thrombin in a ternary complex.


Figure 7: Effect of the heparin pentasaccharide on the acceleration of the antithrombin-thrombin reaction by low affinity heparin. kvalues for the reaction of 20 n M antithrombin with 1 n M thrombin were measured as a function of the low affinity heparin concentration in the absence () or presence of 0.5 µ M () or 1 µ M () pentasaccharide. Data obtained in the absence of pentasaccharide are reproduced from Fig. 4. Solid curves are fits of data by the ternary complex-bridging model described under ``Materials and Methods'' in which K, K, and k were the fitted parameters.



The effect of the pentasaccharide on the low affinity heparin-accelerated antithrombin-factor Xa reaction was similarly studied. Saturation of antithrombin with the pentasaccharide produced a 230 ± 30-fold enhancement of the antithrombin-factor Xa reaction rate constant, in fair agreement with earlier reports (11, 13) . Addition of low affinity heparin to the pentasaccharide-saturated antithrombin at levels as high as 50 µ M had no significant effect on k(), suggesting that the pentasaccharide blocks the low affinity heparin acceleration of this reaction. These results support a requirement for low affinity heparin binding to the pentasaccharide site on antithrombin for enhancing the rate of the antithrombin-factor Xa reaction.


DISCUSSION

The purpose of these studies was to determine whether low affinity heparin lacking the pentasaccharide recognition sequence for antithrombin was capable of accelerating antithrombin-proteinase reactions and if so, whether the mechanism of acceleration was similar to that of high affinity heparin molecules possessing the pentasaccharide. Several pieces of evidence argue that the acceleration of antithrombin-proteinase reactions by low affinity heparin is an intrinsic property of the polysaccharide and not due to the presence of a small amount of high affinity polysaccharide. First, the extent of exchange of labeled high affinity heparin into the low affinity heparin was far too low to account for the observed activity of the low affinity polysaccharide. Second, dextran sulfate produced concentration-dependent accelerating effects similar in kind to, although of lower magnitude than, those of low affinity heparin, indicating that an anionic polysaccharide lacking the specific pentasaccharide is capable of producing rate enhancement. Third, the observation of similar saturation curves for the acceleration of the antithrombin-factor Xa reaction by low affinity heparin when either the polysaccharide or the inhibitor concentrations were varied in a reciprocal fashion indicated the acceleration could not be due to contaminating high affinity heparin.

Several observations indicate that low affinity heparin accelerates antithrombin-proteinase reactions by mechanisms which resemble those previously elucidated for high affinity heparin. The similar bell-shaped curves characterizing high affinity heparin, low affinity heparin, and dextran sulfate accelerations of the antithrombin-thrombin reaction suggest that the two latter polysaccharides act by the same ternary complex bridging mechanism previously shown for high affinity heparin acceleration of thrombin inhibition (14, 15, 16, 17, 18, 19, 20, 21) . However, the different relative binding affinities of antithrombin and thrombin demonstrated for low and high affinity heparins imply a different order of assembly of the ternary complex for the two reactions. The greater affinity of antithrombin than of thrombin for high affinity heparin thus was previously shown to result in ternary complex assembly primarily through an antithrombin-heparin binary complex intermediate (19, 22) . Diagnostic of this mode of assembly is the correspondence of the Kvalues for the ascending and descending limbs of the bell-shaped curve characterizing the high affinity heparin accelerated reaction to those of antithrombin-heparin and thrombin-heparin binary complex interactions, respectively. It follows that the greater affinity of thrombin than of antithrombin for low affinity heparin should result in a reverse order of ternary complex assembly primarily through a thrombin-heparin binary complex intermediate. This expectation is borne out by the observations that 1) the bell-shaped curve for the low affinity heparin-accelerated reaction is displaced to higher polysaccharide concentrations and 2) the ascending and descending limbs of this curve correspond to Kvalues similar to those measured for thrombin-heparin and antithrombin-heparin binary complex interactions, respectively.

Rapid kinetic studies provided direct evidence for a heparin-thrombin-antithrombin ternary complex intermediate on the low affinity heparin-accelerated reaction pathway. Such studies demonstrated that the difference in accelerating effects of low and high affinity heparins arises from a 100-fold weaker affinity of thrombin for antithrombin in forming the ternary complex and not from a different rate of reaction of inhibitor with proteinase within the ternary complex (Fig. S1). Comparison with the Kof 1 m M for the initial encounter complex interaction of thrombin with antithrombin and rate constant of 10 sfor the subsequent reaction of inhibitor with proteinase in this complex measured in the absence of heparin (15) , indicates that both heparins act by promoting the encounter of enzyme and inhibitor in the ternary complex without significantly affecting the rate of conversion of this complex to a stable complex. The 10,000-fold enhancement of the enzyme-inhibitor encounter complex interaction by high affinity heparin was previously shown to arise from a thrombin-heparin bridging interaction increasing the affinity of thrombin for antithrombin in the ternary complex (20) . The 100-fold promotion of the enzyme-inhibitor encounter by low affinity heparin can similarly be accounted for by an antithrombin-heparin bridging interaction stabilizing the association of antithrombin with heparin-bound thrombin in the ternary complex. The antithrombin-heparin binary complex Ksuggests more than sufficient binding energy to produce a 100-fold enhancement in the ternary encounter complex K. The excess binding energy may arise from the need to correct the binary complex Kfor the anticipated large number of overlapping interaction sites on the polysaccharide for the inhibitor and/or to different numbers of ionic interactions involved in binary and ternary complexes (20, 40) . Our results are thus consistent with the reduced rate-enhancing effect of low affinity heparin relative to high affinity heparin resulting from the reduced affinity of the polysaccharide for antithrombin diminishing the ability of heparin to bridge antithrombin and thrombin in the ternary complex intermediate.

Low affinity heparin also appears to accelerate the antithrombin-factor Xa reaction by a mechanism similar to that previously shown for high affinity heparin, but different from the mechanism by which these heparins accelerate the antithrombin-thrombin reaction. This conclusion is evident from the simple sigmoidal saturation curve, instead of a bell-shaped curve, which characterizes the polysaccharide or antithrombin concentration dependence of high affinity heparin, low affinity heparin and dextran sulfate accelerations of the antithrombin-factor Xa reaction (Figs. 4 and 6) (11, 35, 38) . The displacement of the low affinity heparin saturation curve to 1000-fold higher polysaccharide concentrations than the high affinity heparin curve and the correspondence of the apparent Kvalues describing these saturation curves to the Kvalues for the binding of the two heparins to antithrombin indicate the rate enhancements produced by both heparins arise from an interaction of the polysaccharide with antithrombin. Moreover, the failure to observe any diminution of the accelerating effect of either heparin at the highest concentrations of polysaccharide examined implies that a factor Xa-heparin bridging interaction contributes minimally or not at all to heparin rate enhancement.

Previous studies which compared pentasaccharide and full-length high affinity heparin accelerations of the antithrombin-factor Xa reaction demonstrated that an activating conformational change induced in antithrombin by the pentasaccharide, rather than heparin bridging, was primarily responsible for the heparin rate enhancement. That activation of antithrombin by a heparin-induced conformational change is also responsible for the low affinity heparin rate enhancement of the factor Xa reaction is supported by observations in this and prior studies. Binding of low affinity heparin and dextran sulfate to antithrombin thus induces changes in the intrinsic fluorescence, near ultraviolet absorbance and circular dichroism spectra of antithrombin, as well as in the fluorescence of an extrinsic probe covalently linked to the reactive center P1 residue, which are indicative of the activating conformational change (6, 7, 23) . Moreover, these spectroscopic changes are reduced in magnitude relative to those produced by pentasaccharide or full-length high affinity heparins to an extent which correlates with the diminished rate enhancements of the low affinity polysaccharides. Together, these observations are consistent with the reduced acceleration of the antithrombin-factor Xa reaction produced by low affinity heparin being due to both the lower affinity of the polysaccharide for antithrombin as well as the failure of the polysaccharide to induce a full activating conformational change in antithrombin.

NMR difference spectroscopy and the high affinity heparin pentasaccharide fragment were used to further probe the mechanism by which low affinity heparin accelerates antithrombin-proteinase reactions. NMR difference spectra of antithrombin complexes with low and high affinity heparins and competitive binding studies together demonstrated that low and high affinity heparins bind to the same site on antithrombin, in agreement with the findings of earlier studies (41) . In keeping with such findings, blocking the high affinity heparin binding site on antithrombin with pentasaccharide abolished the acceleration of the antithrombin-factor Xa reaction, indicating a requirement for low affinity heparin to bind at this site and induce a conformational change to mediate its accelerating effect. In the case of the antithrombin-thrombin reaction, the pentasaccharide markedly reduced but did not abolish the rate-enhancing effect of low affinity heparin nor did it affect the bell-shaped dependence of the residual rate enhancement on polysaccharide concentration. These observations suggested that low affinity heparin can still bridge thrombin and antithrombin when antithrombin is complexed with pentasaccharide, although to a reduced extent. Such bridging can be explained by a nonspecific electrostatic association of low affinity heparin with antithrombin outside the pentasaccharide binding region, similar, although not necessarily identical, to that which occurs with high affinity heparin (8, 11, 25) . This explanation would be consistent with the reduced affinity of pentasaccharide-blocked antithrombin for low affinity heparin inferred from the bell-shaped curve.

Our results conflict with a previous report which suggested that the small pentasaccharide acceleration of the antithrombin-thrombin reaction could be increased by low affinity heparin to an extent comparable to that of high affinity heparin (25) . This observation was taken as evidence for the pentasaccharide-induced conformational change in antithrombin being principally responsible for heparin acceleration of the antithrombin-thrombin reaction, with heparin bridging of antithrombin and thrombin playing only a minor role. While our results show that low affinity heparin can bridge pentasaccharide-activated antithrombin with thrombin, they also demonstrate that this bridging does not increase the rate enhancement beyond that expected from a bridging effect alone. Our results thus argue against a significant role for conformational activation of antithrombin in the mechanism of heparin acceleration of the antithrombin-thrombin reaction and are in keeping with a predominant bridging mechanism of rate enhancement for this reaction (20, 21) .

In summary, the results of this study indicate that low affinity heparin possesses an intrinsic ability to accelerate antithrombin-proteinase reactions and that the mechanism of this accelerating effect is identical to that of high affinity heparin having a pentasaccharide recognition sequence for antithrombin. Our studies thus establish that the pentasaccharide is not absolutely required for heparin anti-proteinase activity but does play an important role in mediating the high affinity binding of antithrombin to the polysaccharide and in the polysaccharide's ability to induce a full activating conformational change in the inhibitor. These findings suggest that nonspecific heparin sequences may contribute to the anticoagulant effects of clinically administered heparin, as well as of heparan sulfate glycosaminoglycans present in the blood vessel wall. The high levels of low affinity heparin required to produce an anticoagulant effect do not preclude such a contribution, since such heparin levels are also required for physiologic activation of heparin cofactor II (42, 43) . Although heparin cofactor II inhibits thrombin with a 10-fold greater bimolecular rate constant than antithrombin at optimal levels of low affinity heparin, the 2-fold greater plasma concentration of antithrombin over heparin cofactor II (42) would imply that one-fifth of the free thrombin generated in plasma would be inhibited by antithrombin in the presence of low affinity heparin. The observation that low affinity heparin possesses significant antithrombotic activity exceeding that of the specific heparin cofactor II activator, dermatan sulfate (44) , supports the possibility that this antithrombotic activity is not only mediated by activation of heparin cofactor II but also in part by activation of antithrombin.

  
Table: Second-order rate constants (k) for polysaccharide-accelerated antithrombin-proteinase reactions


  
Table: Effect of low affinity heparin on the apparent dissociation constant for pentasaccharide binding to antithrombin

Dissociation constants were measured from fluorescence titrations performed as described under ``Materials and Methods'' in I 0.15, pH 7.4 sodium phosphate buffer at 25 °C.



FOOTNOTES

*
Supported by National Institutes of Health grants HL-39888 (to S. T. O.) and HL-49234 (to P. G. W. G.) and Swedish Medical Research Council Grant 4212 (to I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Gillette Research Institute, 401 Professional Dr., Gaithersburg, MD 20879.

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

The dependence of heparin anticoagulant activity measured by clotting assay on polysaccharide molecular weight may partially account for the lower anticoagulant activity of our 7900 molecular weight low affinity heparin as compared to that reported for higher molecular weight preparations of the low affinity polysaccharide (6, 22, 44).

The abbreviations used are: AT, antithrombin; T, thrombin; H, heparin; P, p-aminobenzamidine.

It is predicted that 3 µg of high affinity heparin is present in 3 mg of low affinity heparin if the former accounts for the observed anti-factor Xa activity of the latter. Addition of 30 µg of fluorescent-labeled high affinity heparin should thus result in the exchange of (30/33) 3 or 2.7 µg of labeled high affinity heparin into the low affinity heparin, assuming the affinity separation procedure results in 0.1% residual high affinity heparin in the low affinity heparin.

P. G. W. Gettins, unpublished data.


ACKNOWLEDGEMENTS

We thank Ann Marie Francis-Chmura and Rick Swanson for technical assistance in these studies.


REFERENCES
  1. Olson, S. T., and Björk, I. (1994) Semin. Thromb. Hemostasis 20, 373-409 [Medline] [Order article via Infotrieve]
  2. Olson, S. T., and Björk, I. (1994) Perspect. Drug Disc. Des. 1, 479-501
  3. Thunberg, L., Bäckström, G., and Lindahl, U. (1982) Carbohydr. Res. 100, 393-410 [CrossRef][Medline] [Order article via Infotrieve]
  4. Choay, J., Petitou, M., Lormeau, J. C., Sina&;, P., Casu, B., and Gatti, G. (1983) Biochem. Biophys. Res. Commun. 116, 492-499 [Medline] [Order article via Infotrieve]
  5. Atha, D. H., Stephens, A. W., and Rosenberg, R. D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1030-1034 [Abstract]
  6. Nordenman, B., Danielsson, Å., and Björk, I. (1978) Eur. J. Biochem. 90, 1-6 [Abstract]
  7. Nordenman, B., and Björk, I. (1978) Biochemistry 17, 3339-3344 [Medline] [Order article via Infotrieve]
  8. Stone, A. L., Beeler, D., Oosta, G., and Rosenberg, R. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7190-7194 [Abstract]
  9. Olson, S. T., and Shore, J. D. (1981) J. Biol. Chem. 256, 11065-11072 [Free Full Text]
  10. Gettins, P. (1987) Biochemistry 26, 1391-1398 [Medline] [Order article via Infotrieve]
  11. Olson, S. T., Björk, I., Sheffer, R., Craig, P. A., Shore, J. D., and Choay, J. (1992) J. Biol. Chem. 267, 12528-12538 [Abstract/Free Full Text]
  12. Lane, D. A., Denton, J., Flynn, A. M., Thunberg, L., and Lindahl, U. (1984) Biochem. J. 218, 725-732 [Medline] [Order article via Infotrieve]
  13. Atha, D. H., Lormeau, J. C., Petitou, M., Rosenberg, R. D., and Choay, J. (1987) Biochemistry 26, 6454-6461 [Medline] [Order article via Infotrieve]
  14. Laurent, T. C., Tengblad, A., Thunberg, L., Höök, M., and Lindahl, U. (1978) Biochem. J. 175, 691-701 [Medline] [Order article via Infotrieve]
  15. Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895 [Free Full Text]
  16. Griffith, M. J. (1982) J. Biol. Chem. 257, 7360-7365 [Free Full Text]
  17. Nesheim, M. E. (1983) J. Biol. Chem. 253, 14708-14717
  18. Danielsson, Å., Raub, E., Lindahl, U., and Björk, I. (1986) J. Biol. Chem. 261, 15467-15473 [Abstract/Free Full Text]
  19. Olson, S. T. (1988) J. Biol. Chem. 263, 1698-1708 [Abstract/Free Full Text]
  20. Olson, S. T., and Björk, I. (1991) J. Biol. Chem. 266, 6353-6364 [Abstract/Free Full Text]
  21. Gan, Z.-R., Li, Y., Chen, Z., Lewis, S. D., and Shafer, J. A. (1994) J. Biol. Chem. 269, 1301-1305 [Abstract/Free Full Text]
  22. Jordan, R., Beeler, D., and Rosenberg, R. D. (1979) J. Biol. Chem. 254, 2902-2913 [Abstract]
  23. Gettins, P. G. W., Fan, B., Crews, B. C., Turko, I. V., Olson, S. T., and Streusand, V. J. (1993) Biochemistry 32, 8385-8389 [Medline] [Order article via Infotrieve]
  24. Nordenman, B., Nordling, K., and Björk, I. (1980) Thromb. Res. 17, 595-600 [Medline] [Order article via Infotrieve]
  25. Evans, D. L., Marshall, C. J., Christey, P. B., and Carrell, R. W. (1992) Biochemistry 31, 12629-12642 [Medline] [Order article via Infotrieve]
  26. Olson, S. T., Björk, I., and Shore, J. D. (1993) Methods Enzymol. 222, 525-560 [Medline] [Order article via Infotrieve]
  27. Bock, P. E., Craig, P. A., Olson, S. T., and Singh, P. (1989) Arch. Biochem. Biophys. 273, 375-388 [Medline] [Order article via Infotrieve]
  28. Nordenman, B., Nyström, C., and Björk, I. (1977) Eur. J. Biochem. 78, 195-203 [Abstract]
  29. Griffin, J. H., and Cochrane, C. G. (1976) Methods Enzymol. 45, 56-65 [Medline] [Order article via Infotrieve]
  30. Petitou, M., Duchaussoy, P., Lederman, I., Choay, J., Jacquinet, J. C., Sina&;, P., and Torri, G. (1987) Carbohydr. Res. 167, 67-75 [CrossRef][Medline] [Order article via Infotrieve]
  31. Nagasawa, K., and Uchiyama, H. (1978) Biochim. Biophys. Acta 544, 430-440 [Medline] [Order article via Infotrieve]
  32. Uchiyama, H., and Nagasawa, K. (1981) J. Biochem. ( Tokyo) 89, 185-192 [Abstract]
  33. Jacques, L. B. (1977) Methods Biochem. Anal. 24, 203-212 [Medline] [Order article via Infotrieve]
  34. Latallo, Z. S., and Hall, J. A. (1986) Thromb. Res. 43, 507-521 [CrossRef][Medline] [Order article via Infotrieve]
  35. Craig, P. A., Olson, S. T., and Shore, J. D. (1989) J. Biol. Chem. 264, 5452-5461 [Abstract/Free Full Text]
  36. Gettins, P., Choay, J., Crews, B. C., and Zettlmeissl, G. (1992) J. Biol. Chem. 267, 21946-21953 [Abstract/Free Full Text]
  37. Fan, B., Turko, I. V., and Gettins, P. G. W. (1994) FEBS Lett. 354, 84-88 [CrossRef][Medline] [Order article via Infotrieve]
  38. Jordan, R. E., Oosta, G. M., Gardner, W. T., and Rosenberg, R. D. (1980) J. Biol. Chem. 255, 10081-10090 [Abstract/Free Full Text]
  39. Owen, B. A., and Owen, W. G. (1990) Biochemistry 29, 9412-9417 [Medline] [Order article via Infotrieve]
  40. Olson, S. T., Halvorson, H. R., and Björk, I. (1991) J. Biol. Chem. 266, 6342-6352 [Abstract/Free Full Text]
  41. Danielsson, Å., and Björk, I. (1978) Eur. J. Biochem. 90, 7-12 [Medline] [Order article via Infotrieve]
  42. Tollefsen, D. M. (1989) in Heparin. Chemical and Biological Properties. Clinical Applications (Lane, D. A., and Lindahl, U., eds) pp. 257-273, Edward Arnold, London
  43. Bray, B., Lane, D. A., Freyssinet, J. M., Pejler, G., and Lindahl, U. (1989) Biochem. J. 262, 225-232 [Medline] [Order article via Infotrieve]
  44. Gray, E., Cesmeli, S., Lormeau, J. C., Davies, A. B., and Lane, D. A. (1994) Thromb. Haemostasis 71, 203-207 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.