©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of the -

D

-Xyloside Naroparcil on Circulating Plasma Glycosaminoglycans

AN EXPLANATION FOR ITS KNOWN ANTITHROMBOTIC ACTIVITY IN THE RABBIT (*)

(Received for publication, July 25, 1994; and in revised form, November 14, 1994)

Philippe J. Masson (§) Dominique Coup Jean Millet Neil L. Brown

From the From Centre de Recherche et Développement, Laboratoires Fournier S.C.A., 50 rue de Dijon, BP 90, 21121 Daix, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-D-Xylosides are known to initiate or prime free glycosaminoglycan (GAG) chain synthesis in cell and tissue culture. As such, the effect of the venous antithrombotic beta-D-xyloside, naroparcil, was investigated on the plasma GAG profile in the rabbit after oral administration. Using dose-response experiments, we showed that antithrombin activity via antithrombin III and heparin cofactor II was increased in parallel with GAG plasma levels compared to control. A more detailed qualitative examination of plasma GAGs by cellulose acetate electrophoresis and ion-exchange chromatography, following oral administration of naroparcil at 400 mg/kg, revealed the presence of higher density charged molecules compared to control. The extracted GAGs were found to activate inhibition of thrombin by heparin cofactor II and contained approximately 25% of a dermatan sulfate-like compound (undetectable in control), which could be responsible for the antithrombotic effect. Using radiolabeled naroparcil, we found radiolabeled GAG fractions and the fact that naroparcil was a substrate for galactosyltransferase I, the second enzyme responsible for GAG chain polymerization, suggested that the compound could initiate in vivo the biosynthesis of antithrombotic free GAG chains. This is, to our knowledge, the first description of the in vivo effect of a beta-D-xyloside on GAG biosynthesis; furthermore, this is correlated with an antithrombotic action.


INTRODUCTION

Recently, we have reported the venous antithrombotic effects of two novel beta-D-xylosides following their oral administration to experimental animals(1, 2) . The product LF 1351 or (RS-4-(hydroxy(4-nitrophenyl)methyl)phenyl-beta-D-xylopyranoside was seen to prevent the formation of Wessler stasis type venous thrombi in rats following a single oral administration. This antithrombotic effect occurred without increases in either activated partial thromboplastin time or thrombin time(1) . Further chemical modification of LF 1351 gave rise to a more potent compound, naroparcil or 4-(4-cyanobenzoyl)phenyl)-1,5-dithio-beta-D-xylopyranoside, which demonstrated venous antithrombotic activity in the rabbit following single oral administration(2) . Similarly to LF 1351, the antithrombotic effect of naroparcil was not accompanied by prolongation of activated partial thromboplastin time or of thrombin time. Naroparcil treatments did, however, produce a dose-related reduction in thrombin generation via the intrinsic pathway in platelet-poor plasma, while at a high dose (400 mg/kg), it caused a reduction in sensitized thrombin times(3) .

Both these novel beta-D-xylosides produced their antithrombotic effects hours after administration, even following intravenous injection, suggesting that the compounds were probably not acting directly. For example, maximum antithrombotic activity was observed 2 h after intravenous administration and 4 h after oral administration for both compounds, independently of the animal species employed. In addition, both compounds were without effect in classical in vitro coagulation tests, even at high concentrations(1, 2) . Due to their structure, it was hypothesized that the compounds may exert their antithrombotic actions, at least partially, via the induction of free endogenous GAG (^1)biosynthesis. beta-D-Xylosides have been known for some considerable time to inhibit or block the in vitro formation of GAG chains on proteoglycans in favor of the induction of free GAG chains(4, 5, 6) . This is thought to occur through the exogenous beta-D-xyloside entering cells and competing with xylosated serines on the core proteins of the endogenous proteoglycans undergoing biosynthesis. In this way, the first galactose of the so-called linkage region is transferred by galactosyltransferase I to the exogenous beta-D-xyloside sugar, the final result being the biosynthesis of free GAGs on the exogenous beta-D-xyloside precursor(7, 8) .

The antithrombotic effects of GAG can be achieved by the catalysis of thrombin inhibition by ATIII alone, by HCII alone, or by both(9) . Fernandez et al.(10) have suggested that the catalysis of thrombin inhibition by in vitro or ex vivo GAGs could provide an index for estimating the antithrombotic potential of agents that have no or little effect on classical coagulation parameters, such as the activated partial thromboplastin time. Employing the technique of Fernandez et al.(10) , it was demonstrated that, 4 h following naroparcil treatment of rabbits, there was a dose-related increase in the formation of ex vivo complexes between human thrombin and (rabbit) HCII at the expense of complexes formed between thrombin and ATIII(3) . This GAG-like activity was further quantified in an assay (11) for HCII-mediated thrombin inhibition and expressed as dermatan sulfate-like material through the use of the appropriate standard curve(3) . These observations strengthened the hypothesis that treatment of rabbits with the beta-D-xyloside naroparcil induced an increase in plasma GAG levels and that these latter were, at least in part, responsible for the observed antithrombotic activity.

The purpose of the present study was to perform a more detailed analysis of the effects of naroparcil on the plasma GAG profile in the rabbit and on the precise nature of the GAGs formed. This study also represents, to our knowledge, the first description of the in vivo effects of a beta-D-xyloside on GAG biosynthesis.


EXPERIMENTAL PROCEDURES

Chemicals

All chemicals were of analytical grade. Carbazole, Pronase E type XIV, porcine skin DS, cetylpyridinium chloride, and Alcian blue 8GX were purchased from Sigma. D-Glucurono--lactone was from Merck. Chondroitin ABC and ACII lyases and unsaturated disaccharide standards were from Seikagaku Kogyo Co. Heparin (150 IU/mg) was from Terdobliate, Novara, Italy, and human ATIII was from Kabi. Naroparcil [4-(4-cyanobenzoyl)phenyl]-1,5-dithio-beta-D-xylopyranoside was synthesized by Laboratoires Fournier S.C.A, Dijon, France and [^14C]naroparcil (83.8 mCi/mM) by Isotopchim, Grenoble, France.

Treatment of Animals

All experiments were performed on male New Zealand White rabbits (1.7-2.3 kg, Charles River, France). Animals were used following 1 week of quarantine. Naroparcil (doses from 25 to 400 mg/kg) was suspended in polyethylene glycol 400 (Prolabo) and administered by gastric intubation in a volume of 3 ml/kg. Control animals received only the vehicle. Four h after compound administration, the animals were anesthetized (6%, w/v, sodium pentobarbital, Sanofi Santé Animale, France, 25 mg/kg intravenous), the carotid artery catheterized, and blood collected over citrate (1 volume of 3.8% (w/v) sodium citrate for 9 volumes of blood). After centrifugation (2400 times g, 20 min, 20 °C), the platelet-poor plasma was frozen (-70 °C) to await further analyses.

Isolation of Plasma GAGs

Plasma (20 ml) was incubated with a solution of Pronase E type XIV (8 mg in 2 ml of 1 M Tris-HCl buffer, pH 7.5, 50 mM CaCl(2)) for 48 h at 50 °C. An equal amount of Pronase was added at 24 h. The Pronase had been previously incubated for 10 min at 50 °C to eliminate susceptible glycosidic activity. Total proteolytic digestion was assessed by using the protein assay of Bradford(13) . Cold trichloracetic acid was added (10%, w/v, final concentration), and samples were kept overnight at 4 °C. Supernatant was recovered by centrifugation (15 min, 8000 times g, 4 °C), the pellet was washed with 1 ml of 10% (w/v) trichloracetic acid, and supernatants were pooled and dialyzed (Spectrapor 3, Polylabo) against 100 volumes of 0.1 M phosphate buffer, pH 6.5, for 48 h at 4 °C. The phosphate buffer was renewed three times during dialysis. Cetylpyridinium chloride was added to the dialysate (0.1% w/v final concentration), mixed, and allowed to stand overnight at room temperature. After centrifugation (15 min, 1500 times g, 20 °C), the precipitate was dissolved in 2 M NaCl. The sample was centrifuged again and supernatant removed. The last procedure was repeated twice. The three resulting supernatants were pooled and precipitated by addition of 5 volumes of 95% (v/v) ethanol for 24 h at 4 °C. The alcoholic precipitate was recovered by centrifugation (15 min, 1500 times g, 4 °C), dried under vacuum at room temperature, and dissolved in water or 0.9% NaCl. Aliquots were desalted on PD10 columns (Pharmacia Biotech Inc.) and lyophilized. The accuracy of the method was assessed by recovery of standard DS added to normal pooled plasma and was found to be 80%.

Uronic Acid Assay

The uronic acid (UA) content of the final GAG extract was determined using a modified method described by Bitter and Muir(14) . To frozen samples (0.2 ml) in 20 times 100-mm Pyrex® tubes kept in an ice bath, 2.5 ml of a carbazole reagent (0.0125%, w/v, carbazole, 0.9536% sodium tetraborate, w/v, in concentrated H(2)SO(4)) was added. Tubes were placed in a boiling water bath for 20 min. After cooling, the absorbance was measured at 530 nm. A standard curve was made with D-glucurono--lactone from 1 to 40 µg.

Sulfate Analysis

The sulfate content of purified GAGs was determined using the method of Terho and Hartiala(15) . Briefly, 500 µl of 1 N HCl was added to an equivalent of 30 µg of UA as a lyophilized sample. After hydrolysis for 90 min at 100 °C, the sample was dried under vacuum, then the residue was dissolved in 500 µl of H(2)O prior to the assay.

Cellulose Acetate Electrophoresis

GAGs, as well as their degradation products obtained after specific enzyme or chemical treatment, were visualized by electrophoresis on cellulose acetate strips (Sartorius) in 0.2 M calcium acetate buffer, pH 5 (16) . Separation was obtained by electrophoresis for 3 h at constant current (20 mA) on a Shandon apparatus. Samples (1 mg/ml UA) were deposited with a sample applicator (Pratiga) to obtain reproducible results. Strips were stained for 5 min with 0.5% (w/v) Alcian blue in 3% (v/v) acetic acid and then destained in 10% (v/v) acetic acid. After washing in deionized water and drying, stained strips were scanned using an autoscanner (Flur/Vis, Helena). Extracted GAGs (30 µg of UA/30 µl) were depolymerized by chondroitinase treatment for 2 h at 37 °C (0.06 unit of chondroitinase ABC in 50 mM Tris, 60 mM sodium acetate buffer, pH 8, 0.06 unit of chondroitinase ACII in 80 mM sodium acetate buffer, pH 6). The reaction was stopped by a 3-min incubation at 100 °C and the degradation products recovered by centrifugation (5 min at 10,000 times g, 20 °C).

Catalysis of Thrombin Inhibition

The Stachrom DS kit (Diagnostica Stago) was used to quantify thrombin inhibition mediated by HCII for both plasma and for extracted GAGs following the principle originally described by Dupouy et al.(11) . The procedures followed were those described in the kit except, when assessing the antithrombin potential of extracted GAGs, the DS standard curve was prepared in Michaelis buffer (25 mM sodium barbital, 40 mM sodium acetate, 100 mM NaCl, pH 7.3). The measurement of the catalysis of thrombin inhibition by ATIII was performed using the same technique by replacing the bovine HCII of the Stago kit with human ATIII (final concentration 1 IU/ml) and using unfractionated heparin (150 IU/mg) for the standard curve(12) . All assays were performed on an automated coagulation apparatus (ACL 300R, Instrumentation Laboratory) giving intra- and interassay coefficients of variations of <10%. The sensitivity of the assays were 0.05 µg/ml with a DS standard curve and 0.08 µg/ml with a heparin standard curve. All values are expressed in DS µg/ml for DS-like.

Detection of I-Thrombin/Thrombin Inhibitor Complexes

The catalysis of antithrombin-thrombin complex formation in plasma or by purified GAGs was investigated by the technique of Fernandez et al.(10) . Briefly, purified human alpha-thrombin (1120 NIH units/mg) was iodinated by an IODOGEN technique (17) to a specific activity of 1-4 µCi/NIH unit. Samples of defibrinated plasma or extracted GAGs in defibrinated plasma (18) were incubated with I-thrombin (1 NIH unit/ml final concentration) in 150 µl for 30 s at 37 °C. After stopping the reaction, samples (corresponding to about 30,000 cpm) were submitted to electrophoresis on 7.9% polyacrylamide gels containing 0.1% SDS, followed by autoradiography, to separate the complexes formed between the various endogenous antithrombins (ATIII, HCII, and alpha(2)-macroglobulin) and I-human alpha-thrombin.

Ion-exchange Chromatography

Isolated GAGs were chromatographed on a 10 times 50-mm DEAE-cellulose column (DE52 microgranular, Whatman) and eluted with a linear gradient of sodium chloride (0-1 M) in 50 mM Tris-HCl buffer, pH 6.5, at a flow rate of 18 ml/h. The separation was monitored by recording the absorbance at 214 nm with the aid of a UV1/214 detector (Pharmacia Biotech) or scintillation counting of aliquots. Two-milliliter fractions were collected and, after exhaustive dialysis, analyzed for uronic acid content, HCII-mediated antithrombin activity, and characterization by cellulose acetate electrophoresis.

Gel Filtration Chromatography

Purified fractions from ion-exchange chromatography were analyzed on a 16 times 100-mm Sephacryl S200HR column (Pharmacia Biotech) eluted with 0.1 M phosphate buffer, pH 6.5, 0.2 M NaCl at a flow rate of 20 ml/h and absorbance monitored at 214 nm. V(o) and V(t) were determined by chromatography of dextran blue and glucose respectively. A molecular mass calibration curve was obtained by chromatography of standard dextrans (Pharmacia Biotech) followed by assay of neutral sugars(19) .

Disaccharide Analysis

Analysis of disaccharides produced by enzymatic digestion of purified GAGs was performed by HPLC using a modified technique developed by Linhardt et al.(20) . HPLC was performed on a Dionex Bio L.C. consisting of a EDM degasser, a GPM pump and a VDM-II UV detector. Data were processed using a Dionex A.I. 450 data station with a Dionex model II advanced computer interface and an IBM PS/2 model 55SX computer. Samples (10-20 µl) were injected by a Kontron 765 autosampler. First, strong anion exchange-HPLC was performed on a 250 times 4.6-mm Partisil 10 column (Whatman), eluted by a linear gradient of sodium chloride (0-0.5 M over 90 min) in water adjusted to pH 3.5, at a flow rate of 1.5 ml/min. Second, polystyrene pellicular resin-based anion exchange columns from Dionex were used to increase resolution and to reduce elution times. A Carbopac PA-1 (4 times 250 mm, 10 µm) eluted isocratically with a mixture of sodium carbonate/sodium bicarbonate (0.6 mM/0.3 mM) allowed the separation of DeltaDi-0S and DeltaDi-0S in 15 min. DeltaDi-4S, DeltaDi-6S, and DeltaDi-UA2S were separated on an AS5A 5-µm column (4 times 250 mm) eluted isocratically with 12 mM sodium carbonate/6 mM sodium bicarbonate at a flow rate of 1 ml/min. Compounds were detected by absorbance at 232 nm.

Galactosyltransferase I Assay

The ability of naroparcil to initiate glycosaminoglycan synthesis was investigated by the technique described by Robinson and Robinson(21) , which measured the rate of galactose transfer from UDP-galactose to the beta-D-xyloside by the galactosyltransferase I. Briefly, the microsomal enzyme was prepared from embryonic chicken cartilage and, after incubation of the microsomal enzyme preparation with UDP-[^14C]galactose (DuPont NEN) and naroparcil, the reaction products were separated by paper chromatography. Sugars were detected on chromatograms by alkaline silver nitrate reagent and radiolabeled products and autoradiography. The latter were then cut out and assayed for radioactivity. The occurrence of the disaccharide (Gal-naroparcil) was confirmed by its beta-galactosidase susceptibility(21) . Galactosyltransferase I K(m) value for naroparcil was determined by dose-response experiments.

Statistical Analysis

Results were obtained in two separate studies regrouping a total of 37 untreated (control) and 34 treated animals. Statistical analysis of each parameter measured (analysis of variance) revealed no significant differences between the data obtained in the two studies; hence, the values reported are those of the two studies pooled together. Values are reported as means ± S.E. of n observations. Comparisons of parameters between naroparcil and vehicle-treated (control) animals were made by analysis of variance followed by Student's t test, if applicable, for comparisons of group means.


RESULTS

Antithrombin Activities and GAG Content

Oral administration of the beta-D-xyloside naroparcil produced dose-related increases in GAG content and in HCII-mediated antithrombin activity (DS-like activity) in plasma (Fig. 1). Furthermore, the endogenous HCII-mediated antithrombin activity of extracted GAGs showed a similar increase (Fig. 1). We found that there was an excellent linear correlation between HCII-mediated antithrombin activity in the plasma and increased GAG content (r = 0.984) as well as the extracted GAG-mediated antithrombin activity found in plasma (r = 0.983).


Figure 1: Dose-response curves of HCII-mediated antithrombin activity found in plasma (µg/ml DS, box) or from extracted plasma GAGs (µg/ml DS, ) and GAG content (µg/ml UA, ) obtained 4 h after oral administration of naroparcil.



A more detailed examination of the effects of naroparcil was made 4 h after the oral administration of a single dose of 400 mg/kg. This treatment produced a highly significant increase in plasma GAG content, in HCII-mediated antithrombin activity (DS-like activity), and in ATIII-mediated antithrombin activity (heparin-like activity) compared to vehicle-treated control animals (Table 1). Also, as seen for the dose response, there was also an increase in the DS-like activity of isolated GAGs expressed as µg of DS/ml of plasma (Table 1). Thus the calculated specific activity of isolated GAGs needed to inhibit thrombin via HCII (expressed as µg of DS/µg of uronic acid) was found to increase significantly compared to control values. The degree of sulfation of total plasma GAGs, undetectable in vehicle-treated animals, was increased by naroparcil treatment to about 2.5% or 2.5 µg/100 µg of uronic acid (Table 1).



Cellulose Acetate Electrophoresis

Extracted plasma GAGs from control and treated rabbits were analyzed by cellulose acetate electrophoresis with or without prior specific degrading treatments. Comparison of the electrophoresis pattern from control and treated animals (400 mg/kg naroparcil) clearly showed that the circulating GAG was modified 4 h after naroparcil treatment, with the appearance of higher density charged polysaccharides (Fig. 2). In control animals (panelA), enzymatic digestion by either chondroitinase ABC or AC abolished much of the GAG material obtained, whereas in naroparcil-treated animals (panelB), a fraction was found to persist after chondroitinase AC digestion, suggesting the presence of a DS-like moiety (Fig. 2). This was evaluated as representing about 18% of the total GAGs on the basis of Alcian blue staining intensity.


Figure 2: Cellulose acetate electrophoresis of plasma GAG before or after enzymatic digestion with chondroitinases ABC (+ABC-ase) or AC (+AC-ase) from control (A) or naroparcil-treated rabbits (B). In control rabbits (panel A) concentrations of GAG were 1 mg/ml UA without treatment and 500 µg/ml UA after chondroitinase treatments. In treated rabbits (panel B), the concentrations were 870 µg/ml UA (without treatment) and 470 µg/ml UA after enzymatic digestion (arrows indicate origin of loading).



Detection of I-Thrombin/Thrombin Inhibitor Complexes

Following incubation of citrated platelet-poor plasma obtained from naroparcil-treated rabbits with I-human alpha-thrombin, SDS-PAGE analysis revealed the presence of HCII-thrombin complexes (Fig. 3, panelA). In agreement with that seen for the cellulose acetate electrophoresis pattern (Fig. 2), HCII complexes were absent if the plasma was previously treated with chondroitinase ABC, but were unaffected by chondroitinase AC, indicating, once more, the presence of a DS-like molecule (Fig. 3, panel A). I-Thrombin-HCII complexes were poorly visualized when plasma obtained from vehicle-treated control animals was used, the major complex formed being that between thrombin and ATIII (Fig. 3, panel A, lane b).


Figure 3: SDS-PAGE analysis of antithrombin complexes. In panel A, rabbit plasma loaded with DS (50 µg/ml) and heparin (15 IU/ml) (lanes a and f), plasma from control and naroparcil-treated rabbits (400 mg/kg) (lanes b and c), and effect of chondroitinase ABC and AC on plasma from treated animals (lanes d and e). In panel B, rabbit plasma loaded with DS (100 µg/ml), extracted GAG from plasma of naroparcil-treated rabbits (200 µg/ml UA) and heparin (15 IU/ml) (lanes a, d, and g). Action of chondroitinases ABC and AC on HCII-thrombin complex potentialized by DS (lanes b and c) or GAG from treated rabbits (lanes e and f).



However, in Fig. 3(panel B), it is seen that the addition of isolated GAGs from treated rabbits to plasma from control animals catalyzed complex formation between I-thrombin and HCII at the expense of those formed with ATIII (Fig. 3). Furthermore, the enzymatic treatment of DS (lanes b (chondroitinase ABC) and c (chondroitinase AC)) gave a similar profile with that of the digestion of extracted GAGs from treated plasma (lanes e (chondroitinase ABC) and f (chondroitinase AC)), indicating the presence of DS-like material.

Chromatography

Isolated plasma GAGs were chromatographed on a DEAE-cellulose column (Fig. 4). GAGs from control animals showed only one principal peak eluted by 0.2 M NaCl (Fig. 4, panel A), whereas chromatography of GAGs from naroparcil-treated rabbits revealed the presence of four peaks eluted at 0.08, 0.18, 0.27, and 0.48 M NaCl, respectively (Fig. 4, panel B). The latter peak (0.48 M NaCl peak) contained more than 90% of HCII-mediated antithrombin activity such that the specific activity of this peak, 1.25 µg of DS/µg of UA, was 3-fold greater than that obtained by the total plasma. In addition, electrophoretic analyses following enzymatic digestion revealed that a DS-like moiety (i.e. chondroitinase ABC-sensitive but chondroitinase AC-insensitive) was only present in the higher charged peak (results not shown).


Figure 4: Ion-exchange chromatography of plasma GAG from control (A) and naroparcil-treated rabbits (B). Absorbance at 214 nm (box) and NaCl gradient (- - -).



Gel filtration of the 0.48 M NaCl peak on Sephacryl S200HR gave heterogeneous molecular masses ranging from 70,000 to 1,000 Da. By arbitrarily cutting the chromatogram into three fractions, giving average molecular masses of 52,000, 13,000, and 1,500 Da, it was seen that HCII-mediated antithrombin activity was principally distributed in the middle molecular mass fraction (61%) and to a lesser extent in the high molecular mass fraction (38.7%).

In order to investigate the involvement of naroparcil in GAG synthesis, animals were orally treated with [^14C]naroparcil (40 µCi) + 400 mg/kg unlabeled naroparcil. This led to the formation of extracted radiolabeled material, which on ion-exchange chromatography gave one broad radioactive peak with the maximum being eluted at 0.45 M NaCl (Fig. 5). This suggested that [^14C]naroparcil had been incorporated into GAG chains (Fig. 5).


Figure 5: Ion-exchange chromatography of plasma GAG from radiolabeled naroparcil-treated rabbits. radioactivity count in fractions (cpm/ml, box) and NaCl gradient(- - -).



Disaccharide Analysis

Fig. 6shows typical chromatograms from control (panel A) and treated animals (panel B) of chondroitinase ABC-digested plasma GAGs obtained on an AS5A column. Treatment with naroparcil increased the proportion of DeltaDi-4S and DeltaDi-6S in circulating GAGs, whereas the non-sulfated disaccharide was diminished ( Fig. 6and Table 2). The DS content, which was undetectable in control animals, was increased to 25% of total GAGs in treated animals.


Figure 6: HPLC chromatograms on AS5A column of disaccharides obtained after chondroitinase ABC digestion of plasma GAGs from control (A) or naroparcil-treated rabbits (B).





Galactosyltransferase I Assay

Naroparcil was found to act as an acceptor for the transfer of a galactose residue from UDP-galactose by a microsomal enzyme preparation of galactosyltransferase I. A typical Michaelis-Menten curve was obtained, giving a calculated K(m) value for naroparcil of 0.085 mM. The corresponding value of beta-D-xylose was 191 mM, while the alpha-D analog of naroparcil was not found to be an acceptor, suggesting a high degree of spatial or conformational restriction.


DISCUSSION

Oral administration of the beta-D-xyloside naroparcil into rabbits produced a clear increase in both plasma GAG content and antithrombin activity. This latter effect was essentially due to the catalysis of HCII-mediated thrombin inhibition, although ATIII-mediated thrombin inhibition was also increased, but giving only moderate levels of heparin-like activity. The heparin-like activity observed in the plasma from treated animals despite being low (0.76 µg/ml or 0.11 IU/ml), could be sufficient, as observed with low dose heparin administration(12, 22) , to cause an antithrombotic effect. This activity is unlikely to be due to heparan sulfate molecules being released into the plasma by the vessel wall, as observed after DS administration(23) . No heparan disaccharides could be detected by HPLC after heparinase digestion of the extracted GAG fraction (results not shown), although we could not exclude the possibility that the GAG extraction method may denature some activity or result in loss of low molecular weight material. Such heparin-like activity has been reported for pentosan polysulfate, DS, and chondroitin sulfate preparations and is probably not specific(24, 25) .

Of further importance was the observed correlation between the increase in HCII-mediated thrombin inhibitory capacity (DS-like activity) and plasma GAG (uronic acid) concentration in naroparcil-treated animals. It was apparent from our results that the former was due to an increase in the latter. Fernandez et al.(10) previously demonstrated dose-related increases in ex vivo alpha-thrombin-HCII complex formation occurred at the expense of alpha-thrombin-ATIII complexes following administration of dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate to rabbits. This capacity to catalyze thrombin inhibition was considered to provide evidence of the antithrombotic potential of glycosaminoglycans and other sulfated polysaccharides(10, 26) . Enzymatic digestion of plasma from rabbits having received naroparcil (400 mg/kg) revealed that the circulating material potentiating thrombin-HCII complex formation was chondroitinase ABC-sensitive and chondroitinase AC-insensitive, indicating the material was dermatan sulfate-like(27, 28) .

Plasma GAGs were quantitatively and qualitatively modified by naroparcil treatment, and the HCII-mediated antithrombin activity was found to be localized among these GAGs. Qualitative differences in plasma GAG profiles following naroparcil treatment were observed by cellulose acetate electrophoresis. The major differences in GAG profile between treated (400 mg/kg, naroparcil) and untreated rabbit plasma were the chondroitinase AC insensitivity, and the appearance of more highly charged molecules. This latter finding confirmed the observation that the SO content of extracted GAG, expressed as a percentage of uronic acid (µg of SO/100 µg of UA), increased from undetectable levels in control animals (<1%) to 2.5% following naroparcil treatment.

A similar pattern in GAG profile to that obtained by cellulose acetate electrophoresis was observed following ion-exchange chromatography. Naroparcil treatment induced the appearance of more highly charged material, notably a fraction eluting at 0.48 M NaCl, which contained about 90% of HCII-mediated antithrombin activity. This finding was further confirmed, employing radiolabeled naroparcil, in that only a peak eluting at 0.45 M NaCl was found. It was assumed that this material corresponded to that eluted at 0.48 M NaCl when using non-radiolabeled naroparcil.

This observation is in accordance with the assumption that newly synthesized free GAG chains were formed on the exogenous beta-D-xyloside naroparcil (see Introduction and below). However, the presence of less highly charged elements (eluting at 0.09 M, 0.18 M, and 0.27 M NaCl) in the plasma of treated animals as compared to control animals is more difficult to explain. These could be due to modifications of existing GAG chains, caused for example by naroparcil-induced inhibition of GAG metabolism, or modification of elimination rate. Alternatively, they could represent catabolized naroparcil-induced free GAG chains that have lost their terminal xyloside primer. Due to the extraction procedure employed, it is unlikely that these less charged elements represent fragments of the linkage region.

Whatever the exact nature of the material eluting at 0.45-0.48 M NaCl, the range of molecular mass from 1,000 to 70,000 Da is indicative of a heterogeneous mixture of similarly charged GAG that contain catalytic activity for thrombin inhibition. However, the activity is more apparent in the higher (>10,000 Da) molecular mass material. This observation is in agreement with the template or ternary complex model of oligosaccharide-catalyzed thrombin inhibition by HCII, whereby in addition to certain charge requirements, appreciable catalytic effects are only observed with polysaccharides of molecular mass geq 8,000 Da(29) . Certainly this is true for heparin, although for DS, the specific activity does not increase above tetradeca- (3,500 Da) or hexadecasaccharide (4,000 Da)(30, 31) .

HPLC analysis of the disaccharides resulting from the digestion of plasma GAG clearly demonstrate the modifications induced by naroparcil. The presence of the sulfated disaccharide DeltaDi-6S constituted the major modification, with a corresponding loss in DeltaDi-0S. Furthermore, 4S-DS, which was undetectable in untreated animals, increased to 25% following naroparcil treatment and thus represented more than half of the DeltaDi-4S present in treated plasma. The disaccharide profile observed in control animals was in complete accordance with recently published data(32) .

The present in vivo findings are in full agreement with previously published in vitro observations on free GAGs obtained following beta-D-xyloside priming. In the present study, in vitro experiments demonstrated that naroparcil was an acceptor for galactose, considerably more efficient than the natural xylose and hence had the potential of being a potent GAG primer. Numerous studies have demonstrated that, when provided with exogenous beta-D-xyloside, mammalian cells and tissues produce a majority of chondroitin sulfate type chains and relatively smaller amounts of heparan sulfate-like material(33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) . However, such generalized patterns are complicated by the use of protein synthesis inhibitors and can vary with differing concentrations of xyloside. For example, Rapraeger (45) demonstrated that low concentrations of methylumbelliferyl-beta-D-xyloside prevented chondroitin sulfate biosynthesis on the proteoglycan syndecan, whereas 10-fold higher concentrations also blocked that of heparan sulfate. Furthermore, Esko and co-workers (46, 47) recently observed that estradiol-beta-D-xyloside and 2-naphthol-beta-D-xyloside were efficient primers for heparan sulfate biosynthesis in wild type and mutant (pgsA-745 cells, lacking xylosyltransferase) Chinese hamster ovary cells, while, at low concentrations, mostly chondroitin sulfate was synthesized.

In the present study, a full analysis of plasma GAG was only performed at a single time point following a single dose (400 mg/kg) of naroparcil. However, as discussed, it is plausible that other doses or time points would provide different GAG profiles. These dose and time points were selected because they corresponded to the maximum observed antithrombotic and antithrombin effects. What remains consistent in all studies is that heparin GAG synthesis is only weakly induced by beta-D-xylosides, even in cells that normally produce heparin(43, 44) . These in vitro observations are confirmed by the present in vivo findings.

The GAG chains initiated on naroparcil probably modified the balance between procoagulant and anticoagulant activities present in the circulation or in the vessel wall. However, much of the mechanism still remains to be elucidated since beta-D-xyloside decreased ATIII binding on cultured endothelial cells, thus leading to a procoagulant effect and increased thrombotic potential(48, 49) .


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 33-80-44-75-42; Fax: 33-80-44-75-20.

(^1)
The abbreviations used are: GAG, glycosaminoglycan; ATIII, antithrombin III; DS, dermatan sulfate; HCII, heparin cofactor II; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; UA, uronic acid; DeltaDi-0S, 2-acetamido-2-deoxy-3-O-(betaD-gluco-4-enepyranosyluronic acid)-D-galactose; DeltaDi-0S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose; DeltaDi-4S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyl uronic acid)-4-O-sulfo-D-galactose; DeltaDi-6S, 2-acetamido-2-deoxy-3O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; DeltaDi-UA2S, 2-acetamido-2-deoxy-3-O-(2-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Béatrice Cremers for SDS-PAGE analysis and Anne Prigent for typing the manuscript.


REFERENCES

  1. Millet, J., Theveniaux, J., and Brown, N. L. (1992) Thromb. Haemostasis 67, 176-179 [Medline] [Order article via Infotrieve]
  2. Millet, J., Theveniaux, J., Vaillot, M., and Brown, N. L. (1992) Thromb. Res. 65, Suppl. 1, S158
  3. Millet, J., Theveniaux, J., and Brown, N. L. (1994) Thromb. Haemostasis 72, 874-879 [Medline] [Order article via Infotrieve]
  4. Okayama, M., Kimata, K., and Suzuki, S. (1973) J. Biochem. (Tokyo) 74, 1069-1073 [Medline] [Order article via Infotrieve]
  5. Schwartz, N. B., Galligani, L., Ho, P.-L., and Dorfman, A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4047-4051 [Abstract]
  6. Robinson, H. C., Brett, M. J., Tralaggan, P. J., Lowther, D. A., and Okayama, M. (1975) Biochem. J. 148, 25-34 [Medline] [Order article via Infotrieve]
  7. Galligani, L., Hopwood, J., Schwartz, N. B., and Dorfman, A. (1975) J. Biol. Chem. 250, 5400-5406 [Abstract]
  8. Schwartz, N. B. (1979) J. Biol. Chem. 254, 2271-2277 [Medline] [Order article via Infotrieve]
  9. Hemker, H. C., and Béguin, S. (1991) Semin. Thromb. Hemostasis 17, 29-34 [Medline] [Order article via Infotrieve]
  10. Fernandez, F. A., Buchanan, M. R., Hirsh, J., Fenton, J. W., and Ofosu, F. A. (1987) Thromb. Haemostasis 57, 286-293 [Medline] [Order article via Infotrieve]
  11. Dupouy, D., Sié, P., Dol, F., and Boneu, B. (1988) Thromb. Haemostasis 60, 236-239 [Medline] [Order article via Infotrieve]
  12. Amar, J., Caranobe, C., Sié, P., and Boneu, B. (1990) Br. J. Haematol. 76, 94-100 [Medline] [Order article via Infotrieve]
  13. Bradford, M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  14. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330-334
  15. Terho, T. T., and Hartiala, K. (1971) Anal. Biochem. 41, 471-476 [Medline] [Order article via Infotrieve]
  16. Seno, N., Anno, K., Kondo, K., and Saito, S. (1970) Anal. Biochem. 37, 197-201 [Medline] [Order article via Infotrieve]
  17. Jandrot-Perrus, M., Didry, D., Guillin, M.-C., and Nurden, A. T. (1988) Eur. J. Biochem. 174, 359-367 [Abstract]
  18. Ofosu, F. A., Modi, G. J., Smith, L. M., Cerskus, A. L., Hirsh, J., and Blajchman, M. A. (1984) Blood 64, 727-747 [Abstract]
  19. Scott, A., and Melvin, E. H. (1953) Anal. Biochem. 25, 1656-1661
  20. Linhardt, R. J., Gu, K. N., Loganathan, D., and Carter, S. R. (1989) Anal. Biochem. 181, 288-296 [Medline] [Order article via Infotrieve]
  21. Robinson, J. A., and Robinson, H. C. (1981) Biochem. J. 194, 839-846 [Medline] [Order article via Infotrieve]
  22. Gensini, G. F., Bonechi, F., Gori, A. M., Fortini, A., Paniccia, R., Lamberti, R., Attanasio, M., Martini, F., Prisco, D., and Neri Serneri, G. G. (1990) Haemostasis 20, Suppl. 1, 129-131 [Medline] [Order article via Infotrieve]
  23. Van Ryn-McKenna, J., Ofosu, F. A., Gray, E., Hirsh, J., and Buchanan, M. R. (1989) Ann. N. Y. Acad. Sci. 556, 304-312 [Abstract]
  24. Fischer, A.-M., Barrowcliffe, T. W., and Thomas, D. P. (1982) Thromb. Haemostasis 47, 104-108 [Medline] [Order article via Infotrieve]
  25. Scully, M. F., Ellis, V., Seno, N., and Kakkar, V. V. (1988) Biochem. J. 254, 547-551 [Medline] [Order article via Infotrieve]
  26. Ofosu, F. A., Fernandez, F., Anvari, N., Caranobe, C., Dol, F., Cadroy, Y., Petitou, M., Mardiguian, J., Sié, P., and Boneu, B. (1988) Thromb. Haemostasis 60, 188-192 [Medline] [Order article via Infotrieve]
  27. Murata, K. (1980) in Methods in Carbohydrate Chemistry (Whistler, R. L., and BeMiller, J. N., eds) Vol. 8, pp. 81-88, Academic Press, New York
  28. Linhardt, R. J., Galliher, P. M., and Cooney, C. L. (1986) Appl. Biochem. Biotechnol. 12, 135-176 [Medline] [Order article via Infotrieve]
  29. Bray, B., Lane, D. A., Freyssinet, J.-M., Pejler, G., and Lindahl, U. (1989) Biochem. J. 262, 225-232 [Medline] [Order article via Infotrieve]
  30. Tollefsen, D. M., Peacock, M. E., and Monafo, W. J. (1986) J. Biol. Chem. 261, 8854-8858 [Abstract/Free Full Text]
  31. Sié, P., Dupouy, D., Caranobe, C., Petitou, M., and Boneu, B. (1993) Blood 81, 1771-1777 [Abstract]
  32. Imanari, T., Toyoda, H., Yamanashi, S., Shinomiya, K., Koshiishi, I., and Oguma, T. (1992) J. Chromatogr. 574, 142-145 [Medline] [Order article via Infotrieve]
  33. Murray, E., Scott-Burden, T., Ferguson, P., and Gevers, W. (1983) Biochim. Biophys. Acta 763, 299-308 [Medline] [Order article via Infotrieve]
  34. Robinson, J., and Gospodarowicz, D. (1984) J. Biol. Chem. 259, 3818-3824 [Abstract/Free Full Text]
  35. Gordon, P. B., Zanger, D. R., and Hatcher, V. B. (1986) Carbohydr. Res. 151, 121-134 [CrossRef][Medline] [Order article via Infotrieve]
  36. Nagasaka, T., Sobue, M., Niwa, M., Yasui, C., Nara, Y., Fukatsu, T., Nakashima, N., and Takeuchi, J. (1989) Exp. Hematol. 17, 923-928 [Medline] [Order article via Infotrieve]
  37. Kolset, S. O., Sakurai, K., Ivhed, I., Øvervatn, A., and Suzuki, S. (1990) Biochem. J. 265, 637-645 [Medline] [Order article via Infotrieve]
  38. Tagaki, K., Nakamura, T., Kon, A., Tamura, S., and Endo, M. (1991) J. Biochem. (Tokyo) 109, 514-519 [Abstract]
  39. Cöster, L., Hernnäs, J., and Malmström, A. (1991) Biochem. J. 276, 533-539 [Medline] [Order article via Infotrieve]
  40. Fransson, L.-A., Havsmark, B., Sakurai, K., and Suzuki, S. (1992) Glycoconjugate J. 9, 45-55 [Medline] [Order article via Infotrieve]
  41. Kanwar, Y. S., Hascall, V. C., Jakubowski, M. L., and Gibbons, J. T. (1984) J. Cell Biol. 99, 715-722 [Abstract]
  42. Sobue, M., Habuchi, H., Ito, K., Yonekura, H., Oguri, K., Sakurai, K., Kamohara, S., Ueno, Y., Noyori, R., and Suzuki, S. (1987) Biochem. J. 241, 591-601 [Medline] [Order article via Infotrieve]
  43. Stevens, R. L., and Austen, K. F. (1982) J. Biol. Chem. 257, 253-259 [Abstract/Free Full Text]
  44. Stevens, R. L., Razin, E., Austen, K. F., Hein, A., Caulfield, J. P., Seno, N., Schmid, K., and Akiyama, F. (1983) J. Biol. Chem. 258, 5977-5984 [Free Full Text]
  45. Rapraeger, A. (1989) J. Cell Biol. 109, 2509-2518 [Abstract]
  46. Lugemwa, F. N., and Esko, J. D. (1991) J. Biol. Chem. 266, 6674-6677 [Abstract/Free Full Text]
  47. Fritz, T., Lugemwa, F. N., Sarkar, A. K., and Esko, J. D. (1994) J. Biol. Chem. 269, 300-307 [Abstract/Free Full Text]
  48. Shimada, K., and Ozawa, T. (1987) Arteriosclerosis 7, 627-636 [Abstract]
  49. Shimada, K., Kobayashi, M., Kimura, S., Nishinaga, M., Takeuchi, K., and Ozawa, T. (1991) Jpn. Circ. J. 55, 1016-1021 [Medline] [Order article via Infotrieve]

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