(Received for publication, July 25, 1994; and in revised form, November 14, 1994)
From the
-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
-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
-D-xyloside on GAG biosynthesis; furthermore, this is
correlated with an antithrombotic action.
Recently, we have reported the venous antithrombotic effects of
two novel -D-xylosides following their oral
administration to experimental animals(1, 2) . The
product LF 1351 or (RS-4-(hydroxy(4-nitrophenyl)methyl)phenyl-
-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-
-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
-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 (
)biosynthesis.
-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
-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
-D-xyloside
sugar, the final result being the biosynthesis of free GAGs on the
exogenous
-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 -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
-D-xyloside on GAG biosynthesis.
Figure 1:
Dose-response curves of HCII-mediated
antithrombin activity found in plasma (µg/ml DS, ) 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).
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).
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.
Figure 4:
Ion-exchange chromatography of plasma GAG
from control (A) and naroparcil-treated rabbits (B).
Absorbance at 214 nm () 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 [C]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
[
C]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, ) and NaCl gradient(- -
-).
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).
Oral administration of the -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 -thrombin-HCII complex formation
occurred at the expense of
-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
-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 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 Di-6S constituted the major
modification, with a corresponding loss in
Di-0S. Furthermore,
4S-DS, which was undetectable in untreated animals, increased to 25%
following naroparcil treatment and thus represented more than half of
the
Di-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
-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
-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-
-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-
-D-xyloside and
2-naphthol-
-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
-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 -D-xyloside decreased ATIII binding on
cultured endothelial cells, thus leading to a procoagulant effect and
increased thrombotic potential(48, 49) .