THROMBOSIS RESEARCH 67; 201-211,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
PHARMACOLOGICAL
F.M.
PROFILE
Santoro
(l),
OF
R.
A
NATIVE
Alvarez,
DERMATAN
F.
SULFATE
Fussi.
y Avellaneda Research Laboratories, Syntex S.A. - Prayones 1838 Luis Guill6n - Pcia. Buenos Aires - Argentina.
(Received
20.9.1991;
accepted
in revised form 56.1992
s/nr.
by Editor M.B. Donati)
ABSTRACT
We performed different "in vivo" investigations to study native DS: antithe pharmacolagical, properties of a model, bleeding potential by thrombosis by the stasis bleeding time and template bleeding tail transection and profibrinolysis by a growing thrombus model and time, by an established thrombus model. The results suggest that DS is a safe antithrombotic drug by i.v. administradoses tion without bleeding potential, even at very high shown a protective index of at (up to 16 mg/Kg). DS has protectleast 4 in contrast to heparin that has shown a models so far studied ive index of 1. The profibrinolytic did not evidence a clear profibrinolytic contribution to prothe antithrombotic properties of DS, but showed a action that cannot be explained longed antithrombotic only by the heparin cofactor II potentiation.
INTRODUCTION
Dermatan Sulfate (DS) is a natural glycosaminoglycan widely distributed in tissues, which is constituted by repeating units of uranic acids (mainly L-iduronic acid) and N-acetyl-D-galactosamine (1). DS strongly accelerates the inhibition of thrombin
Key words: Dermatan fibrinolysis.
sulfate,
heparin,
(1) Correspondence must be addressed rios Andromaco - Av. Ingeniero Huergo Argentina.
201
antithrombosis,
to Dr. Santoro 1145 - Buenos
bleeding,
Laborato(1107) Aires
202
NATIVE DERMATAN SULFATE
Vol. 67, No. 2
serpin called Heparin 111 (21. by a plasma Cofactor II (HC DS has a weak anticoagulant activity comparison with heparin, is almost exclusively supported by plasma (31. This activity Il. DS has been shown to be effective in the prevention thrombosis by the Wessler-type model (venous stasis/ lability model) (4 - 61.
In in HC
of venous hypercoagu-
Nevertheless, little information has been published regarding the effects of DS in a rat venous stasis model (71 and, as far as we index has know, no thera#eutic been settled down in this model. On the other hand, the effect thrombus of DS on the established and on the growing thrombus has not been fully analysed. The aim of the present work was to study the effect thrombus dynamics as well as its therapeutic index.
MATERIALS
AND
of DS on
the
METHODS
Animals Male Wistar rats of 200/300 g. body weight were used. The animals light/dark cycle (7 a.m. to 7 p.m. were maintained on a 12 h. deg.C. light) with free access to food and water, at constant 22 temperature. the animals Before being used, antithrombotic model the animals 100 mg/Kg and xilasine 10 mg/Kg,
were were ip.
ovtrnight. fasted anaesthetized with
the For ketamine
Materials The DS extracted from bovine mucosa was produced by Syntex S.A.. the method of according to electrophoretic analysis, performed exa single band. Sulphur content Capelletti et al (81, showed pressed as S was 6%; dS= -600. The DS thus obtained was not submitted to nitrous acid treatment. Epsilon aminocaproic acid (EACAl Unfractionated available sources. produced by Syntex S.A..
Venous
thrombosis
was obtained heparin from
commercially from was bovine mucosa
model
disinborder of the thigh was The ventral skin from the costal abdoma 4 cm long incision was made on the fected with alcohol; border. from the costal median line, starting inal wall on the the length under cava was isolated for 1 cm The inferior vena placed for 1 min, then removed A tight clip was renal arteries. (surgical and a tight ligature was applied using a cotton thread
Vol. 67, No. 2
NATIVE DERMATAN SULFATE
thread) of 0,521 mm, immediately The abdominal wall was subsequently
below the closed.
203
renal
vein
branches.
at the chosen interval (2 For monitoring of thrombus formation, the hours) the abdomen was reopened under anaesthesia (ether), vena cava (2 cm below the ligature) and the collateral branches clamped. longitudinally with scissors The vascular segment was then cut and the thrombus removed, rinsed in distilled water, blotted on filter paper and placed 24 hours in a vacuum essicator for measwere or physiological salt solution urement of dry weight. DS administered ten minutes before vena cava ligature by intravenous route (i.v.).
Bleeding
models
placed in a Template bleeding time: Unanaesthetized rats were the from one of which metallic cylinder with several openings using Template bleeding time was measured animal's tail emerged: was a standardized device (Simplate-Organon-Teknika). The device part of the tail between 4 applied longitudinally on the dorsal care to avoid large veins. and 6 cm from the tip, taking with filter The blood was blotted every 30 seconds bleeding completely stopped (no rebleeding within 30 or physiological salt solution were administered ten fore the standardized incision by i.v.. Tail transection bleeding time: Unanaesthetized with several openings in a metallic cylinder the animal's tail emerged.
paper until seconds). DS beminutes
rats were from one of
from one mm and minutes until paper seconds).
The tail was cut transversely, using a bistoury, at for three the end of the tail. The rats were left 30 seconds with filter then blood was blotted every bleeding completely stopped (no rebleeding within 30
Profibrinolytic Established bosis model Six hours with DS.
placed which
models thrombus with the
after
the
model (9): It is based following modifications: vena
The thrombus formation tion according to the
cava
ligature,
the
on
the
animals
was monitored one hour after venous thrombosis model.
Growing thrombus model (10): It is based model with the following modifications:
on
the
venous
were
the
venous
throm-
in.iected
DS
injec-
thrombosis
204
NATIVE DERMATAN SULFATE
Two hours after the vena with DS or physiological g/ Kg.
the animals were injected cava ligature, without EACA 1 solution with or salt
The
thrombus formation was (6 hours) according to the
monitored at the chosen venous thrombosis model.
Statistical analyses: To analyse the following tests: Student "t" Keuls test. The
results
are
expresed
Vol. 67, No. 2
as
interval
time
the present results we have used Student-Newman test, Anova, and
g i/-
SE.
RESULTS was 1) Venous thrombosis model: The antithrombotic effect of DS thrombotic evaluated by the thrombus weight reduction and by the protection factor (TPF). The TPF (11) is obtained by using the (%I thromformula: thrombotic occurrence of control animals botic occurrence of treated animals (%)/thrombotic occurrence of control animals (%I. The incidence of thrombosis in the control tight up to 95% due to the inclusion of a vena cava ligature (12). which adds another to the stasis venous model.
increased groups was clip previous to the challenge thrombotic
THRQMBUSINHIBITION BY DS
0 0
2
6
4
D'S(t.iGl(
PHARMACOLOGICAL
F.M.
PROFILE
Santoro
(l),
OF
R.
A
NATIVE
Alvarez,
DERMATAN
F.
SULFATE
Fussi.
y Avellaneda Research Laboratories, Syntex S.A. - Prayones 1838 Luis Guill6n - Pcia. Buenos Aires - Argentina.
(Received
20.9.1991;
accepted
in revised form 56.1992
s/nr.
by Editor M.B. Donati)
ABSTRACT
We performed different "in vivo" investigations to study native DS: antithe pharmacolagical, properties of a model, bleeding potential by thrombosis by the stasis bleeding time and template bleeding tail transection and profibrinolysis by a growing thrombus model and time, by an established thrombus model. The results suggest that DS is a safe antithrombotic drug by i.v. administradoses tion without bleeding potential, even at very high shown a protective index of at (up to 16 mg/Kg). DS has protectleast 4 in contrast to heparin that has shown a models so far studied ive index of 1. The profibrinolytic did not evidence a clear profibrinolytic contribution to prothe antithrombotic properties of DS, but showed a action that cannot be explained longed antithrombotic only by the heparin cofactor II potentiation.
INTRODUCTION
Dermatan Sulfate (DS) is a natural glycosaminoglycan widely distributed in tissues, which is constituted by repeating units of uranic acids (mainly L-iduronic acid) and N-acetyl-D-galactosamine (1). DS strongly accelerates the inhibition of thrombin
Key words: Dermatan fibrinolysis.
sulfate,
heparin,
(1) Correspondence must be addressed rios Andromaco - Av. Ingeniero Huergo Argentina.
201
antithrombosis,
to Dr. Santoro 1145 - Buenos
bleeding,
Laborato(1107) Aires
202
NATIVE DERMATAN SULFATE
Vol. 67, No. 2
serpin called Heparin 111 (21. by a plasma Cofactor II (HC DS has a weak anticoagulant activity comparison with heparin, is almost exclusively supported by plasma (31. This activity Il. DS has been shown to be effective in the prevention thrombosis by the Wessler-type model (venous stasis/ lability model) (4 - 61.
In in HC
of venous hypercoagu-
Nevertheless, little information has been published regarding the effects of DS in a rat venous stasis model (71 and, as far as we index has know, no thera#eutic been settled down in this model. On the other hand, the effect thrombus of DS on the established and on the growing thrombus has not been fully analysed. The aim of the present work was to study the effect thrombus dynamics as well as its therapeutic index.
MATERIALS
AND
of DS on
the
METHODS
Animals Male Wistar rats of 200/300 g. body weight were used. The animals light/dark cycle (7 a.m. to 7 p.m. were maintained on a 12 h. deg.C. light) with free access to food and water, at constant 22 temperature. the animals Before being used, antithrombotic model the animals 100 mg/Kg and xilasine 10 mg/Kg,
were were ip.
ovtrnight. fasted anaesthetized with
the For ketamine
Materials The DS extracted from bovine mucosa was produced by Syntex S.A.. the method of according to electrophoretic analysis, performed exa single band. Sulphur content Capelletti et al (81, showed pressed as S was 6%; dS= -600. The DS thus obtained was not submitted to nitrous acid treatment. Epsilon aminocaproic acid (EACAl Unfractionated available sources. produced by Syntex S.A..
Venous
thrombosis
was obtained heparin from
commercially from was bovine mucosa
model
disinborder of the thigh was The ventral skin from the costal abdoma 4 cm long incision was made on the fected with alcohol; border. from the costal median line, starting inal wall on the the length under cava was isolated for 1 cm The inferior vena placed for 1 min, then removed A tight clip was renal arteries. (surgical and a tight ligature was applied using a cotton thread
Vol. 67, No. 2
NATIVE DERMATAN SULFATE
thread) of 0,521 mm, immediately The abdominal wall was subsequently
below the closed.
203
renal
vein
branches.
at the chosen interval (2 For monitoring of thrombus formation, the hours) the abdomen was reopened under anaesthesia (ether), vena cava (2 cm below the ligature) and the collateral branches clamped. longitudinally with scissors The vascular segment was then cut and the thrombus removed, rinsed in distilled water, blotted on filter paper and placed 24 hours in a vacuum essicator for measwere or physiological salt solution urement of dry weight. DS administered ten minutes before vena cava ligature by intravenous route (i.v.).
Bleeding
models
placed in a Template bleeding time: Unanaesthetized rats were the from one of which metallic cylinder with several openings using Template bleeding time was measured animal's tail emerged: was a standardized device (Simplate-Organon-Teknika). The device part of the tail between 4 applied longitudinally on the dorsal care to avoid large veins. and 6 cm from the tip, taking with filter The blood was blotted every 30 seconds bleeding completely stopped (no rebleeding within 30 or physiological salt solution were administered ten fore the standardized incision by i.v.. Tail transection bleeding time: Unanaesthetized with several openings in a metallic cylinder the animal's tail emerged.
paper until seconds). DS beminutes
rats were from one of
from one mm and minutes until paper seconds).
The tail was cut transversely, using a bistoury, at for three the end of the tail. The rats were left 30 seconds with filter then blood was blotted every bleeding completely stopped (no rebleeding within 30
Profibrinolytic Established bosis model Six hours with DS.
placed which
models thrombus with the
after
the
model (9): It is based following modifications: vena
The thrombus formation tion according to the
cava
ligature,
the
on
the
animals
was monitored one hour after venous thrombosis model.
Growing thrombus model (10): It is based model with the following modifications:
on
the
venous
were
the
venous
throm-
in.iected
DS
injec-
thrombosis
204
NATIVE DERMATAN SULFATE
Two hours after the vena with DS or physiological g/ Kg.
the animals were injected cava ligature, without EACA 1 solution with or salt
The
thrombus formation was (6 hours) according to the
monitored at the chosen venous thrombosis model.
Statistical analyses: To analyse the following tests: Student "t" Keuls test. The
results
are
expresed
Vol. 67, No. 2
as
interval
time
the present results we have used Student-Newman test, Anova, and
g i/-
SE.
RESULTS was 1) Venous thrombosis model: The antithrombotic effect of DS thrombotic evaluated by the thrombus weight reduction and by the protection factor (TPF). The TPF (11) is obtained by using the (%I thromformula: thrombotic occurrence of control animals botic occurrence of treated animals (%)/thrombotic occurrence of control animals (%I. The incidence of thrombosis in the control tight up to 95% due to the inclusion of a vena cava ligature (12). which adds another to the stasis venous model.
increased groups was clip previous to the challenge thrombotic
THRQMBUSINHIBITION BY DS
0 0
2
6
4
D'S(t.iGl(
