1171
Inhibitory Effect of Clopidogrel on Platelet
Adhesion and Intimal Proliferation After
Arterial Injury in Rabbits
J.M. Herbert, A. Tissinier, G. Defreyn, and J.P. Maffrand
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The possible activity of ticlopidine and its analogue clopidogrel in early atherogenesis was investigated.
Incubation of rabbit platelets with the extracellular matrix produced by endothelial cells in culture
induced massive platelet adherence in vitro. This phenomenon was strongly reduced when platelets were
isolated from rabbits that had been treated with a single dose of clopidogrel (10 mg/kg PO) or three doses
of ticlopidine (each 200 mg/kg PO) (94% and 56% inhibition of platelet adhesion, respectively). Three
doses of aspirin (each 200 mg/kg PO) were ineffective. Air-drying injury of the rabbit carotid artery
resulted in platelet adherence to the underlying subendothelium. This platelet accumulation on the
damaged vessel wall was greatly reduced by clopidogrel: 95% (P<.001) inhibition of platelet adhesion after
a single oral dose of 25 mg/kg. Ticlopidine (200 mg/kg) was also effective (71% inhibition; / > <.001),
whereas aspirin (100 mg/kg PO) failed to reduce platelet adhesion to the subendothelium. The effect of
clopidogrel on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to air-drying
endothelial injury was then investigated. After a 16-day treatment, clopidogrel (25 mg/kg per day PO)
inhibited the development of intimal thickening (48% inhibition; P<.01). This effect was dose dependent
and increased with the duration of the treatment. Under the same experimental conditions, ticlopidine
(200 mg/kg per day PO) inhibited myointimal thickening (57%; P<.001), whereas aspirin was ineffective.
These results show that clopidogrel and ticlopidine, two ADP-selective antiplatelet agents, can reduce
myointimal thickening after endothelial injury. This effect can be due to the inhibition of platelet adhesion
and aggregation to the exposed subendothelium. (Arteriosclerosis and Thrombosis 1993;13:1171-1179)
KEY WORDS • clopidogrel •
injury • rabbits
platelets •
T
he phenomenon of restenosis, which can occur
after mechanical dilatation of a coronary stenosis by means of a percutaneous transluminal
coronary angioplasty (PTCA), needs to be studied to
determine the clinical success of this intervention therapy in ischemic heart disease.1-3 To aid in the prevention
of restenosis occurring after PTCA, a number of antiplatelet drugs such as dipyridamole/aspirin, aspirin
alone, or ticlopidine have been widely administered
during or shortly after PTCA.4 7 Until now, none of
these compounds have reduced the restenosis that
occurs in 30% to 40% of the PTCA procedures performed in humans.
The process of intimal thickening in an injured artery
is the consequence of smooth muscle cell (SMC) proliferation and migration from the media to the intima.8
The specific role of platelets in this cellular proliferation
has been suggested by work on drugs affecting platelet
aggregation, although the general contribution of platelets and thrombosis to the development of atherosclerotic plaques has been acknowledged for a long time.8
Received November 11, 1992; revision accepted February 18,
1993.
From the Haemobiology Research Department, Sanofi Recherche, Toulouse, France.
Correspondence to Haemobiology Research Department,
Sanofi Recherche, 195, Route d'Espagne, 31036 Toulouse Cedex,
France (Dr Herbert).
adhesion •
smooth muscle cells •
proliferation
•
Previous investigations suggested that platelet adherence to the thrombogenic subendothelium, followed by
extensive aggregation and secretion of platelet factors,
may provide the mitogenic stimulus for dormant SMCs
in the arterial wall, and it has been recently suggested
that platelets regulate the movement of SMCs into the
intima but that they do not play a role in the initiation
of proliferation.9
The hypothesis that intimal hyperplasia results from
vessel injury and platelet responses to the exposed
subendothelium is supported by experiments in animals.
Rabbits on a normal diet developed fibromuscular
plaques in response to de-endothelialization with a
balloon catheter. This was completely inhibited if the
animals were made severely thrombocytopenic with
antiplatelet antiserum.10 SMC proliferation induced in
animals by endothelial injury was reported to be inhibited by treatment with antiaggregating drugs such as a
thiazole compound1112 or ticlopidine.13 Antiplatelet
therapy with a combination of dipyridamole and aspirin
has also been effective in reducing intimal hyperplasia
in response to coronary artery and femoropopliteal
bypass grafts in dogs and nonhuman primates 1418 and in
slowing the progression of peripheral arterial disease.19
These observations are therefore inconsistent with the
effect of these same compounds on restenosis occurring
after PTCA in humans. There may be a number of
explanations for the difference between these re-
1172
Arteriosclerosis and Thrombosis Vol 13, No 8 August 1993
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sponses, a possible explanation being the low antiadhesive effect of all of these compounds in humans. Indeed,
this latter effect appears to be of particular importance
in the initiation of restenosis, since another model
frequently cited to support platelet involvement in
atherosclerosis is the swine homozygous for von Willebrand's disease. These pigs have prolonged bleeding
times20 and a deficiency of the von Willebrand factor
necessary for normal platelet adherence to the subendothelial tissue.21 These swine are less susceptible to
aortic atherosclerosis either induced by atherogenic diet
alone22 or associated with balloon endothelial injury.21
The close interactions between platelets and the
response to vascular injury suggest that drugs that
strongly inhibit platelet functions, such as adhesion
and/or aggregation, should reduce the extent of lesion
development after injury. The aim of this study was to
examine the effect of clopidogrel, a ticlopidine analogue23 and a powerful inhibitor of ADP-induced platelet aggregation, on platelet adherence to the subendothelium in vitro and in vivo and to evaluate its activity
on myointimal proliferation after air-drying injury of
the rabbit carotid artery.
Methods
In Vitro Experiments
Corneal endothelial cell cultures and preparation of the
extracellular matrix (ECM). Cultures of bovine corneal
endothelial cells were established from steer eyes as
described.24 Cells were routinely cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with
5% newborn calf serum, 5% fetal calf serum (FCS), 4
mmol/L glutamine, 100 /ug/mL streptomycin sulfate,
and 100 IU/mL penicillin at 37°C in 10% CO2 humidified incubators. Cells were passaged weekly at a split
ratio of 1:4, and 1 ng/mL basic fibroblast growth factor
(Amersham, France) was added every other day. Corneal endothelial cells at the third passage were plated at
an initial density of 1.8x10" cells per well in 24-well
cluster plates (Nunc, Roskilde, Denmark) and cultured
under the same conditions as described above except
that 5% dextran T-40 (Sigma) was added in the growth
medium. Eight days after confluency was reached, the
cell layer was washed twice with phosphate-buffered
saline (PBS) and exposed to 0.5% Triton X-100 in PBS
(vol/vol) for 30 minutes under gentle shaking. The
remaining cytoskeleton and nuclei were removed by a
2-minute exposure to 0.025 mol/L NH4OH followed by
extensive washing with water. ECM-coated multiwell
plates containing PBS were stored before use at 4°C for
periods of up to 3 months.
Platelet adhesion to the ECM. Platelet adhesion to the
ECM was measured according to the procedure described by Vlodavsky et al.25 Briefly, rabbit platelets
(~4xlO 9 cells/mL) in acid-citrate-dextrose (ACD; 9
mmol/L citric acid, 1.75 mmol/L sodium dihydrogen
citrate, and 5.6 mmol/L dextrose)-anticoagulated platelet-rich plasma (PRP) were incubated for 15 minutes at
37°C with 1 mCi "'InCl (114 mCi/mmol) (Amersham)
and tropolone (30 fig/mL). Labeling efficiency was 80%.
The labeled platelets were washed once in ACD-PRP to
remove unbound radioactivity and were resuspended in
platelet-poor plasma prepared from citrated blood.
Ninety-five percent of the counts were found to be
associated with the platelet fraction, and the plasma
radioactivity did not exceed 5%, even after 3 hours'
incubation.
To test the ex vivo effect of clopidogrel and ticlopidine on platelet interaction with the ECM, 300 /xL
'"In-labeled platelets (106 cells/;u,L) from control or
treated rabbits were incubated in ECM-coated wells.
After 60 minutes at 25°C, unbound platelets were
aspirated and the ECM was washed extensively with
PBS. The ECM-bound platelets were then solubilized
with IN NaOH (1 mL/well), and the radioactivity of the
lysate was measured in a gamma counter (model MR
480, Kontron).
Rabbit aortic SMC growth in vitro. SMCs were isolated
from the rabbit aorta as described previously.26 Briefly,
media fragments of the thoracic aorta from New
Zealand White rabbits (2.0 to 2.5 kg; Charles River)
were incubated for 16 hours at 37°C in DMEM containing 0.15% collagenase, 5% FCS, penicillin (100 IU/mL),
streptomycin (100 /xg/mL), and glutamine (4 mmol/L)
(Boehringer Mannheim). After incubation, SMCs were
sedimented by gentle centrifugation (400g for 10 minutes), resuspended in DMEM+ 10% FCS, and grown at
37°C in a humidified atmosphere of 5% CO2 in air.
Culture medium (DMEM+10% FCS) was changed
every 3 days and a confluent SMC monolayer was
obtained after about 7 days. Cells were routinely used
from the third to the sixth passage. To determine the
effect of the various compounds on their proliferative
response, cells were plated sparsely (103 cells per well)
in 96-well cluster plates (Nunc) in DMEM+0.5% FCS.
After 3 days, cells in representative dishes were counted
with a Coulter counter (Coultronics), and fresh medium
was added to the remaining dishes (DMEM-t-5% FCS
and the different concentrations of the drugs to be
tested). For growth rate determinations, after 3 days in
culture cells were detached from triplicate wells by
trypsin treatment (0.05% trypsin/0.02% EDTA) and
counted in a Coulter counter. Median inhibitory concentration (IC50) values were calculated on the basis of
the linear regression lines established for each compound tested by using a regression program.
In Vivo Experiments
Air-drying injury. Male New Zealand White rabbits
(Charles River) weighing 2.5 to 3 kg were used. Airdrying injury was induced by applying an air flow
through the carotid artery using a modification of
Fishman's method.2728 Rabbits were anesthetized with
sodium pentobarbitone (30 mg/kg IV), and the left
carotid artery was exposed and ligated at two points 1.5
cm apart. A 27-gauge hypodermic needle was inserted
into the proximal end of the segment by puncturing with
an additional needle. After the lumen had been rinsed
with PBS, a stream of dry air was allowed to flow
through the segment at 240 mL/min for 5 minutes.
Ligatures were then removed, allowing circulation to be
reestablished, hemostasis was ensured, and the incision
was closed.
Tissue preparation and morphological examination.
The antiplatelet drugs were administered orally 2 hours
before carotid injury except for window experiments, in
which clopidogrel was first administered 24 hours after
air-drying injury. Sixteen days after surgery, animals
were anesthetized with sodium pentobarbitone (30
Herbert et al Effect of Clopidogrel on Arterial Injury
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mg/kg IV), and 200 IU/kg heparin (Sigma) was injected. The aorta was then perfused with saline followed
by 4% glutaraldehyde in 0.1 mol/L cacodylate buffer,
pH 7.4. Both common carotid arteries were excised and
fixed by immersion for an additional 4 hours in 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1
mol/L cacodylate buffer, pH 7.4. The vessels were
washed in buffer, postfixed for 2 hours in 2% aqueous
OsO4, and stained in 2% aqueous uracyl acetate en
bloc. They were then dehydrated through graded solutions of alcohol and embedded in Epon 812 for serial
cross sectioning. Epon sections (2 /am) were stained
with toluidine blue. Morphometric analysis of arterial
sections was done by use of the Biocom Imagenia 2000
image analysis system (Biocom, Lyon, France). Plaque
size was determined for each segment by tracing the
luminal perimeter and internal and external elastic
laminae of each section with a digitizing tablet. For
intimal and medial thicknesses, the average of five
equally spaced radial measurements for each section
was used.
Determination ofplatelet deposition on the subendothelium. To quantify platelet adherence to the injured
arterial wall, the rabbits were injected with homologous
'"In-platelets 1 hour before removing the injured vessel
wall segment (1 to 24 hours after air injury). Immediately before removal of the segment, a 5-mL citrated
blood sample was collected from the rabbit for the
determination of whole-blood platelet-specific activity.
Each rabbit was then heparinized (250 IU/kg) to prevent thrombin generation and postmortem platelet accumulation and killed 2 minutes later with an overdose
of sodium pentobarbital. A standard length (1.5 cm) of
each vessel was removed and put in a liquid scintillation
vial containing 1 mL of IN NaOH. One hour later, the
vessel was rinsed with 10 mL of toluene-based counting
fluid, and the number of adherent platelets per square
centimeter of surface area of vessel wall segment was
calculated by measuring the radioactivity of the sample,
the whole-blood platelet-specific activity, and the vessel
wall surface area.
Scanning electron microscopy. In another group of
animals, the inner surface of the left carotid artery was
studied by scanning electron microscopy at 1, 4, and 24
hours after air-drying injury. Animals were anesthetized with sodium pentobarbitone (30 mg/kg IV) and
received an intravenous injection of heparin (200 IU/
kg). The abdomen was opened and a retrograde cannula
was inserted into the abdominal aorta. Carotid arteries
were fixed by retrograde perfusion via the abdominal
aorta at 120 mm Hg pressure with 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 mol/L cacodylate
buffer, pH 7.4. Excised tissues, including right and left
common carotid arteries, were further fixed by immersion in 2% paraformaldehyde and 2.5% glutaraldehyde
for 2 to 4 hours. Arteries were postfixed in 2% OsO4 and
dehydrated using alcohols. They were sectioned at the
midpoint, cut longitudinally, critical-point dried in CO2,
and coated with gold/palladium with a Technics Hummer sputter-coating device. The specimens were examined at 30 kV with a Philips SEM 505 scanning electron
microscope.
Drugs
Ticlopidine (Ticlid), clopidogrel [D-methyl(2chlorophenyl)- 5 -(4,5,6,7-tetrahydrothieno)(3,2-c)pyrid-
1173
Platelets added to the ECM (x10 )
FIG 1. Line graph showing ex vivo effect of clopidogrel on
platelet adhesion to the extracellular matrix (ECM). Increasing concentrations of '"In-labeled platelets prepared from
rabbits treated 2 hours before being administered single oral
doses of clopidogrel (10 mg/kg; •); SR 25989 (50 mg/kg; O);
or the vehicle (•) were incubated with the ECM for 60 minutes
at 25"C. Platelets from the vehicle-treated rabbits were also
incubated under the same experimental conditions without the
ECM (D). The amount of ECM-bound platelets was determined as described in "Methods." Results are expressed as
mean±SD; n=3.
inyl)acetate, hydrogen sulfate] and the corresponding
inactive L-enantiomer (SR 25989) were from Sanofi
Recherche (Toulouse, France). Aspirin was purchased
from Sigma. All compounds were administered orally in
a 5% gum arabic solution in water.
Statistical Analysis
All data are expressed as mean±l SD. The n values
indicate the number of animals studied. Grouped data
were analyzed for significance by comparison with the
vehicle-treated group by using the Kruskal-Wallis or
Mann-Whitney U test as indicated. The level of significance was chosen as P<.05.
Results
Effect of Clopidogrel on Platelet Adhesion
to the ECM
When in In-labeled rabbit platelets were added to
uncoated plastic dishes, little attachment occurred (Fig
1). In contrast, the addition of increasing concentrations
of platelets from untreated rabbits to the ECM produced by bovine corneal endothelial cells resulted in
rapid and massive adherence to the ECM. Platelets
spread on the ECM, developed pseudopods, and
formed a monolayer as visualized by phase-contrast
microscopy. Under these experimental conditions, large
aggregates composed of several layers of platelets were
never found.
Treatment of the animals with a single oral dose of
clopidogrel (10 mg/kg PO) 2 hours before platelet
preparation abolished platelet reactivity, as revealed by
almost complete inhibition of platelet adhesion (94%;
P<.001) (Table 1, Fig 1). Under these conditions,
ADP-induced platelet aggregation was reduced by 86%.
The L-enantiomer of clopidogrel, SR 25989, known to
be inactive on ADP-induced platelet aggregation,29 did
not have any effect on platelet adherence to the ECM.
1174
Arteriosclerosis and Thrombosis
Vol 13, No 8 August 1993
TABLE 1. Effects of Aspirin, Ticlopidine, and Clopidogrel
on the Adhesion of Rabbit Platelets to the Extracellular Matrix
Ex Vivo
Treatment
Dose (mg/kg)
Inhibition (%)
P*
Aspirin
Ticlopidine
200t
loot
200t
4±2
NS
NS
<.001
1
21±5
56±9
12±4
5
41±7
10
50
94±5
.01
•c.001
2±1
NS
Clopidogrel
SR 25989
NS
Inhibition data are expressed as mean±SD. NS, not significant.
* Mann-Whitney U test; n=5 to 6.
tCompounds were administered to the animals for 3 days before
the experiment.
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Under the same experimental conditions, aspirin administered for 3 days at a daily oral dose of 200 mg/kg
induced no activity. Ticlopidine at the same dosage
strongly affected platelet adhesion to the ECM (56%;
P<.001) (Table 1).
When clopidogrel was incubated in vitro (10 /nmol/L)
for 1 hour in the presence of the ECM and platelets
from untreated animals, no effect on platelet adhesion
occurred, therefore confirming that the in vivo metabolization is necessary for the antiplatelet effect of this
class of compounds. Similarly, ticlopidine failed to
reduce platelet adhesion when the ECM was obtained
from endothelial cells grown for 16 days in the presence
of 150 jumol/L ticlopidine.
Effect of Clopidogrel on Rabbit SMC Growth
In Vitro
As shown in Fig 2, neither clopidogrel nor ticlopidine
up to a concentration of 400 jiimol/L altered seruminduced rabbit aortic SMC growth. When used as a
200
100
<D
o
100
200
Concentration
300
400
500
(heparin: |ig/ml{
(Ticlopidine - Clopidogrel: |iM)
FIG 2. Line graph showing effects of clopidogrel and ticlopidine on smooth muscle cell (SMC) growth in vitro. Growtharrested SMCs (20X103 cells per well) were grown in culture
medium plus 5% fetal calf serum in the presence of increasing
concentrations of clopidogrel (•), ticlopidine (A), or heparin
(•). After 3 days in culture, triplicate wells were trypsinized
and cells were counted. Data are expressed as mean percent
inhibition of proliferation compared with replicate cultures
grown without the inhibitor (n=3).
control, standard heparin reduced SMC proliferation
with an IC50 value of 250 /ug/mL. This result is consistent
with already published observations.2630-31
Effect of Clopidogrel on Platelet Adhesion to the
Air-Injured Rabbit Carotid Artery
Scanning electron photomicrographs of the luminal
surface of an uninjured carotid artery are shown in Fig
3, A. One hour after air dessication, both spread and
discoid platelets were found in close contact with the
remaining endothelial surface (Fig 3, B). These cells
were mainly in the margins or between the endothelial
cells, and traces of fibrin were present. This phenomenon becomes more evident in Fig 3, C, which shows the
morphology of the luminal surface 4 hours after airdrying injury. At this time point, all the endothelial cells
had been removed from the full circumference of the
segment of the artery that had been exposed to dessication, and the luminal surface consisted of a monolayer
of spread platelets with some small aggregates and
fibrin. There were also large numbers of polymorphonuclear leukocytes (up to approximately 1000/mm2)
associated with the platelet layer. Twenty-four hours
after air drying, the injured surface was composed of
irregularly shaped red blood cells linked with moderate
to extensive amounts of fibrin and large numbers of
aggregated platelets (Fig 3, D).
Parallel quantification of '"In-labeled platelet adherence to the air-injured artery indicates that after an
initial rise, which occurred within the first 4 hours, the
number of bound lu In-platelets reached a plateau 24
hours after de-endothelialization (Fig 4). At 24 hours,
the injured carotid artery of aspirin-treated animals
showed luIn-labeled platelet adherence of the same
magnitude as that observed in untreated animals (Fig 3,
E, Table 2). However, a single oral administration of
clopidogrel (25 mg/kg) resulted in strong inhibition of
platelet adherence to the subendothelium (Fig 4, Table
2, and Fig 3, F) 24 hours after the air-drying injury. A
similar effect was obtained with daily doses of ticlopidine (200 mg/kg PO) for 6 days.
Effect of Clopidogrel on Myointimal Proliferation
After Air-Drying Injury
In the right carotid artery (which had not been
subjected to air injury) there was no evidence of intimal
proliferation, foam cell formation, or platelet accumulation 16 days after the surgical procedure, indicating
that physical manipulation at the time of surgery without air-drying injury was not sufficient to generate the
lesion. However, the time course of proliferation in the
intimal region of the dessicated left rabbit carotid artery
indicated that after denudation SMCs can be observed
in the intima by day 7 (Fig 5). By 2 weeks the intima had
grown substantially, and it stayed at the same size for
the remainder of the experimental period. Sixteen days
after endothelial denudation, the intimal surface represented 35 ±7% of the tunica media area. Morphometric
analysis revealed that the changes in the increase in the
intimal diameter occurred while the medial diameter
remained mostly unchanged (Fig 5). This neointima,
composed of up to eight layers of SMCs, extended to the
entire circumference and approximately 80% of the
length of the injured area. The SMCs of the neointima
Herbert et al
A
Effect of Clopidogrel on Arterial Injury
1175
D
B
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10 urn
FlG 3. Scanning electron photomicrographs (SEMs) of the luminal surface of an injured control rabbit carotid artery before (A)
and at 1 hour (B), 4 hours (C), and 24 hours (D) after air injury. SEMs of the arterial luminal surface of the carotid artery isolated
24 hours after injury from rabbits treated with aspirin (100 mglkg PO) (E) or clopidogrel (25 mglkg PO) (F). Bar—10 pirn.
1176
Arteriosclerosis and Thrombosis
Vol 13, No 8 August 1993
1=
E
S
jo
Q.
C
O
m
24
12
Time after air injury (h)
10
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FIG 4. Line graph showing effect of clopidogrel on the time
course of platelet adhesion to the de-endothelialized rabbit
carotid artery. Carotid arteries from rabbits treated by a single
oral dose of clopidogrel (25 mg/kg; • ) or vehicle (•) were
de-endothelialized, and "'In-labeled platelet adhesion was
determined at the indicated times as described in "Methods."
Clopidogrel was administered 2 hours before endothelial
injury. Results are mean±SD; n = 7 to 11.
and the superficial media were mainly of the secretory
phenotype, as described by Richardson et al.28
As shown in Fig 5 and Table 3, a 16-day oral
treatment of clopidogrel (25 mg/kg per day) strongly
reduced SMC hyperplasia after de-endothelialization
(48% inhibition; P<.01). A substantial increase in this
effect was observed when the animals were treated for a
23-day period (61% inhibition; P<.001) (Fig 5). This
effect was dose related, because an oral dose of clopidogrel (10 mg/kg per day) resulted in 21% inhibition of
neointimal formation (P<.05). To determine what part
inhibition of platelet adhesion plays in the subsequent
effect of clopidogrel on myointimal proliferation, we
performed window experiments in which clopidogrel
treatment was started 24 hours after de-endothelialization. A 24-hour delay in clopidogrel administration
resulted in a dramatic reduction of its efficacy on either
platelet adhesion or myointimal proliferation (Table 4).
Under treatment with clopidogrel (25 mg/kg per day
PO), the morphology of the SMCs near the medial
surface varied in comparison with the SMCs in the
remaining neointima. Indeed, these SMCs showed decreased amounts of metabolic organelles compared with
SMCs of the neointima from controls. After 23 days, the
neointima of the treated animals was two to three layers
thick, with SMCs showing prominent myofilaments (Fig
TABLE 2. Effects of Aspirin, Ticlopidine, and Clopidogrel
on Platelet Adhesion to the Rabbit Carotid Artery
After De-endothelialization
Treatment
Dose
(mg/kg per day)
30
FIG 5. Line graphs showing effects of clopidogrel on the
myointimal proliferation of the rabbit carotid artery after
endothelial injury. Clopidogrel (25 mg/kg PO; • , o) or the
vehicle (m, a) were administered 2 hours before air injury and
daily for 16 days to rabbits whose carotid artery was deendothelialized at day 0. Intimal (solid symbols) and medial
(open symbols) surfaces were measured as described in
"Methods." Data are mean±SD; n=9. Surface, surface area
of cross sections in square millimeters. **P<.01, ***P<.001
by Kruskal-Wallis test.
6). This morphology is a typical feature of SMCs in a
contractile phenotype.32 Reduction in intimal thickening was also observed after a 16-day administration of
ticlopidine (200 mg/kg per day PO) (57% inhibition;
/><.001) (Table 3), whereas aspirin did not significantly
affect myointimal proliferation after air injury. These
observations have already been shown elsewhere.13-33 It
is noteworthy that none of these compounds affected
re-endothelialization of the denuded area.
Discussion
Restenosis after successful PTCA remains one of the
major unresolved problems with this procedure. Restenosis occurs in 30% to 40% of the dilated lesions,
usually 3 to 4 months after PTCA. 13 Although restenosis can be effectively treated by repeating the PTCA
procedure, the large number of patients requiring second and even third operations adds significantly to
morbidity and cost, and despite numerous clinical trials
and animal studies, no other nonmechanical, effective
treatment has been found.
Several mechanisms were initially proposed as possible causes of restenosis, including organized thrombus
TABLE 3. Effect of Aspirin, Ticlopidine, and Clopidogrel
on the Myointimal Proliferation of the Rabbit Carotid Artery
After De-endothelialization
Inhibition
Treatment
Aspirin
100
4±1
NS
Ticlopidine
200t
25
63±9
•c.Ol
93±7
<.001
Clopidogrel
20
Days after air injury
Inhibition data are expressed as mean±SD. NS, not significant.
*Mann-Whitney U test; n=7 to 11.
tCompound was administered to the animals for 6 days before
the experiment.
Aspirin
Ticlopidine
Clopidogrel
Dose
(mg/kg per day)
Inhibition
100
200
25
17±6
57±11
•c.001
48±9
21±4
<.01
.05
10
NS
Inhibition data are expressed as mean±SD. NS, not significant.
"Kruskal-Wallis test; n=9 to 15.
Herbert et al
TABLE 4. Effect of Delayed Administration of Clopidogrel on
Platelet Adhesion and Myointimal Proliferation
Inhibition of
platelet
adhesion
Inhibition of
intimal
thickening
93±7
48±9
7±3
4±1
Clopidogrel (25 mg/kg per day)
Administered 2 h before injury
and daily for 16 d
Administered 24 h after injury
and daily for 16 d
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or vasospasm, but a number of autopsy studies have
shown that intimal hyperplasia is the main histological
finding of restenotic coronary arteries in patients with
prior successful PTCA.34 During vessel damage, such as
balloon injury, the release and production of growthstimulatory and chemotactic factors occurs, as does the
alteration and destruction of the spatial arrangements
of SMCs and the ECM. SMCs start to proliferate as a
result of exposure to growth factors and the loss of
endothelial inhibitory influences. Platelet adhesion and
aggregation caused by exposure of the thrombogenic
B
\
FlG 6. Scanning electron photomicrographs showing effect
of clopidogrel on the phenotype of intimal smooth muscle
cells (SMCs) of the rabbit carotid artery. Vehicle (A) or
clopidogrel (25 mg/kg PO; B) were administered 2 hours
before air injury and daily for 16 days to rabbits whose carotid
artery was de-endothelialized. A, SMCs present in the neointima of control animals. Cells are in a synthetic state and their
cytoplasm is filled with large amounts offree ribosomes, rough
endoplasmic reticulum, and mitochondria. B, SMCs present
in the neointima of clopidogrel-treated animals. Thick and
thin myofilaments in the cytoplasm are seen in SMCs in a
contractile phenotype. Bar=2 ixm.
Effect of Clopidogrel on Arterial Injury
1177
subendothelium result in the release of growth factors
such as platelet-derived growth factor and epidermal
growth factor and other humoral factors such as serotonin, histamine, and norepinephrine, which are mitogenic and/or chemotactic for SMCs. Other factors, such
as shear stress or the infiltration of inflammatory cells at
the injured site, might also influence the final outcome
of intimal hyperplasia. Although a large number of
factors may contribute to this process, platelets appear
to play a fundamental role in initiating and maintaining
intimal hyperplasia. Therefore, one possible method for
reducing SMC proliferation after arterial injury might
be to administer drugs that inhibit platelet adhesion
and/or aggregation to collagen or other reactive components of the arterial wall.
In this study, we attempted to associate the in vitro
adherence of platelets from rabbits treated with aspirin,
ticlopidine, or clopidogrel to the in vivo deposition of
similar platelets on the exposed carotid artery subendothelial layers. We further extended our investigations to
determine the effect of these same compounds on
myointimal proliferation after air-drying injury of the
vessel wall. Our results demonstrate that, although used
at doses that have been widely described to markedly
reduce platelet functions, aspirin did not alter the in
vitro pattern of platelet interactions with the ECM
produced by endothelial cells in culture. Similar results
have been reported in other models.2535 However, ticlopidine and its potent analogue clopidogrel strongly
counteracted platelet adhesion to the subendothelium.
This effect was dose dependent for both compounds and
was observed at concentrations that have been shown to
reduce ADP-induced platelet aggregation ex vivo.2329
As already shown for platelet aggregation,23'27 the L-enantiomer of clopidogrel (SR 25989) did not significantly
affect platelet adhesion to the ECM, thus demonstrating
the specificity of the observed effect.
Such observations can be compared with results
showing that ticlopidine inhibits platelet adhesion to
foreign surfaces such as glass beads,36 Gore-Tex, or
Dacron grafts37'38 in dogs. Unlike what was observed for
aspirin, ticlopidine and clopidogrel are both potent
inhibitors of platelet adhesion in vitro, but in spite of the
high degree of similarity between the ECM produced by
endothelial cells in culture and the basal lamina of the
vascular endothelium, the possible role that vascular
prostaglandin I2 and rheological parameters could play
in such a phenomenon was disregarded. Thus, the
adherence of platelets to the subendothelium was studied in vivo after exposure of the subendothelium after
injury of the rabbit carotid artery.
Platelet accumulation was examined after extensive
endothelial removal by air-drying injury. Our observations confirmed other work showing that platelet interactions with the exposed subendothelium were minimal
during the first 4 hours after endothelial desquamation.28 The denuded surface was then rapidly covered by
platelets, which spread and discharged a variable
amount of their granule content. In the present study,
although there was minimal medial damage, neutrophils
accumulated on the arterial surface at early time points
and infiltrated the vessel wall. This neutrophil infiltration and associated edema of the media that occurred
shortly after injury are typical of an inflammatory
response and are consistent with other observations.28
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Vol 13, No 8 August 1993
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As already reported,3335 aspirin remained without
effect on platelet deposition on the arterial wall denuded of endothelium, whereas platelet interactions
with the subendothelial connective tissue can be almost
totally prevented by a single dose of clopidogrel (25
mg/kg PO) or repeated daily doses of ticlopidine (200
mg/kg PO) for 6 days. These results therefore confirm
those obtained on platelet adhesion to the ECM in vitro
and in vivo3940 but conflict with data from Cattaneo et
al,41 who showed that ticlopidine did not affect platelet
adhesion in other animal models, such as balloon-deendothelialized rabbit aortas or platelet accumulation
induced by an indwelling catheter in rat carotid arteries.
Nevertheless, although abundant evidence of the effectiveness of antiplatelet drugs in preventing platelet
aggregation is now available, studies on the influence of
such drugs on the in vivo deposition of platelets on
injured vessels have produced contradictory results and
seem to vary with the experimental model.17-35-39-43 To
our knowledge, however, clopidogrel is considered to be
one of the most potent compounds inhibiting platelet
adherence to the subendothelium in vivo.
Since it has been demonstrated that these compounds
interfere directly with platelet adhesion, an important
initial step in restenosis, we decided to determine their
effect on arterial SMC proliferation (intimal hyperplasia) in vivo. As a result of air drying, intimal thickening
reached a maximum of eight SMC concentric layers by
2 weeks and showed no further increase by 2 months
(not shown). After 2 weeks, changes in the phenotype
were also observed for SMCs in the neointima, which
were mainly of the secretory phenotype (Fig 6).
In this model, aspirin showed no effect on the morphology of the lesion or on its development, therefore
confirming data reported previously by Clowes and
Karnovsky.33 Arterial hyperplasia was, however, significantly reduced by a daily dose of ticlopidine (200 mg/kg
PO). Ticlopidine, at a dose similar to that used in our
study, has been reported to inhibit intimal SMC proliferation by balloon catheter endarterectomy in the rabbit
abdominal aorta.13 Under the same experimental conditions, clopidogrel exhibited dose-dependent inhibition
of intimal thickening. As already shown for ADPinduced platelet aggregation, not only was clopidogrel
about 10-fold more active than ticlopidine, but also it
was able to affect the phenotypic state of SMCs in the
fibroproliferative lesions. This observation is of importance because until now heparin or the heparinoids
were the only compounds described as exhibiting such
an effect.30 However, unlike these compounds, which
are known to exhibit potent antiproliferative activity
against vascular SMCs in culture,26-30-31 ticlopidine or
clopidogrel does not alter vascular SMC growth in vitro
(Fig 2). It is therefore assumed that the effect of these
compounds on myointimal proliferation is mainly due to
their antiplatelet effects. This suggestion was further
reinforced by window experiments showing that the first
24 hours after de-endothelialization were critical for the
development of the intimal hyperplasia. Indeed, when
the treatment with clopidogrel was started 24 hours
after de-endothelialization, loss of efficacy of the compound was observed (Table 4). This effect occurred
simultaneously with a reduction of clopidogrel's effect
on platelet adhesion to the injured vessel. These results
suggest that platelets are mainly involved in the early
stages of experimental atherosclerosis and that interruption of platelet adherence may reduce the severity of
this progressive disease.
In summary, the reactivity of platelets to the exposed
subendothelium that develops after air-drying injury
appears to be of great importance in the subsequent
SMC migration and/or proliferation. On the assumption
that antiplatelet drugs like ticlopidine or clopidogrel
might suppress the release of growth or chemotactic
factors from adhering platelets, we found significant
reductions in intimal SMC proliferation after arterial
de-endothelialization. On this basis, one could assume
that compounds related to ticlopidine, such as clopidogrel, with greater potency as ADP-selective inhibitors, may be useful in preventing arterial hyperplasia in
humans.
Acknowledgment
The authors thank A.J. Patacchini for her helpful comments
on the manuscript.
References
1. Leimgruber PP, Roubin GS, Hollman J, Cotsonis GA, Meier B,
Douglas JS, King SB, Gruentzig AR. Restenosis after successful
coronary angioplasty in patients with single-vessel disease. Circulation. 1986;73:710.
2. Shiu MF, Silverton NP, Oakley D, Cumberland D. Acute coronary
occlusion during percutaneous transluminal coronary angioplasty.
Br Heart J. 1985;54:129.
3. Essed CE, Van Den Brand M, Becker AE. Transluminal coronary
angioplasty and early restenosis. Br Heart J. 1983;49:393.
4. Thornton MA, Gruentzig AR, Hollman J, King SB, Douglas JS.
Coumadin and aspirin in prevention of recurrence after transluminal coronary angioplasty: a randomized study. Circulation.
1984;69:721.
5. Kent KM, Bentivoglio JG, Block PC, Cowley MJ, Dorros G,
Gosselin AJ, Myler RK, Simpson J, Williams DO, Fisher L,
Gillespie MJ, Kelsey S, Mullin SM, Mock MB. Percutaneous transluminal coronary angioplasty: report from the registry of the
National Heart, Lung and Blood Institute. Am J Cardiol. 1982;
49:2011.
6. White CW, Chaitman B, Knudtson ML, Chisholm RJ. Antiplatelet
agents are effective in reducing the acute ischemic complications of
angioplasty but do not prevent restenosis. Coronary Arterial Dis.
1991;2:757.
7. White CW, Knudson M, Schmidt D, Chisholm RJ, Vandormael M,
Morton B, Roy L, Khaja F, Reitman M. Neither ticlopidine nor
aspirin-dipyridamole prevents restenosis post PTCA. Circulation.
1987;76:213.
8. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J
Med. 1986;314:488.
9. Fingerle J, Johnson R, Clowes AW, Malesky MW, Reidy MA.
Role of platelets in smooth muscle cell proliferation and migration
after vascular injury in rat carotid artery. Proc NatlAcad Sci U SA.
1989;86:8412.
10. Friedman RJ, Stemerman MB, Wenz B, Moore S, Gauldie L, Gent
M, Tiell ML, Spaet TH. The effect of thrombocytopenia on experimental atherosclerotic lesion formation in rabbits. J Clin Invest.
1977;60:1191.
11. Schaub RG, Keith JC, Simmons CA, Rawlings CA. Smooth muscle
proliferation in chronically injured canine pulmonary arteries is
reduced by a potent platelet aggregation inhibitor U-53059.
Thromb Haemost. 1985;53:351.
12. Schaub RG, Simmons CA. Medial smooth muscle cell proliferation
in the balloon injured rabbit aorta: effect of a thiazole compound
with platelet inhibitory activity. Thromb Haemost. 1984;51:75.
13. Reece AH, Walton PL. Inhibition of intimal proliferation of rabbit
aorta by ticlopidine. Thromb Haemost. 1979;42:367.
14. Chesebro JH, Fuster V, Clements IP, Smith HC, Holmes DR,
Bardsley WT, Wallace RB, Puga FG, Piehler JM, Danielson GK,
Schaff HV, Frye RL. Effect of dipyridamole and aspirin on late
vein-graft patency after coronary bypass operation. N Engl J Med.
1984;310:209.
15. Donaldson DR, Salter MC, Kester RC, Rajah SM, Hall TJ,
Sreeharan N, Crow MJ. The influence of platelet inhibition on the
Herbert et al
Downloaded from http://atvb.ahajournals.org/ by guest on June 15, 2017
patency of femoro-popliteal dacron bypass grafts. Vase Surg. 1985;
19:224.
16. Hagen PO, Wang ZG, Mikat EM, Hackel DB. Antiplatelet therapy
reduces aortic intimal hyperplasia distal to small diameter vascular
protheses in nonhuman primates. Ann Surg. 1982;195:328.
17. Hansen KJ, Howe HR, Edgerton A, Faust KB, Kon ND, Meredith
JH. Ticlopidine versus aspirin and dipyridamole: influence on
platelet deposition and three-month patency of polytetrafluoroethylene grafts. J Vase Surg. 1986;4:174.
18. Metke MP, Lie JT, Fuster V, Josa M, Kaye MP. Reduction of
intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin. Am J Cardiol. 1979;43:1144.
19. Hess H, Mietaschk A, Deichsel G. Drug-induced inhibition of
platelet function delays progression of peripheral occlusive arterial
disease: a prospective double-blind arteriographically controlled
trial. Lancet. 1985;1:415.
20. Mertz ET. The anomaly of a normal Duke's and a very prolonged
saline bleeding time in swine suffering from an inherited bleeding
disease. Am J Physiol. 1942; 136:360.
21. GriggsTR, Reddick RL, Sultzer D, Brinkhous KM. Susceptibility
to atherosclerosis in aortas and coronary arteries of swine with von
Willebrand's disease. Am J Pathol. 1981;102:137.
22. Fuster V, Fass DN, Bowie EJ. Resistance to atherosclerosis in pigs
with genetic and therapeutic inhibition of platelet function.
Thromb Haemost. 1979;42:270. Abstract.
23. Delebassee D, Bernat A, Frehel D, Defreyn G, Maffrand JP.
Antiaggregating and antithrombotic effects in the rat of two
thienopyridine enantiomers structurally related to ticlopidine.
Tenth International Congress on Thrombosis; May 22-27, 1988;
Athens, Greece. Abstract No. 197.
24. Gospodarowicz D, III CR. Extracellular matrix and control of
proliferation of vascular cndothelial cells. J Clin Invest. I98O;65:
1351.
25. Vlodavsky I, Eldor A, Hyam E, Atzmon Y, Fuks Z. Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. Thromb Res. 1982;28:179.
26. Paul R, Herbert JM, Maffrand JP, Lansen J, Modat G, Pereillo
JM, Gordon J. Inhibition of vascular smooth muscle cell proliferation in culture by pentosan polysulfate and related compounds.
Thromb Res. 1987;46:793.
27. Fishman JA, Ryan GB, Karnovsky MJ. Endothelial regeneration
in the rat carotid artery and the significance of endothelial denudation in the pathogencsis of myointimal thickening. Lab Invest.
1975;32:339.
28. Richardson M, Hatton MWC, Buchanan MR, Moore S. Wound
healing in the media of the normolipemic rabbit carotid artery
injured by air drying or by balloon catheter de-endothelialization.
Am J Pathol. 1990; 137:1453.
Effect of Clopidogrel on Arterial Injury
1179
29. Defreyn G, Gachet C, Savi P, Driot F, Cazenave JP, Maffrand JP.
Ticlopidine and clopidogrel (SR 25990C) selectively neutralize
ADP inhibition of PGEl-activated platelet adenylate cyclase in
rats and rabbits. Thromb Haemost. 1991;65:186.
30. Herbert JM, Nuti D, Paul R, Maffrand JP. In vitro and ex vivo
regulation of vascular smooth muscle cell growth and phenotypic
modulation by sulphated polysaccharides. Artery. 1988; 16:1.
31. Hoover RL, Rosenberg RD, Haering W, Karnovsky MJ. Inhibition
of rat arterial smooth muscle cell proliferation by heparin, II: in
vitro studies. Circ Res. 1980;47:578.
32. Chamley-Campbell JH, Campbell GR, Ross R. The smooth muscle
cell in culture. Physiol Rev. 1979;59:1.
33. Clowes AW, Karnovsky MJ. Failure of certain antiplatelet drugs to
affect myointimal thickening following arterial endothelial injury in
the rat. Lab Invest. 1977;36:452.
34. Austin GE, Ratliff NB, Hollman J, Tabei S, Phillips DF. Intimal
proliferation of smooth muscle cells as an explanation for recurrent
coronary artery stenosis after percutaneous transluminal angioplasty. J Am Coll Cardiol. 1985;6:369.
35. Dejana E, Cazenave JP, Groves HM, Kinlough-Rathbone RL.
Richardson M, Packham MA, Mustard JF. The effect of aspirin
inhibition of PGI2 production on platelet adherence to normal and
damaged rabbit aortae. Thromb Res. 1980;17:453.
36. Conard J, Lecrubier C, Scarabin PY, Horellou MH, Samama M,
Bousser MG. Effects of long term administration of ticlopidine on
platelet function and hemostatic variables. Thromb Res. 1980;20:
134.
37. Pumphrey CW, Fuster V, Dewanjee MK, Murphy KP, Vliestra
RE, Kaye MP. A new in vivo model of arterial thrombosis: the
effect of administration of ticlopidine and verapamil in dogs.
Thromb Res. 1982;28:663.
38. Geroulanos S, Walter P, Cogan C, Binkert M, Turina M, Senning
A. Platelet inhibitors. Haemostasis. 1982;12:155. Abstract.
39. Plate G, Stanson AW, Hollier LH, Dewanjee MK, Solis E, Kaye
MP. Effect of platelet inhibitors on platelet and fibrin deposition
following transluminal angioplasty of the atherosclerotic rabbit
aorta. Eur J Vase Surg. 1989;3:127.
40. Orlando E, Cortelazzo S, Nosari I, Lepore G, Pagani G, De Gaetano G, Barbui T. Inhibition by ticlopidine of platelet adhesion to
human venous subendothelium in patients with diabetes. J Lab
ClinMed. 1988;112:583.
41. Cattaneo M, Winocour PD, Somers DA, Groyes HM, KinloughRathbone RL, Packham MA, Mustard JF. Effect of ticlopidine on
platelet aggregation, adherence to damaged vessels, thrombus formation and platelet survival. Thromb Res. 1985;37:29.
42. Escolar G, Bastida E, Castillo R, Ordinas A. Ticlopidine inhibits
platelet thrombus formation studied in a flowing system. Thromb
Res. 1987;45:561.
43. Weiss HJ, Turitto VT. Prostacyclin inhibits platelet adhesion and
thrombus formation on subendothelium. Blood. 1979;53:244.
Downloaded from http://atvb.ahajournals.org/ by guest on June 15, 2017
Inhibitory effect of clopidogrel on platelet adhesion and intimal proliferation after
arterial injury in rabbits.
J M Herbert, A Tissinier, G Defreyn and J P Maffrand
Arterioscler Thromb Vasc Biol. 1993;13:1171-1179
doi: 10.1161/01.ATV.13.8.1171
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