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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1518-1525
A Double-Blind Randomized Comparison of Combined Aspirin and
Ticlopidine Therapy Versus Aspirin or Ticlopidine Alone on Experimental
Arterial Thrombogenesis in Humans
By
Jean-Pierre Bossavy,
Claire Thalamas,
Luc Sagnard,
André Barret,
Kjell Sakariassen,
Bernard Boneu, and
Yves Cadroy
From the Laboratoire de Recherche sur l'Hémostase et la
Thrombose, Pavillon Lefèbvre, CHU Purpan, Toulouse; Service de
Chirurgie Générale et Vasculaire, CHU Purpan, Toulouse;
Centre d'Investigation Clinique, CHU Purpan, Toulouse Cedex, France;
Sanofi Recherche, Gentilly Cedex, France; and the Department of
Biology, the Division of Physiology, University of Oslo, Oslo, Norway.
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ABSTRACT |
No randomized study comparing the effect of combined ticlopidine and
aspirin therapy versus each drug alone in reducing poststenting thrombotic complications has been performed. To compare these three
antiplatelet regimens versus placebo, we conducted a double-blind randomized study using an ex vivo model of thrombosis. Sixteen healthy
male volunteers were assigned to receive for 8 days the following four
regimens separated by a 1-month period: aspirin 325 mg/d, ticlopidine
500 mg/d, aspirin 325 mg/d + ticlopidine 500 mg/d, and placebo. At
the end of each treatment period, native nonanticoagulated blood was
drawn directly from an antecubital vein over collagen- or tissue factor
(TF)-coated coverslips positioned in a parallel-plate perfusion chamber
at an arterial wall shear rate (2,600 s 1 ) for 3 minutes. Thrombus, which formed on collagen in volunteers treated by
placebo, were rich in platelets and poor in fibrin. As compared with
placebo, aspirin and ticlopidine alone reduced platelet thrombus
formation by only 29% and 15%, respectively (P > .2). In
contrast, platelet thrombus formation was blocked by more than 90% in
volunteers treated by aspirin + ticlopidine (P < .01 v placebo or each treatment alone). Furthermore, the effect of
the drug combination therapy was significantly larger than the sum of
the two active treatments (P < .05). Thrombus, which formed
on TF-coated coverslips in volunteers treated by placebo,
were rich in fibrin and platelets. Neither of the three antiplatelet
treatments significantly inhibited fibrin deposition and platelet
thrombus formation on this surface (P > .2). Thus, the
present study shows that combined aspirin and ticlopidine therapy
dramatically potentiates the antithrombotic effect of each drug alone,
but that the antithrombotic effect of the combined treatment depends on
the nature of the thrombogenic surface.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE ROLE OF PLATELETS in arterial
thrombosis has been well-established during the last decades. As a
result, platelet inhibitor therapy has become of considerable interest
for prevention and treatment of acute and chronic arterial diseases.
Among currently available antiplatelet drugs, aspirin blocks platelet
production of thromboxane A2 and ticlopidine inhibits
adenosine diphosphate (ADP)-induced platelet aggregation.
When used as single antithrombotic agents, these drugs have a moderate
clinical efficacy.1 This may be due to the fact that
aspirin and ticlopidine interfere with only one of the various pathways
of platelet activation. Therefore, to improve the antithrombotic
effect, it has been suggested to combine these two drugs. Thus,
ticlopidine potentiated the antithrombotic effect of aspirin in a
recent experimental study performed in rats.2 Furthermore,
this combined antiplatelet therapy was very potent in reducing cardiac
events and vascular complications after coronary artery stent placement
as compared with conventional anticoagulant therapy.3
However, no clinical or experimental study has randomly compared in
humans the antithrombotic effect of the combination of aspirin and
ticlopidine to each drug alone.
The antithrombotic effect of drugs can be experimentally investigated
in humans using an ex vivo model of thrombogenesis, which closely
mimics relevant clinical situations.4-8 In this model,
native blood is drawn from healthy volunteers through a parallel-plate
chamber device where it interacts, in well-established flow conditions,
with a thrombogenic surface. Blood flow conditions mimic wall shear
rates encountered in moderately stenosed arteries (2,600 s1). Two relevant thrombogenic molecules, which are
present in atherosclerotic plaques and primarily responsible for
thrombus formation,9,10 are exposed to blood, ie, collagen
and tissue factor (TF). The efficacy of antithrombotic drugs is
determined by quantifying the respective thrombus content in platelets
and fibrin by immunoenzymatic methods. This model has been used to
investigate the antithrombotic effect of aspirin, linotroban, a
thromboxane A2 receptor inhibitor, and clopidogrel,
respectively.11-14 The latter two drugs moderately reduced
thrombus formation, but no significant effect was found with aspirin.
Using this ex vivo model of acute initial thrombus formation, we
designed a double-blind, randomized study with blood from healthy
volunteers to compare the antithrombotic effect of three different
antiplatelet regimens versus placebo: either combined aspirin + ticlopidine therapy, or aspirin or ticlopidine alone.
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MATERIALS AND METHODS |
Subjects
The study population consisted of healthy white male volunteers aged 20 to 30 years. They had no history or clinical sign of any disease and
did not take any medication known to affect blood coagulation or
platelets during the study period. The volunteers smoked less than 10 cigarettes per day, and they did not smoke the day of the perfusion
experiments. Clinical chemistry, hematologic, and hemostatic laboratory
values were within the normal ranges. All subjects gave written,
informed consent to the protocol, which was approved by the local Human
Subjects Committee (Comité Consultatif de Protection des
Personnes dans la Recherche Biomédicale, Toulouse). All eligible
volunteers were randomly assigned to one of the four sequences of
treatment, as indicated below.
Study Design
This monocentric, randomized, controlled, double-blind study was
performed in the Center for Clinical Investigation at Hôpital Purpan, Toulouse, France. After selection for the trial, each volunteer
satisfying the inclusion and exclusion criteria received one of the
four tested regimens (Table 1): either (I)
placebo for 8 days, or (II) placebo during 5 days associated with
aspirin 325 mg once daily the last 3 days, or (III)
ticlopidine 250 mg twice daily plus placebo for 8 days, or
(IV) ticlopidine 250 mg twice daily for 8 days associated with aspirin
325 mg once daily during the last 3 days. These periods of
administration were chosen to obtain a full pharmacologic effect.
Aspirin was given for a shorter period because its pharmacologic effect
is more quickly reached than that of ticlopidine and because, for
safety reasons, the healthy volunteers enrolled in this study had to
receive the combined treatment for the shortest possible period. The
four regimens were given separately with a wash-out period of 3 to 6 weeks between each of them. All drugs and placebo were supplied by
Sanofi Recherche (Toulouse, France) under blister indicating the day
and time of intake. On the eighth day of treatment, the subjects were
requested to come to the study center to be examined and rest 1 or 2 hours before blood donation. All adverse effects were recorded and
appropriate follow-ups were monitored. Premature withdrawal from the
study, for any reason (adverse effect, wish of the subject, lack of
compliance), led to replacement of the subject by another volunteer who
followed the same sequence so that 16 complete case reports could be
available for analysis at the end of the study.
Preparation of thrombogenic surfaces.
The thrombogenic molecules were coated on Thermanox plastic coverslips
(Miles Laboratories, Naperville, IL) previously washed in 70% ethanol
and rinsed five times in sterile water. Equine collagen
(Collagenreagent Horm; Nycomed, Munchen, Germany) was spray-coated onto
plastic coverslips to a final density of 0.5 µg/cm2. The
collagen-coated coverslips were stored at room temperature for 15 to 20 hours before use as described previously.4,5,8 TF, purified
from human placenta (Thromborel; Behring, Rueil-Malmaison, France), was
used as described previously.6,8 Dilution of 1:50 was
prepared in coating buffer (0.1 mol/L sodium carbonate, pH 9.5).
Thermanox plastic coverslips were incubated in 2 mL of the Thromborel
dilution for 17 hours at 4°C. The TF-coated coverslips were rinsed
five times with phosphate-buffered saline (PBS; Seromed, BiochromKG,
Berlin, Germany) and stored for less than 7 hours at 4°C before
use.
Perfusion experiments.
Two perfusion experiments were performed after each period of
treatment, with collagen and TF as thrombogenic surface, respectively. Perfusion experiments were performed with a parallel-plate perfusion chamber device at 37°C.5,7,8 After blood sample
collections (see below), native blood was drawn directly from an
antecubital vein of the volunteers through a 19G infusion set (Ohmeda,
Helsingburg, Sweden) over a collagen-coated coverslip positioned in the
parallel-plate perfusion chamber. The blood flow rate was maintained at
10 mL/min by a peristaltic roller pump (Multiperpex LKB, Pharmacia,
St-Quentin-en-Yvelines, France) placed distal to the chamber. Given the
cross-sectional dimension of the blood flow channel of the perfusion
chamber, the wall shear rate was 2,600 s1, which
corresponds to that encountered in moderately stenosed arteries. The
blood perfusion experiment lasted for 3 minutes and was followed by a
30-second perfusion of PBS at the same flow rate to wash out blood from
the flow channel. The coverslip covered by thrombotic deposits was
removed from the chamber and divided into two equal parts parallel to
the direction of the blood flow, as described previously.8
One half of the thrombotic deposit was placed in a plasmin solution and
processed as described below. The other half was immersed into freshly
prepared fixation solution (2.5% glutaraldehyde in 0.1 mol/L
cacodylate, pH 7.4) at 4°C for 90 minutes, and stored in 0.1 mol/L
cacodylate 7% sucrose at 4°C until embedded in Epon. A second
perfusion experiment was subsequently performed with blood directly
drawn from a contralateral cubital vein over a TF-coated coverslip.
Evaluation of the Efficacy of the Different Drug Regimens
The efficacy of each drug treatment was evaluated by morphometry,
immunology, and by the determination of platelet activation and
thrombin formation downstream to the site of thrombus formation. As
previously described, three methods of quantification were used because
they give complementary information about thrombus size, thrombus
composition, and mechanisms of thrombus formation.8
Morphometrical determination of thrombotic deposits.
Microscopic evaluation of thrombotic deposits was performed on epoxy
embedded semithin sections (1 µm thick) stained with toluidine blue
and basic fuchsin, as previously described.8,15 The
sections were prepared at an axial position of 2 mm downstream from the
upstream edge of the coverslip and perpendicular to the direction of
the blood flow. Standard morphometry,15 performed by light
microscopy at 1,000 × magnification, was used to
quantify the percent coverage with platelets adherent to collagen or
fibrin (% platelets). These morphometric evaluations were performed at 10-µm intervals along the surface by moving the section along an
eye-piece micrometer in the microscope ocular.
Immunological determination of fibrin and platelet deposition.
Fibrin deposition was quantified by immunologic determination of fibrin
degradation products of plasmin-digested thrombi, as described
previously.16,17 After perfusions with blood and PBS, the
thrombus was immediately incubated in 2 mL of a plasmin solution
(Chromogenix, Mölndal, Sweden, 0.7 IU/mL, in Tris-buffered saline, pH 7.4) for 30 minutes under gentle shaking and at 37°C. Plasmin digestion was stopped by aprotinin (2,000 KIU/mL, Sanofi, Gentilly, France). The solution was centrifuged (4°C,
4,300g, 15 minutes) and the supernatant frozen at  80°C
for measurement of fibrin degradation products and P-selectin levels
(see below). Fibrin degradation products were measured using an
immunoenzymatic assay (Asserachrom D-Di; Stago, Asnières,
France). The amount of deposited fibrin is directly determined from the
levels of fibrin degradation products expressed in fibrin equivalent
units as indicated by the manufacturer: this unit corresponds to the quantity of clotted fibrinogen that leads to the observed fibrin degradation products level.
Platelet deposition was quantified by measurement of a specific
platelet  granule membrane protein, P-selectin.8 After centrifugation of the plasmin-digested thrombus, the pellet was dissolved in 400 µL of a lytic buffer, three times frozen and thawed,
and then sonicated (4°C, 20 kHz) for 270 seconds. The lytic buffer
is made of PBS containing 1% Triton X-100 (Merck, Chelles, France), 16 mmol/L octyl- -D glucopyranoside (Boehringer Mannheim, Meylan,
France), 1 mmol/L EDTA (Merck), 20% sodium azide (Merck), 10 µmol/L
pepstatin A (Sigma, Saint-Quentin-Fallavier, France), 10 µmol/L
leupeptine (Sigma), 100 KIU/mL aprotinin, 0.1 mmol/L phenylmethyl
sulfonyl fluoride (PMSF) (Sigma). All samples of dissolved pellets were
stored at 80°C until assayed for P-selectin measurement by
immunoenzymoassay (Bender MedSystems, Vienna, Austria). The level of
P-selectin was measured both in the dissolved pellet and in the
supernatant of the plasmin-digested thrombus. Total number of platelets
deposited was calculated from the amount of P-selectin present in the
thrombus and that present in corresponding nonactivated platelets of
each blood donor determined before the ex vivo perfusion experiment.
Results were expressed as number of platelets
deposited/cm2. The area of coverslip surface exposed to the
blood flow is 0.9 cm2.
Determination of platelet activation and thrombin formation.
Platelet activation, thrombin generation, and fibrin formation were
determined by measuring plasma levels of thromboglobulin (TG),
thrombin-antithrombin complexes (T-AT), and fibrinopeptide A (FPA),
respectively. TG, T-AT, and FPA were measured in blood (3.2 mL)
collected in 0°C precooled tubes or syringes containing a mixture
(0.8 mL) of platelet inhibitors and anticoagulants (sodium citrate,
citric acid, theophylline, adenosine, dipyridamole, heparin, and
aprotinin), as described previously.7 Blood samples were collected first at the flow inlet of the chambers at the beginning of
the perfusion experiment to determine whether the different drug
regimens influenced the basal plasma levels of markers of platelet
activation and thrombin formation. Then, to measure the platelet
activation and thrombin formation secondary to blood interaction with
the thrombogenic surfaces, blood samples were collected at the flow
outlet of the chambers between 2.5- and 3-minute perfusions, by a
syringe pump (Harvard Apparatus, South Natick, MA), as previously
described.7 Blood samples were immediately centrifugated
(4,300g, 4°C, 30 minutes) and aliquots of plasma were
stored at 80°C until assayed. The plasma concentrations of
TG, FPA, and T-AT were measured by immunoenzymoassays (Assera-TG and Assera-FPA, Stago; Enzygnost-T-AT, Behring, respectively).
Other Laboratory Procedures
Red blood cell, leukocyte and platelet count, hemoglobin,
and hematocrit were measured by an electronic counting device (Model S
plus; Coulter Electronics, Hialeah, FL) during and after each period of
treatment. Subjects' compliance was checked by performing platelet
aggregation tests: blood was collected into a citrated vacutainer
(Becton Dickinson, ref 367704, Meylan, France) containing 0.5 mL of
0.129 mol/L trisodium citrate for 4.5 mL of blood. Platelet-rich plasma
was obtained after a centrifugation at 150g, 15 minutes, at
room temperature and platelet-poor plasma was obtained after a second
centrifugation at 1,500g, 15 minutes. Platelet count was
adjusted to 250 × 109/L by appropriate dilution of
the platelet rich-plasma with autologous platelet-poor plasma. Platelet
aggregation was performed with a platelet aggregometer (Coulter
Electronics) at 37°C and 1,000 rpm following calibration with
platelet-rich plasma (10% optical transmission) and platelet-poor
plasma (90% optical transmission). The aggregating agents were ADP
(2.5 and 5 µmol/L final concentration; Stago), equine collagen (3.3 and 10 µg/mL final concentration; Nycomed) and arachidonic acid (1 mmol/L final concentration, BioData Corp, Horsham, PA). The maximum
amplitude of platelet aggregation was measured and expressed as a
percentage of the difference between platelet-rich plasma and
platelet-poor plasma.
Statistical Analysis
Results were expressed as mean ± 1 standard error of mean
(SEM). A four-treatment, four-period, cross-over design,
called William's design and an analysis of variance
(ANOVA) mixed model (unstructured matrix when compound
symetry not appropriate) with terms for subject, period, first order
carry-over, and treatment were used for statistical analysis. In either
situation, the 95% confidence interval for each treatment difference
has been calculated. In all statistical hypothesis tests, the level of
significance P was < .05.
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RESULTS |
Study Population
Twenty-one male, white subjects, ages 20 to 30 years (mean age, 24 years) were enrolled in the study and randomly assigned to the
different treatments from February 1996 through July 1996. Five stopped
the trial prematurely. In 2 cases, the trial was stopped because of an
error in the treatment allocation and a technical problem in the
perfusion experiment, respectively. In 3 other cases, the trial was
stopped for adverse events: 2 subjects had low neutrophil count (1.78 × 109/L and 1.75 × 109/L under
ticlopidine and aspirin treatment, respectively; normal values: 2.0 to
7.0 × 109/L) and 1 had pruritus with ticlopidine.
Other minor adverse events, notably bleeding and gastrointestinal
disorders, were rare, very moderate, and they did not lead to the
interruption of treatment. In particular, the bleeding disorders were 1 purpura, 5 epistaxis, 2 rectorragia, and they were evenly distributed
within the three groups with active treatment (3 bleeding disorders
with aspirin, 3 with ticlopidine, and 2 with aspirin + ticlopidine).
Sixteen volunteers completed the study according to the protocol.
Effect of Treatment on Platelet Aggregation
Platelet aggregation was evaluated on blood samples drawn from
volunteers before each perfusion experiment. Results are shown in
Table 2. Ticlopidine inhibited ADP-induced
(P < .01), but not collagen-induced platelet aggregation. In
contrast, aspirin inhibited collagen-induced (P < .01), but
not ADP-induced platelet aggregation. Collagen-induced platelet
aggregation was significantly more inhibited by the combination
aspirin + ticlopidine than by aspirin alone (72% and 43% reduction
v 48% and 16% reduction as compared with placebo at 3.3 and
10 µg/mL, respectively, P < .01). In contrast, the
inhibitory effect of ticlopidine on ADP-induced platelet aggregation
was not enhanced by aspirin. Finally, aspirin, alone or associated with
ticlopidine, fully inhibited arachidonic acid-induced platelet
aggregation in all 16 volunteers.
Effect of Treatment on Baseline Level of Hemostatic System Activation
Baseline values of plasma markers of platelet (TG) and coagulation
activation (T-AT, FPA) were within normal ranges in the volunteers
treated by placebo (24.5 ± 2.9, 2.8 ± 0.2, and 2.0 ± 0.4 µg/mL, respectively). These values remained unchanged when the
volunteers were treated by aspirin, ticlopidine, or aspirin + ticlopidine (P > .2, data not shown).
Effect of Treatment on Thrombus Formation on Collagen
The respective effects of the three antithrombotic treatments on
collagen-induced thrombus formation are shown in
Fig 1. Representative light micrographs are
shown in Fig 2. In volunteers treated with placebo, and as previously found,8 thrombi formed on
collagen were rich in platelets and poor in fibrin. Compared with
placebo, aspirin and ticlopidine alone had a modest antithrombotic
effect. Aspirin and ticlopidine decreased platelet deposition by 29%
and 15%, respectively, (P > .2) and fibrin
deposition by 42% (P = .01) and 37% (P = .04),
respectively. In contrast, platelet thrombus formation and fibrin
deposition were almost totally blocked in all 16 volunteers treated by
aspirin + ticlopidine (P < .01 v placebo or each
treatment alone). Furthermore, the combination of aspirin + ticlopidine
was significantly more efficient in reducing thrombus formation than
the sum of the two active treatments (P < .05).
Interestingly, whereas platelet adhesion, which represents the percent
of surface covered with platelets, was enhanced by aspirin (24%
enhancement as compared with placebo, P < .04, Table 3), there was a slight, but
nonsignificant, decrease of platelet adhesion in volunteers treated by
aspirin + ticlopidine (17% reduction, P = .13).
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| Fig 1.
Effect of antiplatelet treatment on deposition of
platelets ( ) and fibrin ( ) on collagen-coated coverslips
evaluated by immunoenzymology. The surface was exposed for 3 minutes to
nonanticoagulated blood at a shear rate of 2,600 s1 from
volunteers given either aspirin (ASA), ticlopidine (T), or the
combination of both (ASA + T) or placebo (P). Values are means ± SEM (n = 16). *P < .05, **P < .01 versus
placebo.
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| Fig 2.
Representative light micrographs of blood-collagen
interactions. The surface was exposed at a shear rate of 2,600 s1 during 3 minutes to nonanticoagulated blood drawn
from volunteers given either placebo (A), aspirin (B), ticlopidine (C),
or the combination of both (D). The sections were prepared
perpendicular to the direction of the blood flow. Note that the
collagen coat is not visible because the staining procedure does not
contrast the fibrils. Original magnification × 1,000. Bar represents
10 µm.
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The plasma levels of markers of platelet activation (TG), thrombin
(T-AT), and fibrin (FPA) formation in volunteers treated by placebo
were, respectively, 7, 1.5, and 3 times higher in effluent blood
sampled distal to the site of thrombus formation than their respective
baseline levels (Table 4). Aspirin,
ticlopidine, and aspirin + ticlopidine prevented the release of -TG
in a comparable manner (P < .05). However, neither treatment
significantly decreased distal levels of T-AT and FPA.
Effect of Treatment on Thrombus Formation on TF
The results of thrombi formed on TF after the three therapeutic
regimens are shown in Fig 3. Thrombus,
which formed on TF-coated coverslips in volunteers treated by placebo,
were rich in fibrin and platelets, with platelets deposited almost
exclusively on top of fibrin meshes (data not shown). Neither treatment
significantly inhibited fibrin deposition or platelet thrombus
formation on this surface (P > .2).
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| Fig 3.
Effect of antiplatelet treatment on deposition of
platelets () and fibrin () on TF-coated coverslips evaluated by
immunoenzymatic method. The surface was exposed at a shear rate of
2,600 s1 during 3 minutes to nonanticoagulated blood
drawn from volunteers given either aspirin (ASA), or ticlopidine (T),
or the association of both (ASA + T) or placebo (P) according to the
protocol. Values are means ± SEM (n = 16).
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TF-dependent thrombus formation resulted in a much higher activation of
coagulation, but also of platelets, than collagen-dependent thrombus
formation: plasma levels of TG, T-AT, and FPA were, respectively, 4, 42, and 55 times higher with TF than with collagen (P < .05)
(Tables 4 and 5). Neither treatment
prevented the formation of these markers of platelet and coagulation
activation (P > .2).
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DISCUSSION |
Subacute and acute arterial thrombosis are main concerns, which have
promoted a search for new antithrombotic regimens. The combination of
aspirin and ticlopidine has been tested in several trials, notably
after coronary artery stent placement.3,18-22 Most of the
studies indicate a better efficacy3,20-22 of the antiplatelet drug combination on bleeding complications, hospital stay
duration, and stent thrombotic closure rates. Nevertheless, the most
recent study has suggested that stent thrombosis may be equally
prevented by aspirin or aspirin + ticlopidine.19 Thus, the
exact respective pharmacologic value of aspirin, ticlopidine, and the
combined therapy remains unknown.
The primary goal of the present study was to quantify the
antithrombotic effect of the combination of aspirin and ticlopidine on
thrombus formation in an ex vivo model of human thrombogenesis and to
compare it with the antithrombotic effect of aspirin and ticlopidine
alone in a double-blinded and randomized manner. We demonstrate that
the combination of ticlopidine and aspirin dramatically potentiated the
antithrombotic effect of each drug alone: aspirin plus ticlopidine
almost totally blocked thrombus formation on collagen substrate,
whereas aspirin or ticlopidine alone were only modestly effective.
However, the antithrombotic effect of the combination aspirin + ticlopidine depended on the nature of the thrombogenic surface, as it
was totally ineffective in preventing arterial thrombus formation
elicited by TF.
The ex vivo model of human thrombogenesis used in the present work
allows the study of thrombosis directly in man under various and
well-controlled blood flow conditions and on different types of
relevant vascular thrombogenic surfaces.4-8 Both collagen and TF are present in human atherosclerotic plaques and both have been
shown to be major determinants of thrombus formation at human atherosclerotic lesions after plaque disruption.9,10
Furthermore, mechanisms involved in thrombus formation in relation to
the antithrombotic efficacy of different antithrombotic regimens may
also depend on these substrates. Therefore, we have chosen to study
antithrombotic drugs by using these two major thrombogenic substrates
because they give different and complementary information of
antithrombotic efficacy and because they are relevant to various
clinical situations, which are complicated by thrombosis. Shear
conditions were controlled with wall shear rates of 2,600 s1, mimicking those encountered in moderately
stenosed coronary arteries.
Both morphometric and immunologic approaches were used to measure
thrombus formation, because together they give optimal and complementary information about thrombus formation.8
Morphometric analysis allows direct visualization of the thrombus (Fig
2) and it allows the determination of platelet-surface interaction, ie, platelet adhesion (Table 3). Besides its simplicity, the immunologic methods allow quantitative measurements of both platelets and fibrin of
the whole thrombi formed on the thrombogenic surface. The two methods
were performed on the same specimen by dividing the thrombotic deposits
into two equal large parts in parallel with the direction of the blood
flow, as deposition of platelets and fibrin varies in an
axial-dependent manner.23,24
Thrombi on the collagen substrate were rich in platelets, but poor in
fibrin. This platelet thrombus formation was modestly prevented by
aspirin, although not significantly different from the placebo group
(P > .2). Comparable data from previous studies using the
same thrombosis model are available, and these studies showed
furthermore that the antithrombotic effect of aspirin increases with
increasing shear rate.11,12 Ticlopidine shows a moderate antithrombotic effect, as well (P > .2). Comparable results
were obtained with a newer and more potent thienopyridine derivative, clopidogrel.14 In this model, significant antithrombotic
effect of clopidogrel on platelet thrombus formation at 2,600 s1 appeared after a longer period of drug intake,
ie, between 1 and 2 weeks of daily oral ingestion. The antithrombotic
effect obtained in subjects treated by the combination aspirin + ticlopidine was quite remarkable, as platelet thrombus formation was
almost totally blocked in all 16 volunteers treated by this therapeutic regimen. Statistical analysis indicated furthermore that there was a
synergistic effect of the association because the effect was exceeding
the sum of the effects of each treatment alone. Taken together, these
results indicate that both ADP and thromboxane A2 play a
major and complementary role in mediating collagen-induced platelet
thrombus formation. However, to achieve a significant antithrombotic
effect, one needs to block simultaneously both pathways, as either
pathway is able to compensate the other one.
Aspirin + ticlopidine blocked thrombus formation (Fig 1), but had no
effect on platelet adhesion (Table 3), indicating that different
mechanisms are involved in these two subsequent processes of thrombus
formation: ADP and thromboxane A2 are not involved in
platelet adhesion, whereas they are important mediators of platelet
aggregation. However, it is interesting to note that the total
inhibition of platelet thrombus formation by aspirin + ticlopidine did
not result in enhanced platelet adhesion. In the present study and in
other works performed with comparable models, antithrombotic agents,
which decreased platelet-platelet interactions, increased platelet
adhesion.12-14,25 There is indeed a balance between the
platelet supply to the reactive surface and the consumption of
platelets by growing thrombi23,24: when the platelet
consumption by the growing thrombi decreases, there is concomitantly an
increase in the platelet concentration in the blood layers streaming
adjacent to the collagen surface. This results in more platelets
available for adhering to collagen. Two steps are necessary for an
optimal platelet adhesion to collagen at this high shear condition: an
initial reversible glycoprotein Ib-mediated platelet adhesion
subsequently followed by a glycoprotein IIb/IIIa activation, which
allows to firmly tether platelets to the collagen surface through its
interaction with adhesive proteins.26 Thus, it is possible
that the simultaneous ingestion of aspirin and ticlopidine inhibited
platelet activation, and consequently glycoprotein IIb/IIIa activation
much more efficiently than aspirin alone, so that firm adhesion after
the initial weak collagen attachment did not take place because
platelets were subsequently washed out by the blood flow.
The three antiplatelet drug regimens affected coagulation as well, as
fibrin deposition was significantly reduced. In a previous comparable
study, clopidogrel significantly inhibited fibrin deposition on
collagen, notably at low shear rate.14 The apparent
anticoagulant effect provided by antiplatelet regimens may be the
direct consequence of a reduction in platelet deposition, as fibrin
deposition on collagen substrate occurs generally subsequent to
platelet thrombus formation.27 It is also possible that
this finding is a result of reduced platelet activation because
activated platelets amplify the coagulation cascade by binding
activated coagulation factors to form the tenase and prothrombinase
complexes.28
Mechanisms involved in thrombus formation elicited by TF substrate are
totally different from those on collagen. In contrast to collagen, TF
is a procoagulant surface where thombi are composed of both fibrin and
platelets with the latter deposited on top of the fibrin
mesh.6 In addition, platelet activation and thrombin formation occurring on this surface are very high, as indicated by the
plasma levels of TG, T-AT, and FPA, which are 4, 42, and 55 times
higher than with collagen, respectively (Tables 4 and 5). Moreover, on
TF, thrombin is a prime mediator involved in thrombus formation, as
indicated by a previous study using a selective inhibitor of factor Xa,
the recombinant tick anticoagulant peptide.29 We found that
neither antiplatelet regimen inhibited the thrombotic process on TF,
indicating thereby that ADP and thromboxane A2 were not
primarily involved in TF-induced thrombogenesis. It is possible that,
on TF, the platelet inhibitor effect of aspirin and ticlopidine may
have been overcome by high thrombin concentrations. The effect of the
antiplatelet regimen on thrombus formation induced by surfaces coated
with different levels of TF, which would generate different levels of
thrombin, remains therefore to be determined.
We compared the efficacy of different antiplatelet and anticoagulant
therapies in man using this experimental model. Criticisms with respect
to its significance and clinical relevance may be raised. Whereas TF
and types I and/or III collagens are important determinants of
the thrombogenicity of ruptured human atherosclerotic lesions,9,10 the respective amount and the respective role of each of these substrates may be highly variable depending on the
stage and depth of the lesion. Stent implantation results in blood
exposure to a mixture of thrombogenic materials including collagen, TF,
and foreign material. In our study, thrombus formation was only
determined on a collagen- or a TF-coated surface. But, as discussed
above, our thrombosis model has previously been shown to be useful in
evaluating different antithrombotic agents.8,13,14,28-30 One can note that results obtained with aspirin and ticlopidine on
collagen-coated surfaces appear consistent with clinical data, ie,
these drugs used alone moderately prevent arterial thrombus formation.
It is important to note that we examined the effect of antithrombotic
drugs on early acute platelet thrombus formation, as perfusion times
lasted only 3 minutes. This perfusion time was chosen because platelet
deposition and thrombus formation in this model are maximum at 3 minutes.8 In addition, studies of antithrombotic drug
effects on very early events of thrombus formation are important, as
these events have profound impact on later ones.31,32
Longer perfusion times give additional information on thrombus growth and thrombus stabilization. For example, whereas thrombin is not involved in initial thrombus formation on collagen, it plays a major
role in thrombus growth and thrombus stabilization.33 Thus,
whereas our experiments show that ADP and thromboxane A2 do
not play a major role in mediating initial thrombin-driven platelet
thrombus formation, whether these agonists play a role in thrombus
stabilization is unknown. The late antithrombotic effects of aspirin,
ticlopidine alone, or the combined aspirin + ticlopidine therapy remain
to be determined.
In the present study, aspirin and ticlopidine were administered for 3 and 8 days, respectively. It is possible that the antithrombotic effect
observed in volunteers treated by aspirin + ticlopidine may be present
earlier than day 8. We chose these times of administration with regard
to ticlopidine, which is known for its delayed onset of
action.34 Thus, pharmacologic studies have indicated that significant inhibition of ADP-induced platelet aggregation occurs only
after 3 to 5 days of oral administration of 250 mg twice daily of
ticlopidine.34,35 However, it is not known whether the
combination of aspirin + ticlopidine may shorten this delay of action.
In a recent clinical study, the beneficial effect of the combined
aspirin + ticlopidine therapy on thrombotic stent occlusions was
observed within 3 days after stenting.3 Therefore, because
the main clinical goal of antithrombotic therapy, notably in patients
undergoing coronary stent implantation, is to be effective as soon as
possible to prevent early stent thrombosis, a time-course of the
antithrombotic effect of the combined aspirin + ticlopidine therapy is
warranted.
Finally, ticlopidine has side effects, especially a risk of
neutropenia. A new thienopyridine derivative, chemically related to
ticlopidine, clopidogrel, has been recently developed. A large recent
study showed that this product was quite safe.36 Whether the addition of clopidogrel to aspirin yields equivalent antithrombotic effects as ticlopidine has not been established. However, our study
shows that ADP and thromboxane A2 have important and
complementary roles in mediating acute arterial platelet thrombus
formation and therefore that the combined ADP and thromboxane
A2 antagonism is a promising therapeutic approach.
| |
FOOTNOTES |
Submitted January 12, 1998;
accepted April 21, 1998.
Address reprint requests to Yves Cadroy, MD, PhD, Laboratoire de
Recherche sur l'Hémostase et la Thrombose, Pavillon
Lefèbvre, CHU Purpan, 31059 Toulouse CEDEX, France;
e-mail: cadroy.y{at}chu-toulouse.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank S. Claudel for her expert assistance in the
statistical analysis.
| |
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Abstract
Introduction
Abstract