© 1999 American Heart Association, Inc.
Thrombosis |
Antithrombotic Efficacy of the Vitamin K Antagonist Fluindione in a Human Ex Vivo Model of Arterial Thrombosis
Effect of Anticoagulation Level and Combination Therapy With Aspirin
From Laboratoire de Recherche sur l'Hémostase et la Thrombose, Pavillon Lefèbvre, CHU Purpan, Toulouse France (B.B., Y.C.); Service de Chirurgie Générale et Vasculaire CHU Purpan, Toulouse, France (J.-P.B.); Department of Biology, Division of Physiology, University of Oslo, Oslo, Norway (K.S.S.); and Centre d'Investigation Clinique, CHU Purpan, Toulouse, France (C.T.).
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Abstract |
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Abstract—Thrombin is a main mediator of arterial thrombus formation, and its inhibition is an important antithrombotic strategy. However, the place of vitamin K antagonists among the different therapeutic strategies for preventing arterial thrombus formation is still debated. We studied the antithrombotic efficacy of the vitamin K antagonist fluindione in a human ex vivo model of arterial thrombosis and determined whether aspirin enhances fluindione efficacy. Ten healthy male volunteers were randomly assigned to receive fluindione, alone or in combination with aspirin (325 mg/d). Fluindione was given at increasing doses to give a stable international normalized ratio (INR) between 1.5 and 2.0 and between 2.1 and 3.0. We induced arterial thrombus formation ex vivo by exposing collagen- or tissue factor (TF)–coated coverslips in a parallel-plate perfusion chamber to native blood for 3 minutes at an arterial wall shear rate of 2600 s-1. Platelet and fibrin deposition were measured by immunoenzymatic methods. Fluindione inhibited thrombus formation on TF-coated coverslips in a dose-dependent manner by 50% and 80% at INR 1.5 to 2.0 and INR 2.1 to 3.0, respectively (P<0.05). Fluindione in combination with aspirin inhibited TF-induced thrombus formation in a comparable manner. Collagen-induced thrombus formation was not reduced in subjects treated by fluindione. It was reduced by 50% to 60% in those treated with fluindione plus aspirin, regardless of the level of anticoagulation (P<0.05). Thus, the effectiveness of fluindione for preventing arterial thrombosis is dependent on the nature of the thrombogenic trigger. Fluindione is very effective in preventing TF- but not collagen-triggered thrombus formation. Aspirin enhances the antithrombotic effectiveness of fluindione, because combined treatment interrupts both TF- and collagen-induced thrombus formation.
Key Words: thrombosis • aspirin • anticoagulants
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Introduction |
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Oral vitamin K antagonists are effective and widely used agents for the treatment of venous thrombosis and the prevention of systemic embolism in patients with prosthetic heart valves and atrial fibrillation.1 However, the place of vitamin K antagonists among the different therapeutic strategies for preventing arterial thrombosis is still debated, despite the fact that thrombin is a primary mediator of arterial thrombus formation and that inhibition of thrombin generation is an important arterial antithrombotic strategy.2 Few studies suggest that vitamin K antagonists are useful in this setting,1 3 4 5 6 but information regarding the effectiveness of this therapy and the relationship between the level of anticoagulation as expressed by the international normalized ratio (INR) and the antithrombotic effect is still lacking.
Aspirin is widely used for the prevention and treatment of acute and chronic arterial diseases, but it has moderate clinical efficacy.7 Because the pathogenesis of thrombosis is complex and multifactorial, a combination of anticoagulant and antiplatelet therapy represents an attractive alternative strategy. Some studies have shown the clinical benefit of such combined treatment among mechanical heart valve recipients.8 9 Data regarding acute ischemic heart disease are more controversial.6 10 11 12 Thus, the important questions that remain are whether the combination of aspirin and oral anticoagulant therapy improves the antithrombotic efficacy of either drug alone in the setting of arterial thrombosis and whether this efficacy appears at low or high levels of anticoagulation.
The antithrombotic effect of drugs can be experimentally investigated in humans by use of an ex vivo model of thrombogenesis that closely mimics relevant clinical situations.13 14 15 16 17 In this model, native blood is drawn from volunteers through a parallel-plate chamber device, where it interacts with a thrombogenic surface in well-established blood flow conditions, mimicking wall shear rates encountered in moderately stenosed arteries (2600 s-1). Two relevant thrombogenic molecules, collagen and tissue factor (TF), which are present in atherosclerotic plaques and primarily responsible for thrombus formation in vivo,18 19 are exposed to blood. The efficacy of antithrombotic drugs is determined by quantification of the respective thrombus content in platelets and fibrin by immunoenzymatic methods.17 20 This model has been used to investigate numerous antithrombotic strategies, and results appear consistent with clinical data.17 21 22 23 24 25 26 27 28 29
In the present study, we used this ex vivo model of acute initial arterial thrombus formation to determine the efficacy of the vitamin K antagonist fluindione in preventing arterial thrombosis, the level of anticoagulation required to obtain an antithrombotic effect, and whether fluindione in combination with aspirin improves the antithrombotic efficacy of fluindione alone. We designed an open, randomized study in healthy male volunteers. Fluindione was administered either alone or in combination with aspirin, and it was given to obtain a low (INR 1.5 to 2.0) and a conventional (INR 2.1 to 3.0) level of anticoagulation, respectively.
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Methods |
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Subjects
The study population consisted of 10 healthy white male volunteers aged 18 to 40 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 <10 cigarettes per day, and they did not smoke on the day of the perfusion experiments. Clinical chemistry, hematological, 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, France).
Study Design
This monocentric, randomized controlled study was performed in
the Center for Clinical Investigation at Hôpital Purpan,
Toulouse, France. After selection for the trial, each volunteer
followed 3 sessions, in random order, in which either no treatment,
fluindione alone (Procter and Gamble Pharmaceuticals), or combined
fluindione plus aspirin (325 mg/d, UPSA) was given. These 3 sessions
were separated by a washout period of 3 to 6 weeks. For the control
session, volunteers performed only the blood donation for experimental
thrombogenesis. For the fluindione sessions, blood donation was
performed when INR was stable within the respective target ranges, ie,
1.5 to 2.0 and 2.1 to 3.0. The initial dose of fluindione was set at 15
mg/d. INR was measured 3 times per week, and dosage was adjusted until
INR had stabilized first between 1.5 and 2.0 and then between 2.1 and
3.0 for 
3 consecutive days. In the fluindione plus aspirin session,
aspirin treatment was initiated on the first day fluindione was given.
A clinical follow-up was done on each visit at the study center, and at
the end of each period of treatment, 10 mg of vitamin K was
administered to reverse the anticoagulant treatment. All adverse
effects were recorded, and appropriate follow-ups were
scheduled.
Preparation of Thrombogenic Surfaces
The thrombogenic molecules were coated on Thermanox plastic
coverslips (Miles Laboratories) as previously
described.14 15 17 26 Equine collagen (Collagen Reagent
Horm, Nycomed) was spray-coated onto plastic coverslips to a
final density of 0.5 µg/cm2. TF, purified from
human placenta (Thromborel, Behring), was diluted 1:133 in coating
buffer (0.1 mol/L sodium carbonate, pH 9.5), and coverslips were
incubated in 2 mL of the Thromborel dilution for 24 hours at 4°C.
Perfusion Experiments
Two perfusion experiments were performed after each period of
treatment, with collagen and TF as the thrombogenic surface,
respectively. Perfusion experiments were performed with a
parallel-plate perfusion chamber device at
37°C.14 15 17 26 After blood samples were collected (see
below), native blood was drawn directly from an antecubital vein of the
volunteers through a 19-gauge infusion set (Ohmeda) 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) placed distal to
the chamber. The wall shear rate at the thrombogenic surface was 2600
s-1. The blood perfusion experiment lasted for 3
minutes and was followed by a 30-second perfusion of PBS to wash out
blood from the flow channel. The coverslip covered by thrombotic
deposits was removed from the chamber and divided into 2 equal parts
parallel to the direction of the blood flow, as described
previously.17 26 One half 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 epoxy resin. A second perfusion experiment was
subsequently performed with blood drawn directly from a contralateral
cubital vein over a TF-coated coverslip.
Determination of Thrombus Formation
Immunological Determination of Fibrin and Platelet
Deposition
Fibrin deposition was quantified by immunological determination
of fibrin-degradation products of plasmin-digested thrombi, as
described previously.20 After perfusion, the thrombus was
immediately incubated in 2 mL of a plasmin solution (Chromogenix; 0.7
IU/mL in Tris-buffered saline, pH 7.4) for 30 minutes under gentle
shaking at 37°C. Plasmin digestion was stopped by the addition of
aprotinin (2000 KIU/mL, Sanofi). The solution was centrifuged
(4°C, 4300g, 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 by an immunoenzymatic assay (Asserachrom D-Di, Stago). The
amount of deposited fibrin was determined directly from the levels of
fibrin-degradation products expressed in fibrin equivalent units as
indicated by the manufacturer.
Platelet deposition was quantified by measurement of a specific
platelet 
-granule membrane protein, P-selectin.17
After centrifugation of the plasmin-digested thrombus,
the pellet was dissolved in 400 µL of a lytic buffer, frozen and
thawed 3 times, and then sonicated (4°C, 20 kHz) for 270 seconds. The
lytic buffer was composed of PBS containing 1% Triton X-100 (Merck),
16 mmol/L octyl ß-D-glucopyranoside (Boehringer
Mannheim), 1 mmol/L EDTA (Merck), 20% sodium azide (Merck),
10 µmol/L pepstatin A (Sigma), 10 µmol/L leupeptin
(Sigma), 100 KIU/mL aprotinin, and 0.1 mmol/L PMSF (Sigma). All
samples of dissolved pellets were stored at -80°C until assayed for
P-selectin measurement by immunoenzymatic assay (Bender MedSystems).
The level of P-selectin was measured both in the dissolved pellet and
in the supernatant of the plasmin-digested thrombus. We calculated the
total number of platelets deposited by dividing the amount of
P-selectin present in the thrombus by that present in
nonactivated platelets of healthy blood donors (321±14
ng/108 platelets, n=26).
Morphometric Determination of Platelet Adhesion
Microscopic evaluation of platelet adhesion was performed on
epoxy-embedded semithin sections (1 µm thick) stained with
toluidine blue and basic fuchsin, as previously
described.17 26 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,30 performed by light microscopy at
1000x magnification, was used to quantify the percent coverage with
fibrin recognized as circular spots associated or not associated with
platelets (percent fibrin deposition) and with platelets
adherent to collagen or fibrin (percent platelet adhesion). We
performed these morphometric evaluations at 10-µm intervals along the
surface by moving the section along an eyepiece micrometer
in the microscope ocular.
Determination of Platelet Activation and Thrombin
Formation
Platelet activation and thrombin generation were determined
by measurement of plasma levels of
ß-thromboglobulin (ßTG) and
thrombin-antithrombin complexes (T-AT), respectively. ßTG and T-AT
were measured in blood (3.2 mL) collected in 0°C precooled 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.16 17
Blood samples were collected at the flow outlet of the chambers between
2.5 and 3 minutes of perfusion by a syringe pump (Harvard
Apparatus). Blood samples were immediately
centrifuged (4300g, 4°C, 30 minutes), and aliquots
of plasma were stored at -80°C until assayed. Plasma concentrations
of ßTG and T-AT were measured by immunoenzymatic assays
(Assera-ßTG, Stago, and Enzygnost-T-AT, Behring,
respectively).
Other Laboratory Procedures
Red cell, leukocyte, and platelet counts, hemoglobin, and
hematocrit were measured by an electronic counting device (model S
plus, Coulter Electronics) during and after each period of treatment.
Coagulation times were determined with fresh blood samples collected in
citrated evacuated container tubes (0.5 mL of 0.129 mol/L trisodium
citrate for 4.5 mL of blood; Becton Dickinson) by use of a coagulometer
(STA, Stago). Prothrombin times (PTs) were measured with Thromborel
(Behring; international sensitivity index [ISI] 1.03) and expressed
as INRs according to the formula INR=(patient PT/control
PT)ISI. One-stage assays of factors II, VII, and
X were performed with plasmas deficient in factor II
(Biomérieux), VII (Stago), or X (Stago), respectively. One-stage
assays of coagulation factor IX were performed with automated APTT
(Organon Teknika) and factor IX–deficient plasmas (Behring). We
checked subjects' compliance to aspirin treatment by performing
platelet aggregation tests with arachidonic acid
(1 mmol/L final concentration; BioData Corporation) and a
platelet aggregometer (Coulter Electronics).
Statistical Analysis
Statistical analyses were performed with the PCSM
program (Deltasoft). Volunteers were randomized according to a
3-treatment, 3-period crossover design. Results were expressed as
mean±SEM. Statistical comparison was performed with an ANOVA followed
by a Neuman-Keuls test when the P value was 
0.05. In
comparisons of 2 groups, probability values were calculated by the
Wilcoxon test. Least squares regression analysis was
used to determine the correlation coefficient (r) and the
significance level (P) for relationships between
variables. To assess the independence of relationships, a multiple
regression analysis with calculation of the standardized
partial regression coefficient (ß) was performed. Probability values
<0.05 were considered significant.
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Results |
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Study Population
Ten white male subjects aged 22 to 31 years (mean age 24 years) were enrolled in the study and randomly assigned to the different treatments from January 1997 through January 1998. None prematurely stopped the trial. Minor adverse events, notably bleeding and gastrointestinal disorders, were rare and moderate and did not lead to interruption of treatment. One small cutaneous hematoma was observed in each of 2 subjects with a conventional anticoagulation level of fluindione combined with aspirin.
Blood Parameters
Table 1
summarizes the mean
doses and durations of fluindione treatment required to obtain INRs
within the target range. The respective target INRs, ie, 1.5 to 2.0 and
2.1 to 3.0, were obtained in all patients (Table 1) except 1 who
received fluindione plus aspirin therapy, in whom INR was 3.2 when
blood donation for experimental thrombogenesis was performed. Oral
anticoagulant treatment led to a decline in the activity of factors II,
VII, IX, and X that was dependent on the level of anticoagulation. All
these parameters were comparable, even with the combination
of aspirin and fluindione (P=NS). Finally,
platelet aggregation to arachidonic acid was fully
inhibited (>90%) in all 10 volunteers given aspirin plus fluindione,
indicating good compliance with aspirin treatment.
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Effect of Treatment on TF-Induced Thrombus Formation
Thrombi formed on TF-coated coverslips in control
experiments were rich in fibrin and platelets, with platelets
deposited almost exclusively on top of fibrin meshes. Fluindione
inhibited platelet and fibrin deposition, as measured by
immunoenzymatic methods (Figure 1), in a
dose-dependent manner. At low anticoagulation levels (INR 1.5 to 2.0),
platelet and fibrin deposition were prevented by 50% and 55%,
respectively (P<0.05 versus control). Conventional
fluindione treatment (INR 2.1 to 3.0) reduced platelet and fibrin
deposition by 78% and 84%, which was significantly more potent than
the lower fluindione anticoagulant treatment (P=0.02). When
aspirin was combined with fluindione, platelet and fibrin
deposition were reduced in a dose-dependent manner comparable to
that obtained with fluindione alone (Figure 1).
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Morphometric analysis confirmed that fibrin deposition and thus
platelet adhesion to fibrin meshes were reduced in a dose-dependent
manner by fluindione (Table 2). However,
in subjects treated with the combination of aspirin and
conventional-intensity fluindione, platelet adhesion to fibrin
meshes was significantly more reduced than in those treated with
fluindione alone (P<0.05; Table 2).
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Fluindione also prevented ßTG release and T-AT formation in a
dose-dependent manner (P<0.05 versus control; Figure 2). A comparable effect was found in
subjects treated with the combination of aspirin and fluindione.
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The antithrombotic effect in the presence or absence of aspirin was
significantly correlated to INR and to the plasma levels of factors X
and II but not of factors VII and IX (Table 3). Due to interrelationships between INR
and the plasma levels of factors II, VII, IX, and X, we performed a
multiple regression analysis to analyze the
independence of the association. Using this test, we found that the
antithrombotic effect of fluindione was more specifically correlated to
plasma levels of factor X (ß=0.60, P<0.001 for
platelet reduction and ß=0.57, P<0.001 for fibrin
reduction).
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Effect of Treatment on Collagen-Induced Thrombus Formation
Thrombi that formed on collagen-coated coverslips in controls were
rich in platelets and poor in fibrin (Figure 3). Platelet deposition was not
reduced in subjects treated by fluindione. In contrast, it was reduced
by 48% in those treated with fluindione plus aspirin, regardless of
the level of anticoagulation (P<0.05 versus control).
Similarly, fibrin deposition was not prevented by fluindione alone,
whereas it was decreased by 60% when fluindione was combined with
aspirin (P<0.05 compared with fluindione alone). Probably
due to large interindividual variations in fibrin deposition within
control experiments (range 0.08 to 8.34
µg/cm2), the effect of an aspirin adjunct on
fibrin deposition was not statistically significant compared with
controls. The antithrombotic effect was correlated neither to INR nor
to plasma levels of factors II, VII, IX, or X (P>0.5).
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Whereas platelet adhesion to collagen, as measured by morphometry,
was not altered by fluindione, there was a slight but significant
increase of platelet adhesion in volunteers treated with aspirin
plus fluindione (13% enhancement compared with fluindione INR 2.1 to
3.0, P<0.05; Table 2
).
Collagen-dependent thrombus formation resulted in a lower activation of
platelets and coagulation than TF-dependent thrombus formation:
plasma levels of ßTG and T-AT were respectively 2 and 8 times lower
with collagen than with TF (Figures 2 and 4). ßTG release was not prevented by
fluindione alone but was prevented by 55% when fluindione was combined
with aspirin (P<0.01 versus control and fluindione alone).
Neither treatment prevented T-AT formation.
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Discussion |
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The place of vitamin K antagonists among the different therapeutic strategies for preventing arterial thrombus formation and the clinical benefit of adding aspirin to oral anticoagulants in this setting are still debated.1 In the present study, we demonstrated (1) that fluindione is an effective agent for preventing experimental arterial thrombosis in humans, but that its efficacy is related to the plasma anticoagulation level and is dependent on the thrombogenic trigger, and (2) that combined aspirin plus fluindione therapy is effective regardless of the thrombogenic trigger, which indicates that the antithrombotic potency of combined therapy is higher than that of fluindione alone.
Fluindione reduced fibrin and platelet deposition on TF in a
dose-dependent manner (Figure 1). This finding is not
surprising, because thrombin generation plays a key role in the
formation of mixed fibrin- and platelet-rich thrombi on
TF.24 25 However, fluindione appears particularly
effective and much more potent than unfractionated
heparin.17 Because platelets are largely recruited
into arterial thrombi by thrombus-bound thrombin rather
than soluble thrombin,31 32 33 we presume that by decreasing
the functional levels of the vitamin K–dependent coagulation factors,
fluindione reduces the generation of thrombin and thus the amount of
thrombus-bound thrombin.
The antithrombotic effect of fluindione reflected the plasma level of
anticoagulation, as expressed by the INR (Table 3). Fluindione
was most effective in preventing platelet thrombus formation at
conventional anticoagulation levels (INR 2.1 to 3.0). Similarly,
clinical studies3 4 5 have shown that oral anticoagulants
at high levels of anticoagulation were effective in secondary
prevention of myocardial infarction. However, in the present study,
fluindione at low anticoagulation levels (INR 1.5 to 2.0) also was
effective in preventing TF-induced thrombus formation (Figure 1).
This level of anticoagulation is reported to be effective in
primary prevention of ischemic heart disease in
men.6
TF-induced thrombus formation was inhibited by fluindione plus aspirin
in a dose-response manner comparable to that of fluindione alone
(Figure 1). Nevertheless, some data suggest that aspirin does
enhance the antithrombotic efficacy of fluindione. First, conventional
fluindione plus aspirin therapy was effective in all subjects:
platelet and fibrin deposition was
<1.3x107/cm2 and <0.6
µg/cm2 in each subject treated with fluindione
plus aspirin, whereas it was not negligible in 3 subjects treated with
fluindione alone, ie,
>2x107/cm2 and >1.7
µg/cm2, respectively. In addition, the
morphometric analysis indicated that by inhibiting fibrin
deposition, fluindione plus aspirin reduced the percent of
platelets adherent to fibrin meshes significantly more than
fluindione alone (P<0.05; Table 2).
Fluindione exerted no antithrombotic effect on collagen-coated surfaces
(Figure 3). Thus, the present data confirm that thrombin is
not involved in the mediation of collagen-triggered initial thrombus
formation at a wall shear rate of 2600
s-1.17 24 29 However, because
thrombin plays a major role in thrombus growth and
stabilization,28 34 the late antithrombotic effects of
vitamin K antagonists on collagen surfaces remain to be
determined.
On collagen, the combination of aspirin and fluindione, regardless of
the INR, resulted in a significant decrease in platelet and fibrin
deposition (Figure 3). Because fluindione alone had no
antithrombotic effect on this surface and because the effect of
fluindione plus aspirin was comparable with low and conventional levels
of anticoagulation, the antithrombotic efficacy of fluindione plus
aspirin on collagen may reflect the effect of aspirin alone. Thus, in a
previous study,26 platelet and fibrin deposition on
collagen were prevented by aspirin in a comparable manner.
Platelet adhesion on collagen was increased in subjects treated
with fluindione plus aspirin compared with subjects not given aspirin
(Table 2). This also has been found in previous studies using
comparable perfusion models.21 22 23 26 Indeed, when
platelet consumption by growing thrombi is decreased by agents that
interrupt platelet-platelet interactions, such as aspirin,
there is concomitantly an increase in the platelet concentration in
the blood layers streaming adjacent to the collagen surface, which
results in more platelets available to adhere to collagen.
Overall, the antithrombotic effect of fluindione plus aspirin was greater than that produced by either drug used alone, because both TF- and collagen-triggered thrombus formation was prevented when these drugs were combined. The 2 agents exerted independent effects, because fluindione was principally responsible for the antithrombotic effect on TF, whereas aspirin was effective on collagen. These results are in agreement with clinical studies9 in which the addition of aspirin to oral anticoagulants in patients with mechanical heart prostheses was more effective in preventing systemic embolism than oral anticoagulants alone.
The antithrombotic effect of fluindione was correlated to plasma levels
of factors II and X but not to plasma levels of factors VII and IX
(Table 3). In addition, the antithrombotic effect was more
correlated to plasma levels of factor X than to those of factor II. In
rabbits, reduction of prothrombin and possibly of factor X was also
more important than reduction of factor VII and IX for the
anticoagulant effect of vitamin K
antagonists.35 Thus, these results confirm the
importance of the reduction of factors X and II for the antithrombotic
effect of vitamin K antagonists, but it is possible that
the respective roles of factors II and X depend on the type of
thrombosis.
Finally, it has been reported that high doses of aspirin (>1500
mg/d) augment the anticoagulant effect of vitamin K
antagonist.36 We did not observe such results
with a lower dosage of aspirin (325 mg/d), because the daily dose of
fluindione required to reach the target INR (Table 1) or the
time period needed to reach a stable level of anticoagulation (Table 1) was comparable even when fluindione and aspirin were
combined.
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Footnotes |
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Reprint requests to Yves Cadroy, MD, PhD, Laboratoire de Recherche sur l'Hémostase et la Thrombose, Pavillon Lefèbvre, CHU Purpan, 31059 Toulouse Cedex, France.
Received December 21, 1998; accepted February 9, 1999.
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