(Circulation. 1995;91:1568-1574.)
© 1995 American Heart Association, Inc.
Articles |
Activated Clotting Time as an Appropriate Test to Compare Heparin and Direct Thrombin Inhibitors Such as Hirudin or Ro 46-6240 in Experimental Arterial Thrombosis
From the Pharma Division (A.G., T.T., S.R.), F. Hoffmann-La Roche Ltd, Basel, Switzerland; and Service de Chirurgie Cardiaque et de Chirurgie Experimentale (J.P.C.), Nancy, France.
Correspondence to Dr Sébastien Roux, Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4002 Basel, Switzerland.
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Abstract |
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Background Specific thrombin inhibitors are considered to be more potent antithrombotics than heparin. However, the relation between the systemic anticoagulation generated by thrombin inhibitors and their antithrombotic effect is not well defined. In a guinea pig carotid thrombosis model, the activated clotting time (ACT), the activated partial thromboplastin time (aPTT), and thrombin-generation tests were evaluated for their ability to predict the arterial antithrombotic effect of direct thrombin inhibitors such as hirudin and Ro 46-6240 compared with heparin.
Methods and Results Thrombosis of the carotid artery was induced by subendothelial damage in guinea pigs, and the subsequent cyclic flow variations were monitored. The effects of pretreatment with intravenous heparin, hirudin, and Ro 46-6240 were tested. After doubling the baseline aPTT, 1 IU · kg-1 · min-1 heparin was inactive, whereas either hirudin or Ro 46-6240 (30 µg · kg-1 · min-1) prevented thrombus formation by 80%. Heparin (10 IU · kg-1 · min-1) induced the same antithrombotic effect but with indefinite aPTT prolongation. However, for similar prolongation of the ACT, the three compounds had equivalent antithrombotic effects. Thrombin generation was predictive of the antithrombotic effect of the thrombin inhibitors but not of heparin.
Conclusions The arterial antithrombotic effect of direct thrombin inhibitors, when compared with those of heparin, should be evaluated by the ACT and not the aPTT or thrombin-generation assays. For a "therapeutic" aPTT prolongation, thrombin inhibitors induce higher systemic anticoagulation than does heparin and thus might unduly have higher bleeding liability.
Key Words: anticoagulants • coagulation • heparin • thrombosis • hemostasis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The primary role of platelets in arterial thrombotic occlusion has been documented in patients with coronary artery disease1 as well as in animal models.2 3 Besides its central role in the coagulation cascade,4 thrombin is a potent platelet agonist and thus constitutes an interesting target for drugs that would prevent the formation of fibrin- and platelet-rich thrombi induced by thrombin.
Recently, new specific thrombin inhibitors such as hirudin and hirulog, which are derived from Hirudo medicinalis,5 were shown to be promising new antithrombotics in various animal models and tested in clinical situations associated with coronary thrombosis.6 7 8 9 Their superiority over heparin has been predicated on the fact that for a similar prolongation of the activated partial thromboplastin time (aPTT), the specific thrombin inhibitors were more antithrombotic than heparin.10 11 12 13 14 15 16 Today, the aPTT has wide acceptance in clinical use and is used to monitor heparin therapy for the prevention or the treatment of deep venous thrombosis. However, the aPTT has serious limitations when high doses of heparin are required, such as during extracorporeal circulation or percutaneous transluminal coronary angioplasty (PTCA).7 17 18 In these cases, the activated clotting time (ACT) is usually preferred. The ACT is a whole-blood coagulation test that mainly evaluates the intrinsic coagulation pathway and is routinely used for monitoring heparin treatment during cardiac surgery.19 20
In addition, the aPTT might not be adequate for comparing anticoagulants with different mechanisms of action. For example, in the recent GUSTO 2a trial, the effects of heparin in acute coronary syndrome were to be compared with those of hirudin. Dosing was based on a similar prolongation of the aPTT, and this trial had to be interrupted because of excessive major bleedings in the hirudin arm. These adverse events illustrate that both monitoring and dose requirements of new anticoagulants are ill defined.
The goal of the present study was to compare in a guinea pig arterial thrombosis model21 the antithrombotic effects of unfractioned heparin with those of two specific thrombin inhibitors: recombinant hirudin5 and Ro 46-6240, a new piperidine-derived thrombin inhibitor.22 23 24 Furthermore, we evaluated the effects of these three compounds on the aPTT, the ACT, the bleeding time, and a thrombin-generation test.
Unlike clotting times, the thrombin-generation test evaluates the rate of development of thrombin activity through both the intrinsic and extrinsic coagulation pathways.25 This test has proved its value both for testing the mode of action of anticoagulants and to explore certain pathological conditions.26
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Instrumentation
GOHI male guinea pigs (355 to 690 g) (BRL) were used for this study. Guinea pigs were anesthetized with 90 mg/kg ketamine hydrochloride IM (Parke Davis) and 12 mg/kg xylazine IM (Bayer). Arterial femoral catheters were inserted for blood pressure measurement (2F Microtip, Millar) and for blood sampling. A left jugular vein catheter was also inserted for drug infusion with a pump (Infors). The right carotid artery was dissected free, and a 1-mm-diameter Doppler probe (20 MHz, Triton) was secured to monitor the blood flow. Blood pressure (mm Hg), carotid flow (V), and heart rate (beats per minute) derived from an electrotachometer coupled to the arterial pressure signal were recorded on a Graphtec Linearrecorder VII (model WR 3101, Hugo Sachs).
Cyclic Flow Variations
The model generates a platelet-rich
carotid thrombus by
subendothelial injury but without any stenotic device.21
Briefly, 2 mm distal to the Doppler flow probe, the damage of the
subendothelium was induced by pinching a 1-mm segment of the dissected
carotid artery with forceps (BD 311, 3.5 in, Aesculap) over 2 seconds.
Without treatment after the damage, the carotid blood flow started to
decline until complete occlusion, similar to the Folts dog coronary
model when severe stenosis is applied.2 When flow
approached zero, a gentle shaking of the carotid artery dislodged the
occlusive thrombus and restored flow as previously
described.2 When no cyclic flow variations (CFVs) were
observed, the same procedure was repeated 5 minutes later and the
number of pinches necessary to induce the CFVs were counted over the
40-minute observation period. Several pinches are likely to increase
the thrombogenicity of the subendothelial layers.27
Therefore, we assumed there would be a better protection of the animal
by the applied treatment with more frequent pinches needed to initiate
the CFVs. Thus, a thrombosis index was determined as the ratio of the
number of CFVs to the number of pinches.
Coagulation Tests
Blood was collected at baseline and at the
end of the experiment
for blood cell count and hematocrit (Digicell 800, Contraves). Bleeding
time (BT) was measured in duplicate with a 22-gauge needle from two
punctures of ear arterioles and adapted from a technique previously
described.28 At the end of the experiment, BT was measured
again. Blood was collected without anticoagulant for determination of
the ACT (HR-ACT cartridge, Hemotec Inc) and into
vol of 108
mmol/L citrate for determination of the aPTT measured with a
semiautomated fibrometer coagulation timer using ellagic acid as
activating reagent (Becton Dickinson). The ACT cartridge contains 120
mg/mL kaolin. To determine the influence of blood cells on the ACT, we
infused a separate group of guinea pigs with either saline, heparin (10
IU · kg-1 · min-1), or hirudin
(30
µg · kg-1 · min-1) or Ro
46-6240
(30 µg · kg-1 · min-1).
After a
30-minute infusion, arterial blood was withdrawn and the ACT was
measured on whole blood, citrated/recalcified whole blood for
determining the influence of citrate, and citrated/recalcified
platelet-poor plasma for determining the role of platelets in this
assay.
Thrombin-Generation Assay
Guinea Pig Defibrinated
Plasma
Defibrinated plasma was obtained according to a method
previously described25 and adapted to guinea pigs. Blood
from guinea pigs was collected on trisodium citrate (10.8 mmol/L).
Platelet-poor plasma was prepared by two centrifugations performed at
10 000g at 4°C for 10 minutes. The platelet-poor plasma
(<5000 platelets/µL) was prewarmed at 37°C for 10 minutes and
defibrinated by incubating with 1.8 U/mL Reptilase (batroxobin, a
generous gift of Dr K. Stocker, Pentapharm), letting a clot form for 1
hour at 37°C and keeping the clotted plasma on ice for 30
minutes.
Determination of Thrombin Generation
Thrombin generation was measured essentially as described by
Beguin et al25 and adapted to guinea pig plasma. Briefly,
the thrombin inhibitors were incubated with defibrinated platelet-poor
plasma for 5 minutes at 37°C in a microtiter plate (Dynatech M 29 A,
Embrach). Thrombin formation was started by adding 50 µL of a
solution containing CaCl2 (final concentration, 14 mmol/L)
and a mixture of rabbit brain cephalin and ellagic acid (final
dilution, Actin Dade, Dade Diagnostics) for
studying the
intrinsic coagulation pathway and rabbit brain thromboplastin (final
dilution, 
) for the extrinsic coagulation
pathway. In
preliminary experiments, these dilutions of activators were shown to
generate approximately the same peak of thrombin amidolytic activity
(see below). Three concentrations of inhibitors and one control (buffer
instead of inhibitor) were tested simultaneously per experiment. We
tested concentrations of the inhibitors corresponding to those measured
in vivo at steady state in the three dosing groups. The concentrations
of hirudin and Ro 46-6240 were estimated from an aPTT standard curve,
and those of heparin were measured using an antifactor IIa chromogenic
assay.
Thrombin generation was assessed at 43-second intervals by subsampling 10 µL of the activation mixture into 250 µL of the thrombin-specific chromogenic substrate S-2238 (Endotell) at a concentration of 400 µmol/L. The amidolytic activities in the plasma samples were followed at 405 nm for 24 seconds at 37°C in a kinetic microtiter autoreader (Molecular Devices). The thrombin generation was quantified in terms of the peak of thrombin amidolytic activity. The percentage of inhibition of thrombin generation was expressed using the following formula: ([peak mOD/min control - peak mOD/min experiment]/peak mOD/min control)x100. The results shown are the mean of three determinations.
ACT Versus aPTT in Different Species
The in vitro dose
responses of the aPTT versus the ACT were
compared for heparin and Ro 46-6240 in rabbit and human blood.
Increasing concentrations of either heparin (0.01 to 10 U/mL) or Ro
46-6240 (0.01 to 10 µmol/L) were added to recalcified whole blood or
recalcified platelet-poor plasma for the ACT and the aPTT,
respectively.
Design of the In Vivo Study and Statistical Analysis
The
treatment doses were chosen based on a pilot study to
determine a nonantithrombotic dose (albeit prolonging the aPTT), an
intermediate antithrombotic dose, and a fully antithrombotic dose. The
guinea pigs were randomly assigned to receive an infusion (33
µL · kg-1 · min-1) of
either saline
(placebo, n=10), heparin (Liquemine containing 150 IU/mg, F.
Hoffmann-La Roche Ltd) (1, 10, and 30
IU · kg-1 · min-1, n=5
each),
recombinant hirudin (CGP 39393 kindly provided by Dr R. Wallis,
CIBA-GEIGY) (10, 30, and 100
µg · kg-1 · min-1,
n=5
each), and Ro 46-6240 (see below) (10, 30, and 100
µg · kg-1 · min-1,
n=5
each). To shorten the time for reaching plasma concentration at steady
state, each infusion was preceded by a bolus (100 µL/kg). For each
dose, the bolus amounted to the quantity of compound that would have
been delivered in a 3-minute infusion (eg, the low-dose heparin or
hirudin bolus was 3 IU/kg and 30 µg/kg, respectively). Ten minutes
after starting the infusion, the vascular damage was initiated as
described in "Cyclic Flow Variations."
The experimental procedure was approved by the Animal Care and Use Committee of the district of Basel and met the standards set by the US Department of Health and Human Services.
The results are expressed as mean±SEM. The data were analyzed by a two-way ANOVA with treatment and dose as factors. When an F value was significant (<.05), Fisher's protected LSD tests were performed to determine difference between groups.
Ro 46-6240
Ro 46-6240
(N-[N4-[[(S)-1-amidino-3-piperidinyl]methyl]-N2-(2-naphtalene-sulfonyl)-L-asparaginyl]-N-cyclopropylglycine;
F. Hoffmann-La Roche Ltd) is a new synthetic, potent
(Ki, 0.3 nmol/L), active-site, reversible
and selective thrombin inhibitor with a half-life of 10 to 20 minutes
in rats, guinea pigs, and monkeys.22 Ro 46-6240 was
dissolved in 0.2 mol/L lactic acid and 2.5% glucose. The solution was
adjusted to pH 4 with Ringer's lactated solution and diluted with
saline.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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In Vivo Thrombogenesis
Heart rate, mean arterial pressure, bleeding time, hematocrit, and platelet count at baseline were similar among the tested groups and remained stable during the 40-minute observation period (data not shown).
Fig 1A
shows that after a brief pinch in a
control
animal, the carotid blood flow started to decline until interruption of
blood flow. The following CFVs indicated ongoing and stable
thrombogenesis. As seen in Fig 1B, a full antithrombotic effect
prevented further pinches from inducing CFVs.
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All three treatments
inhibited thrombus formation in a
dose-dependent manner (Fig 2a). Ro 46-6240, hirudin at
30 µg · kg-1 · min-1, and
heparin at 10
IU · kg-1 · min-1
induced an 80% reduction of the thrombosis index. The highest dose was
statistically no more efficacious than the intermediate dose.
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Plasma Concentrations, Bleeding Time, and Coagulation
Variables
The plasma concentrations of the inhibitors at low,
intermediate,
and high dose were approximately 0.056, 0.57, and 1.70 µg/mL for Ro
46-6240; 0.7, 9, and 20 µg/mL for hirudin; and 0.20, 2.25, and 7 U/mL
for heparin, respectively.
As seen in Fig 2b
, the three
compounds induced a similar dose-related
prolongation of the ACT (P=.2). At the doses that reduced
the thrombosis index by approximately 80%, the prolongations of ACT
(2.7 times the control value) were similar for the three compounds.
In
contrast to the ACT, the aPTT showed a dramatic interaction between
dose and treatment (P<.001) (Fig 2c). Heparin at 1
IU · kg-1 · min-1 increased
the aPTT
up to twice the control value (43±3 versus 20±1 seconds) but
without
an antithrombotic effect. At intermediate doses, Ro 46-6240 and hirudin
were similarly antithrombotic with equal aPTT prolongation (47±3 and
43±6 seconds, respectively, versus 20±1 seconds as control). In
contrast, heparin (10
IU · kg-1 · min-1)
indefinitely
prolonged the aPTT when inducing a comparable antithrombotic effect to
that of the intermediate dose of the thrombin inhibitors. Thus, the ACT
in whole blood seemed more appropriate than the aPTT to predict the
arterial antithrombotic effect of thrombin inhibitors with different
mechanisms of action.
Fig 2d shows that the bleeding
time was not prolonged except for the
highest doses of the inhibitors studied, where it increased by 43%,
53%, and 40% for heparin, hirudin, and Ro 46-6240, respectively
(P<.05).
Regression analysis shows that the ACT and the
aPTT correlate for
hirudin and Ro 46-6240 for the low and intermediate dose regimen
(R2=.74 and .91 for Ro 46-6240 and hirudin,
respectively; P<.05). No correlation could not be found
with heparin (Fig 3).
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150 seconds was
unmeasurable.
Effects of Blood Cells on the ACT
In a separate group of
guinea pigs, the effects of blood cells on
the ACT were assessed (Table). In whole blood, heparin,
hirudin, and Ro 46-6240 prolonged the ACT to similar extents. In
platelet-poor plasma, heparin indefinitely prolonged the ACT
(Table).
In contrast, neither hirudin nor Ro 46-6240 in the absence of blood
cells indefinitely prolonged the ACT. Accordingly, in platelet-poor
plasma, hirudin and Ro 46-6240 prolonged the ACT by 5 and 4.4 compared
with 2.4 and 2.8 times control in whole blood, respectively.
Citrated/recalcified whole blood ACT was not statistically different
from that in native whole blood. Thus, blood cells influence the ACT
more in the presence of heparin than in the presence of Ro 46-6240 or
hirudin.
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Inhibition of Thrombin Generation
At the lowest concentration
corresponding to a nonantithrombotic
dose in vivo, Ro 46-6240 and hirudin similarly inhibited the thrombin
generated through the intrinsic pathway by approximately 40%, whereas
heparin almost completely blocked it (Fig 4B). The
intermediate concentration of Ro 46-6240 or hirudin fully inhibited the
thrombin generation of this pathway. When the extrinsic pathway was
tested, the low concentration of Ro 46-6240 was ineffective, whereas
the respective effects of hirudin and heparin were the same as through
the intrinsic pathway (Fig 4A). Interestingly, maximal
inhibition of
thrombin generation was equally associated for the three compounds with
at least a twofold increase in the aPTT (Fig 2c).
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Thus, in this thrombin-generation test, full inhibition is predictive of an antithrombotic effect only for the direct thrombin inhibitors such as hirudin or Ro 46-6240.
ACT Versus aPTT in Different Species
Similarly to the guinea
pig, the dose response of heparin in human
and rabbit blood showed a dramatic higher sensitivity in the aPTT
compared with the ACT (Fig 5, left). In contrast, the
dose response of Ro 46-6240 was superimposable for both coagulation
tests in human and in rabbit blood (Fig 5, right).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study shows for the first time that in arterial thrombosis models, the aPTT is not adequate to compare heparin and specific thrombin inhibitors and the ACT is more appropriate than the aPTT to evaluate compounds with different mechanisms of action for blocking thrombin activity. In addition, the study also shows that specific thrombin inhibitors are as effective in this model as heparin infused at supratherapeutic doses.
Predictive Value of the aPTT in Arterial Thrombosis Models
Comparisons between various specific thrombin inhibitors and
heparin of their relative efficacy are usually based on the assumption
that for an equal prolongation of the aPTT, the antithrombotic effect
is more pronounced with the specific thrombin inhibitors than that
observed with heparin. However, this assumption does not address the
question of whether prolongation of the aPTT is reflecting the same
anticoagulation when thrombin is inhibited by different mechanisms.
Although the aPTT is also considered as a gold standard to monitor
conventional heparin treatment, its relevance for comparing various
anticoagulants has recently been questioned.29 The aPTT is
a coagulation test that evaluates the intrinsic coagulation system by
measuring time for recalcified plasma to clot after adding an
activating agent such as ellagic acid or kaolin.30 In
addition to the activating agent, a phospholipid is added that acts as
a substrate for platelet factor 3 activity required for intrinsic
activation.
The prolongation of the aPTT has been shown to be a poor predictor of antithrombotic effect in arterial thrombosis models. Specific inhibitors of factors Xa or IXa, although efficient in arterial models of thrombosis, do not alter the aPTT to the same extent.31 The same phenomenon applies for low molecular weight heparins, which have a high level of inhibitory activity toward free factor Xa but lack appreciable antithrombin activity.32
Despite the fact that the aPTT varies according to both the type of activator and the type of phospholipid used, it is usually indefinitely prolonged when massive doses of heparin are used.33 34 Accordingly, in the present study heparin plasma levels of more than 2 U/mL indefinitely prolonged the aPTT. Thus, in clinical situations where high heparin plasma levels are needed, the ACT is preferred to the aPTT.7 17 18
Predictive Value of the ACT in Experimental Arterial
Thrombosis
In contrast with the aPTT, the changes of the ACT were
predictive
for the antithrombotic effect of all three tested anticoagulants. The
ACT is a test that is performed on fresh whole blood, is independent of
the presence of exogenous platelet factor 3, can be performed at
bedside,18 19 35 and is poorly correlated
to the aPTT in
the presence of heparin.35 The present study also
showed that there is a good correlation between the ACT and aPTT for
specific thrombin inhibitors, whereas that is not the case for heparin.
This finding is in line with a recent study that showed that in human
heparinized blood, an ACT exceeding 200 seconds corresponds to an
unmeasurable aPTT.35 We also sought to determine the role
of blood cells in the ACT with different treatments. We showed that in
platelet-poor plasma, heparin induced an indefinite prolongation of
ACT, whereas it was increased only threefold when tested in whole
blood. Although the absence of procoagulant effect of blood cells
influenced the effect of Ro 46-6240 and hirudin, the latter did not
induce an indefinite ACT as did heparin. Other evidences support the
fact that platelets can mask the heparin activity more than the hirudin
activity in the ACT test.36 Accordingly, Bode et
al36 showed that in the presence of a high concentration
of heparin, the ACT was lowered progressively by the addition of
increasing concentrations of lysed platelets, whereas platelets had
only a small effect on a matched antithrombin concentration induced by
hirudin.
We have extended our finding in the guinea pig to other species. Regardless of the blood used (eg, human, rabbit), the aPTT invariably showed a higher sensitivity toward heparin compared with the ACT, whereas this was not the case for Ro 46-6240.
Other observations support the view that the procoagulant factors present in platelets can modulate the effect of heparin. Heparin contains certain fractions that activate platelets,37 38 the latter being able after activation to release heparin-neutralizing proteins such as platelet factor 4 and ß-thromboglobulin.39 40 The addition of platelet factor 4 can normalize an aPTT prolonged by heparin but not by a direct thrombin inhibitor.14 Therefore, platelet factor 4 could also partially blunt the ACT prolongation induced by heparin.
Thrombin-Generation Test
Unlike the tests that measure
clotting times, the
thrombin-generation test dynamically evaluates thrombin formation from
both the intrinsic and the extrinsic coagulation pathways. Inhibition
of thrombin generation was predictive for an antithrombotic effect for
the thrombin inhibitors but not for heparin. Because this assay
generates only soluble thrombin and not clot-bound thrombin, one
possibility is that in vivo the low dose of heparin could inhibit
soluble thrombin much better than thrombin bound to the carotid
thrombus. Accordingly, Weitz et al41 showed that in
contrast with antithrombin III–independent thrombin inhibitors,
heparin was much more prone to inhibit soluble than clot-bound
thrombin. In addition, at 0.1 µmol/L heparin, a concentration
corresponding to our low dose, less than 20% of clot-bound thrombin is
inhibited.41 Another explanation is that, like in the aPTT
test, the lack of platelets in the thrombin-generation assay precludes
any inhibitory interactions between platelet-derived factors and
heparin. This hypothesis is supported by the finding that the
inhibition of thrombin generation is considerably decreased in the
presence of platelets.42
Clinical Importance
The lack of relevance of the aPTT, at
least in animal models, for
comparing heparin with thrombin inhibitors has some clinical
consequences. The first clinical implication is that the current
guidelines for heparin use after myocardial infarction or angioplasty,
ie, a doubling of the baseline aPTT, might be incorrect. In such urgent
situations, the ACT would probably be a more appropriate test. However,
this statement must be put in perspective with the excessive risk of
bleeding associated with high doses of
heparin.32 43 44 In
addition, our observation supports the view that a "therapeutic"
prolongation of the aPTT induced by specific thrombin inhibitors
induces a more pronounced systemic anticoagulation than heparin. Thus,
ignoring this aPTT mismatch, one could unduly come to the conclusion
that thrombin inhibitors have a higher bleeding complication rate than
heparin.
In conclusion, in this arterial thrombosis model, heparin, hirudin, and Ro 46-6240 showed similar antithrombotic efficacies. The ACT proved to be a better predictable parameter for the antithrombotic effect than the aPTT. The relative antithrombotic effects of anticoagulant drugs with different mechanism of actions should be based on coagulation tests with similar kinetics.
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Acknowledgments |
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This study was supported by F. Hoffmann-La Roche Ltd, Basel, Switzerland. We thank Dr R.B. Wallis for the supply of recombinant hirudin and Loretta Falivene and Heidi Hoffmann Maiocchi for technical help.
Received July 25, 1994; revision received September 19, 1994; accepted September 28, 1994.
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