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Effects of Lepirudin, Argatroban and Melagatran and Additional
Influence of Phenprocoumon on Ecarin Clotting Time
Tivadar Fenyvesi* M.D., Ingrid Jörg* M.D., Christel Weiss + Ph.D., Job Harenberg* M.D.
Key words: direct thrombin inhibitors, ecarin clotti ng time, oral anticoagulants, enhancing effects
*IV. Department of Medicine +Institute for Biometrics and Medical Statistics University Hospital Mannheim
Theodor Kutzer Ufer 1 - 3 68167 Mannheim Germany
*Corresponding author Phone: +49 -621 -383 -3378 E-ma il: tivadar.fenyvesi @med1.ma.uni -heidelberg.de
Version 06.08.03 for Thromb Res
Review Copy
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Abstract
Introduction: Direct thrombin inhibitors (DTI) prolong the
ecarin clotting time (ECT). Oral anticoagulants (OA) decrease
prothrombin levels and thus interacts with actions of DTIs on
the ECT method during concomitant therapy.
Materials and methods: Actions of lepirudin, argatroban, and
melagatran on ECT were investigated in normal plasma (NP) and
in plasma of patients (n = 23 each) on stable therapy with
phenprocoumon (OACP). Individual line characteristics were
tested statistically.
Results: Control ECT in OACP was prolonged compared to NP
(50.1±0.9 vs. 45.7±0.8 sec; p < 0.001). Lepirudin prolonged the
ECT linearly. Argatroban and melagatran delivered biphasic
dose-response curves. OA showed additive effects on the ECT of
lepirudin but not of argatroban and melagatran. Both in NP and
OACP the first and second slopes of melagatran were steeper
compared to argatroban (primary analysis; p<0.001). When using
the same drug, slopes in OACP were steeper than in NP
(secondary analysis; p<0.001). At similar molar concentrations
the crossing points of both slopes were significantly higher
with melagatran (323.1±11.0 s in NP and 333.2±8.2 s in OACP) than withReview argatroban (219.6±14.7 Copyand 248.4±15.2 s) corresponding to ratios of 7.1±0.2 and 6.7±0.2 (melagatran)
versus 4.8±0.3 and 4.9±03 with argatroban (p<0.0001).
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Discussion: The patterns of interactions between vitamin K
antagonists and DTI effects are different for bivalent
(increase of slope without affecting linearity) and monovalent
inhibitors (slight increase or alteration of non-linear
slopes), but there are also differences between the two
monovalent inhibitors on thrombin inhibition as determined by
ECT.
Review Copy
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1. Introduction
Recently, a snake venom based testing method, the ecarin
clotting time (ECT), was refined 1 to overcome the limitations
of traditional monitoring measures such as the activated
partial thromboplastin time (aPTT) methods. A toxin of Echis
carinatus cleaves prothrombin to meizothrombin and other
active intermediates.
Heparins and direct thrombin inhibitors are mostly monitored
by the aPTT 2 3. Limitations of aPTT methods include non-linear
dose-effect relationships with plateau effect, variability
among different testing instruments, reagents and different
lots of the same reagent 4. The ECT, has a linear dose-response
relationship towards the direct thrombin inhibitor lepirudin 1
5 and is therefore more accurate in monitoring of this direct
thrombin inhibitor. ECT is also more sensitive towards new
DTIs like argatroban and melagatran than the aPTT 3 6. ECT is
insensitive against heparins, however, because heparins
require antithrombin which can’t react with meizothrombin or
other intermediates like meizothrombin(desF1) with thrombin
activity of the prothrombin-thrombin conversion 7.
Hirudin is contained in the saliva of the medical leech Hirudo medicinalisReview. It is a tadpole-like proteine Copy molecule occurring in two variants with 65 or 64 amino acids (molecular mass: 6.9
kDa) 8. It is a bivalent inhibitor of the active catalytic site
and the anion binding exosite (also called fibrinogen
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recognition site) of thrombin. Lepirudin is a recombinant
hirudin (r-hirudin). The direct thrombin inhibitors lepirudin 9
10 and argatroban 11 (an arginin derivative, hydrated mole mass
0.526 kD, monovalent active site inhibitor) are established to
maintain effective anticoagulation in patients with heparin-
induced thrombocytopenia without and with thrombosis (HIT type
II). Melagatran (mole mass 0.478 kDa), a synthetic small
molecular active site inhibitor applied in form of an orally
available prodrug, ximelagatran, is currently under
investigation for prophylaxis and treatment of venous
thromboembolism in clinical trials 12 13 14 15. Direct thrombin
inhibitors and heparins increase the anticoagulant effects of
oral vitamin K antagonists in various clotting time
measurement techniques 16. These interactions are of
considerable extent with prothrombin time 17 18 19 and weaker
within aPTT 20 or ECT methods 21 22. There are marked
differences regarding the extent of increasing effects between
direct thrombin inhibitors and vitamin K antagonists within
the ECT method in literature 19 20. Clinical relevance of such
additive effects arises during concomitant treatment periods.
Such periods appear for instance when oral anticoagulants are
discontinued to introduce direct thrombin inhibitors for the
duration of diagnostic or therapeutic interventions. In theReview present study, we describe Copyeffects of three direct thrombin inhibitors (lepirudin, argatroban and melagatran) on
the ECT in normal plasma (NP) and in plasma of patients on
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steady-state oral anticoagulation with phenprocoumon (orally
anticoagulated plasma, OACP). We hypothesised that monovalent
and bivalent inhibitors may interact differently with
decarboxylated precursors of thrombin in OACP.
2. Materials and Methods
Blood samples (~10 ml) of 23 healthy volunteers (normal
plasma, NP) and of 23 patients (INR: 2.63 ± 0.13; mean ±
s.e.m.; range: 1.4 - 3.3 ) on steady-state treatment (orally
anticoagulated plasma, OACP) with the vitamin K antagonist
phenprocoumon (Hoffmann La Roche, Basel, Switzerland) were
collected by clean cubital vein punction into plastic vials
with sodium citrate (3.8 %; plasma/citrate: 9/1, V/V).
All patients were outpatients and were within the therapeutic
range. The time in therapeutic range was comparable. None of
the patients was near to an acute thrombotic event. None of
the patients received any concomitant treatment potentially
interfering with phenprocoumon.
The centrifuged plasma samples (1800g, 10 min) were either
tested immediately or shock frozen in liquid nitrogen and
stored at –80°C for analyses within 4 weeks. Plasma samples
were spiked with concentrations ranging from 300 ng/ml to 3000 ng/ml of Reviewr-hirudin (Lepirudin, Aventis,Copy Frankfurt/Main, Germany; molecular mass 6.9 kDa) and argatroban (by courtesy
of Mitsubishi Chemical Corp., Tokyo, Japan; molecular mass
0.526 kDa), and 30 ng/ml to 1000 ng/ml of melagatran (kindly
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provided by Astra Zeneca, Moelndal, Sweden; molecular mass
~0.478 kDa). All ECT measurements were carried out in a KC 10a
micro device from Amelung Comp. (Lemgo, Germany)23. The Ecarin
reagent® (lot No 8303/116-08) was kindly provided by Pentapharm
Ltd. (Basel, Switzerland). From the two methods currently
described and available 1 5 the method with the higher
detection sensitivity was chosen. This method is carried out
according to 1.
Statistical analyses
All data are given as mean values ± standard deviations of
means (s.e.m.). For all parameters analyzed, Duncan -Scheffe
test was performed using SAS software and level of significance was set at p < 0.001.
Approach to line characteristics
The linear concentration-effect relationships of lepirudin
were characterised by slope and intersection. The values
obtained for NP and OACP were tested with the tests described
above. For both values level of significance was set at p < 0.001. TheReview non -linear curves of argatroban Copy and melagatran were considered to consist of two parts: a linear acceding part and a plateau phase. They were fitted separately to linear equations delivering the characteristic parameter slope. To
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find out the y values assigned to the crossing points of the two slopes, equations of both sections of each individual curve were transformed, equated and dissolved for y. The corresponding y -value was considered as a characteristic value attaching the two curve phases to each other (Fig. 1). The differences of the y values were analyzed between argatroban and melagatran (primary analysis) and between NP and OACP (secondary analysis). Primary and secondary analysis was also carried out for the linear phase and plateau phase slopes of argatroban and melagatran. Quality control for all curve characterising equ ations was R 2 (mean > 0.950).
3. Results
ECT expressed in seconds
All immediate acting thrombin inhibitors affected the ECT in a
concentration dependent manner in both materials (normal and
OA plasma). The dose-response relationship with lepirudin was
linear. Argatroban and melagatran had non-linear
concentration-effect relationships. The steeper initial part
showed a transition to a flatter relationship from about 1000
ng/ml with argatroban and 300 ng/ml with melagatran. Normal
ECT range in our study was 45.7 ± 0.8 sec in NP. In OACP, this
value was prolonged to 50.1 ± 0.9 sec (p < 0.001). Lepirudin Reviewhad a linear dose-response relationshipCopy with NP and OACP (Fig. 2 A). A concentration of 500 ng/ml prolonged ECT to
102.9 ± 3.4 sec in NP and to 123.2 ± 3.0 sec in OACP (p <
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0.001). A higher therapeutic concentration (2000 ng/ml) of
lepirudin delivered ECT values of 271.7 ± 8.0 sec in NP and
341.4 ± 10.3 sec in OACP (p < 0.0001).
500 ng/ml argatroban lead to ECT values of 163.6 ± 9.7 sec in
NP and 171.1 ± 11.2 sec in OACP (p < 0.001; Fig. 3 A). 2000
ng/ml prolonged the ECT to 317.8 ± 18.5 sec and 345.6 ± 19.3 in
NP and OACP, respectively (p < 0.001).
Melagatran at 100 ng/ml delivered clotting times of 179.2 ± 4.0
sec in NP and 190.0 ± 4.0 sec in OACP (p < 0.0001; Fig. 4 A).
300 ng/ml prolonged ECT to 345.7 ± 9.1 sec in NP and 361.4 ±
7.8 sec in OACP (p < 0.0001).
Fitting of concentration-coagulation time relationships
Significant deviations of the slopes and intersections of
dose-response lines of lepirudin were found between NP and
OACP. Also the linear (slope 1) and plateau phase slopes
(slope 2) showed significant deviations between argatroban
(slope 1: 0.24±0.02 s/ng•ml in NP and 0.26±0.02 s/ng•ml in
OACP) and melagatran (slope 1: 1.32±0.04 s/ng•ml in NP and 1.38
s/ng•ml in OACP) in the same group (primary analysis, p <
0.0001; for slope 2 see table 1). The same finding occurred between bothReview groups using the same drug Copy (secondary analysis, p < 0.001; for details see table 1) Within the same group (NP or
OACP) the y-values belonging to the crossing points of the
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slopes of both curve phases were 219.6±14.7 s (NP) and
248.4±15.2 s (OACP) with argatroban and 323.1±11.0 s (NP) and
333.2±8.2 s (OACP) in the case of melagatran (primary analysis,
p < 0.0001). The secondary analysis between NP and OACP using
the same drug resulted in significant deviations, too (p <
0.001; for details see table 1).
Individual normalised ECT ratios
Clotting times were transformed into individual normalised
ratios based on the individual control value of each volunteer
or patient, respectively. Control ratio is 1 in all cases.
Concentration-ECT ratio lines of lepirudin were divergent with
NP and OACP (Fig. 2 B). A concentration of 500 ng/ml increased
the ECT ratio by 2.3 ± 0.1-fold in NP and 2.5 ± 0.1-fold in
OACP. A higher therapeutic concentration (2000 ng/ml) of
lepirudin delivered ECT ratios of 6.0 ± 0.2 in NP and 6.9 ± 0.2
in OACP (p < 0.001).
In the cases of both argatroban and melagatran the
concentration-ECT ratio curves of NP and OACP plasmas were
almost identical, without any tendency towards a divergence
(Fig. 3 B, 4 B). 500 ng/ml argatroban provided ECT ratios of 3.6 ± 0.2 Reviewin NP and 3.4 ± 0.2 in OACP Copy samples (n. s. Fig. 3 B). At 2000 ng/ml it increased the ECT ratio to 7.0 ± 0.4 and
6.9 ± 0.4 in NP and OACP, respectively (n. s.).
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Melagatran (100 ng/ml) increased the ratios by about 4 times
(3.9 ± 0.1 in NP and 3.8 ± 0.1 in OACP; n. s.; Fig. 4 B). A
high therapeutic concentration of 300 ng/ml delivered ECT
ratios of 7.6 ± 0.2 in NP and 7.3 ± 0.2 in OACP (n. s.).
Fitting of concentration-coagulation time ratio relationships
Also in the case of the ratios, significant deviations of the
slopes and intersections of dose-response lines of lepirudin
were found between NP and OACP. The linear and plateau phase
slopes showed significant deviations between argatroban (slope
1: 0.0053±0.0005 and 0.0051±0.0004 ratio/ng•ml in NP and OACP)
and melagatran (slope 1: 0.029±0.001 and 0.028±0.001
ratio/ng•ml in NP and OACP) in the same group (primary
analysis, p < 0.0001; for slope 2 see table 2). The same was
found between both groups using the same drug (secondary
analysis, p < 0.001; for details see table 2). Within the same
group (NP or OACP) the y-values belonging to the crossing
points of the slopes of both curve phases were elevated with a
very high significance in the case of melagatran (7.1±0.24 in
NP and 6.7±0.18) compared to argatroban (4.8±0.33 in NP and
4.9±0.32 in OACP) in OACP; primary analysis, p < 0.0001). The secondary Reviewanalysis using the same drug Copy resulted showed non- significant deviations between NP and OACP, for details see
table 2.
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4. Discussion
Our study demonstrated different patterns of interactions
between vitamin K antagonists and DTIs not only between
bivalent and monovalent inhibitors, but also between the two
monovalent inhibitors. The slow-acting bivalent inhibitor
lepirudin exerted linear concentration-effect relationships,
according to those described in literature 1 5 19 20. The
monovalent compounds argatroban and melagatran, however,
showed non-linear dose-effect relationships, possibly due to
their different mechanism and kinetics24 of action. These
differences might be due to the differing binding modes of the
compounds. Lepirudin is a bivalent ligand for both the
catalytic active site and the anion binding exosite of
thrombin 25 26, while argatroban and melagatran are monovalent
inhibitors of the catalytic active site alone 27. In case of
monovalent active site inhibitors like argatroban and
melagatran, the interaction between enzyme (thrombin) and
inhibitor may come to a saturation state, because here, above
a certain threshold concentration, the coagulation curve is
changing. Increasing concentrations of the inhibitor lead to
much less prolongation of clotting times, compared to the linear accedingReview phase. Lepirudin, a recombinantCopy hirudin, not only inhibits the active catalytic site, but also binds to the
fibrinogen-binding site (= anion binding site or exosite 1).
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The saturation of the active catalytic site would not hinder
further effects of lepirudin on this binding site, possibly
explaining the linear concentration-effect relationship. A
sub-optimal gamma-carboxylation of factor II during vitamin K
antagonism could increase the accessibility of this binding
site to lepirudin 28.
Feedback mechanisms could explain the shape of the monovalent
relationships. This could include a positive feedback of
meizothrombin (the enzyme primarily generated within the ECT
method) on F XI which is responsible for generation of
thrombin (enzyme generated secondarily) within the clot, as
well as feedbacks to F VII, F IX and F X29. At higher
concentrations of argatroban or melagatran the inhibitor
possibly reacts with the thrombin generated by feedback
mechanism. Deficiencies of active factors influenced by stable
oral anticoagulation could influence feedback mechanisms in a
different manner for each combination of direct thrombin
inhibitor and binding sites. The precise mechanistic
backgrounds remain to be investigated, however.
Differing data are reported in literature about the extent of
enhancement of direct thrombin inhibitor effects by oral
anticoagulants 19 20. In the case of lepirudin, with increasing concentrations,Review the results of the Copypresent work indicate enhancements by phenprocoumon effects which might be not
negligible in clinical practice. There should be an effort to
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consider these interactions during overlapping applications,
e.g. when monitoring patients who are switched from vitamin K
antagonists to lepirudin or vice versa. Such therapeutic
switches occur when outpatients receiving oral anticoagulants
are hospitalised for surgical or invasive diagnostic
procedures or the treatment of venous thromboembolism is
switched from a direct thrombin inhibitor to a vitamin K
antagonist.
The aPTT is prolonged by oral anticoagulation due to a
decrease of factors IX, X and II 30 31. Within the test system
of ECT only the decrease in the level of F II affects
coagulation times during OA therapy. There is so far neither a
standardisation of the various aPTT reagents nor an adaptation
of the reesult interpretation on OA effects, probably due to
the varying effects of the reagents. This may not account for
ECT favorising this method for determination of the effects of
DTIs during concomitant oral anticoagulation.
According to the results presented herein, appropriate
clinical studies to validate therapeutic ranges for ECT ratio
using appropriate control samples may be required. One attempt
is currently being made in an international collaborative
study 32.
AcknowledgementsReview Copy
The authors would like to thank to Mrs. Christina Giese, Mrs.
Antje Hagedorn and Mrs. Inge Träger for excellent laboratory
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work and patient care. This study was supported by a grant of
the Faculty of Clinical Medicine Mannheim, University of
Heidelberg.
Legends to figures and tables
Figure 1: Exemplification of the approach to individual line
characteristics of non-linear concentration response
relationships. Individual relationships with argatroban.
Figure 2: Concentration-ECT relationships of lepirudin
expressed in seconds (A) and as ECT ratio (B) in normal plasma
(NP, continuous line, n = 23) and plasma samples of patients
on oral anticoagulation with phenprocoumon (OACP,
discontinuous line, n = 23). Data is given as mean ± s.e.m.
Figure 3: Concentration-ECT relationships of argatroban
expressed in seconds (A) and as ECT ratio (B) in normal plasma
(NP, continuous line, n = 23) and plasma samples of patients
on oral anticoagulation with phenprocoumon (OACP,
discontinuous line, n = 23). Data is given as mean ± s.e.m.
Figure 4: Concentration-ECT relationships of melagatran expressed Reviewin seconds (A) and as ECT ratio Copy (B) in normal plasma (NP, continuous line, n = 23) and plasma samples of patients
on oral anticoagulation with phenprocoumon (OACP,
discontinuous line, n = 23). Data is given as mean ± s.e.m.
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Table 1: Synopsis of important statistical parameters of
individual concentration-ECT [sec] curve characteristic
parameters (slopes [s/ng•ml], intersections, crossing points
of slopes
(n. a.: not applicable).
Table 2: Synopsis of important statistical parameters of
individual concentration-ECT [ratio] curve characteristic
parameters (slopes [ratio/ng•ml], intersections, crossing
points of slopes
melagatran (n. a.: not applicable).
Review Copy
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Table 1:
lepirudin Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.11 0.15 n. a. n. a. n. a. s.e.m. 0.0031 0.005 n. a. n. a. n. a.
argatroban Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.24 0.26 0.08 0.08 219.6 248.4 s.e.m. 0.02 0.022 0.005 0.006 14.7 15.2
melagatran Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 1.31 1.38 0.35 0.41 323.1 333.2 s.e.m. 0.04 0.03 0.01 0.01 11.0 8.2
Table 2:
lepirudin Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.0025 0.003 n. a. n. a. n. a. s.e.m. 0.0001 0.0001 n. a. n. a. n. a.
argatroban Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.0053 0.0051 0.0018 0.0016 4.8 4.9 s.e.m. 0.0005 0.0004 0.0001 0.0001 0.33 0.32
melagatran Parameter Slope part 1 Slope part 2 y-value of transition Group NP OACP NP OACP NP OACP Mean 0.029 0.028 0.0078 0.0082 7.1 6.7 s.e.m. 0.001 0.001 0.0003 0.0003 0.24 0.18
Review Copy
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