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Effect of non-specific reversal agents on

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Article in Thrombosis and Haemostasis May 2012
DOI: 10.1160/TH12-03-0179 Source: PubMed

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Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer 2012

Effect of non-specific reversal agents on anticoagulant activity

of dabigatran and rivaroxaban
A randomised crossover ex vivo study in healthy volunteers
Raphael Marlu1; Enkelejda Hodaj2; Adeline Paris2; Pierre Albaladejo3,5; Jean Luc Crackowski2; Gilles Pernod4,5
1Haemostasis

Unit, University Hospital Grenoble, France; 2Clinical Research Centre, INSERM CIC03, University Hospital Grenoble, France; 3Department of Anaesthesiology,
University Hospital Grenoble, France; 4Vascular Medical Unit, University Hospital Grenoble, France; 5Joseph Fourier Grenoble 1 University / CNRS TIMC-IMAG UMR 5525 / THemas,
Grenoble, France

Summary
The new anticoagulants dabigatran and rivaroxaban can be responsible for haemorrhagic complications. As for any anticoagulant, bleeding management is challenging. We aimed to test the effect of all putative haemostatic agents on the anticoagulant activity of these new
drugs using thrombin generation tests. In an ex vivo study, 10 healthy
white male subjects were randomised to receive rivaroxaban (20 mg) or
dabigatran (150 mg) in one oral administration. After a two weeks
washout period, they received the other anticoagulant. Venous blood
samples were collected just before drug administration (H0) and 2
hours thereafter. Reversal of anticoagulation was tested in vitro using
prothrombin complex concentrate (PCC), rFVIIa or FEIBA at various
concentrations. Rivaroxaban affects quantitative and kinetic parameters, including the endogenous thrombin potential (ETP-AUC and more
pronouncedly the thrombin peak), the lag-time and time to peak. PCC

Correspondence to:
Prof. G. Pernod
Vascular Medical Unit, CHU Grenoble
38043 Grenoble Cx9, France
Tel.: +33 4 76 76 57 17, Fax: +33 4 76 76 55 24
E-mail: GPernod@chu-grenoble.fr

Introduction
New oral antithrombotic drugs that target either thrombin (dabigatran, Boehringer Ingelheim, Ingelheim, Germany) or factor (F)Xa
(rivaroxaban, Bayer, Leverkusen, Germany) are under clinical development. Clinical phase III trials have been conducted with both
drugs which were approved for the treatment of venous thromboembolic disease (VTE), and also for the prevention of systemic
embolism in non-valvular atrial fibrillation (NVAF). Besides their
efficacy, both drugs were responsible for haemorrhagic complications. In the treatment of NVAF, the yearly rate of major bleeding
was 3.11% in the group receiving 150 mg of dabigatran twice daily,
similar to the rate of major bleeding of 3.36% in the warfarin
group (1). Likewise, major bleeding was recently reported in 5.6%
of NVAF patients treated with rivaroxaban, again similar to the
warfarin group, although intracranial haemorrhages were significantly reduced by factor two (2)
As promising drugs, it is expected that these treatments will be
widely used in long-term indications such as AF. However, as for

strongly corrected ETP-AUC, whereas rFVIIa only modified the kinetic

parameters. FEIBA corrected all parameters. Dabigatran specially affects the kinetics of thrombin generation with prolonged lag-time and
time to peak. Although PCC increased ETP-AUC, only rFVIIa and FEIBA
corrected the altered lag-time. For both anticoagulants, lower doses of
FEIBA, corresponding to a quarter to half the dose usually used, have
potential reversal profile of interest. In conclusion, some non-specific
reversal agents appear to be able to reverse the anticoagulant activity
of rivaroxaban or dabigatran. However, clinical evaluation is needed regarding haemorrhagic situations, and a meticulous risk-benefit evaluation regarding their use in this context is required.

Keywords
Anticoagulants, dabigatran, rivaroxaban, reversal, thrombin generation test

Received: March 21, 2012

Accepted after major revision: April 18, 2012
Prepublished online: May 25, 2012
doi:10.1160/TH12-03-0179
Thromb Haemost 2012; 108: 217224

any anticoagulant, the management of any bleeding event will be

challenging. Several haemostatic agents have been proposed to reverse the anticoagulant activity of these new drugs, although only
spurious clinical data, based on some published case reports, are
available (35). Therefore, our aim was to test the ability of all putative non-specific haemostatic agents currently available, at different dosages, to reverse the anticoagulant activity of both dabigatran and rivaroxaban, using thrombin generation tests (TGT) (6)
in an ex vivo study in healthy volunteers.

Material and methods

Subject selection
Ten healthy white male subjects (age 1845 years), with a body
mass index between 18 and 30 kg/m2 were included in this study
(ClinicalTrials.gov, number NCT01210755) between November
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Marlu et al. Reversal of dabigatran and rivaroxaban

2010 and March 2011. Subjects with a personal or family history of

thrombosis or bleeding disorders were excluded, as well as subjects
with renal or liver impairment. All subjects gave written informed
consent, and received financial compensation for their participation on this study. The study was approved by French Ethics
Committee (Comit de Protection des Personnes Sud-Est V, IRB
n6705), and by the French Health Agency (AFSSAPS).

Assays

Venous blood samples were collected in Vacutainer tubes (Becton

Dickinson, Le Pont de Claix, France) containing 0.105 M trisodium citrate just before drug administration (H0) and 2 h thereafter (H2). Following a double centrifugation at 2,500 g for 15 minutes (min), platelet poor plasma was collected, quick-frozen and
stored at 80C until the analyses were performed.

Thrombin generation was measured as previously described

(9) using Calibration Automated Thrombinography (CAT)
reagents (Diagnostica STAGO, Asnires, France), consisting of 5
pM tissue factor and 4 M phospholipids. The fluorescence signal
was read after adding 2.5 mM of fluorogenic substrate Z-Gly-GlyArg-AMC using a Varioskan Ascent fluorimeter (Thermofisher,
Illkirch, France). The raw data of fluorescence measurements were
exported to Sigmaplot version 9.0 for mathematical calculations.
Then, calculations from Sigmaplot were exported to GraphPad
Prism5.0 software to superimpose the different curves. For each
parameter of the TGT analysis, we tested the reversion by comparing results both to H2 (defined as 2 h after anticoagulant administration, without reversal agents) and H0 (baseline, defined as the
time just before drug intake). Quantitative parameters included
the endogenous thrombin potential (ETP-AUC, nM.min), and the
maximum concentration of thrombin (Peak, nM). Kinetic parameters (in seconds [s]) included lag-time (LT) and time to reach the
maximum concentration of thrombin (time to Peak, TTP). The
rate index of the propagation phase (velocity) was calculated according to the formula Peak / (TTP-LT) and expressed in nM x
min-1. Since a calibration curve obtained in Dabigatran-treated patients cannot be used, data were processed by using the calibration
curve of the H0 sample instead of the H2 sample, as previously described (10). In addition, the anticoagulant effect of each drug was
assessed by classical haemostatic tests, which have been described
to correlate with anticoagulant concentrations (11, 12). Chronometric assays were performed with a STA-R coagulometer (Diagnostica STAGO). All reagents were also from Diagnostica STAGO.
Activated partial thromboplastin time (aPTT) using STA PTTA
reagent and prothrombin time (PT) using STA Neoplastin CI
plus were determined by classical methods.

Reversal agents

Statistical analysis

The three reversal agents tested were recombinant factor VIIa

(rFVIIa, Novoseven, NovoNordisk, Copenhagen, Denmark), activated prothrombin complex concentrate (FEIBA, Baxter AG,
Vienna, Austria), and the four factor prothrombin complex concentrate (PCC) Kanokad (LFB, Courtaboeuf, France). Since no
data were available regarding the dose of haemostatic agent to use,
each of these agents was tested at different dosages. Therefore,
rFVIIa was tested at a final concentration of 3 g/ml corresponding to a therapeutic dose of 120 g/kg (8), and also at two lower
doses, 0.5 and 1.5 g/ml final concentration. FEIBA was used at
the final concentrations of 0.25, 0.5, 1 (corresponding to 80 U/kg)
and 2 U/ml, and Kanokad at the final concentration of 0.25, 0.5
(corresponding to 25 U/kg), and 1 U/ml.

Quantitative data were expressed as means standard deviations

(SD).
Paired-t tests were used to analyse the reversal effect for each
parameter expressed quantitatively. We checked the normality assumption with a Shapiro-Wilk's test and the Wilcoxon signedranks test was used if the normality assumption was violated.
Spearman's coefficient of rank correlation (rho) was used to assess
the correlation between the different parameters of the thrombin
generation test. Two-sided significance tests were used throughout.
All statistical analyses were performed using Stata software
(V.11, College Station, TX, USA). A p-value < 0.05 was considered
as statistically significant.

Drug administration
At the therapeutic dose, rivaroxaban is usually administered at 20
mg once a day, whereas dabigatran is classically administered at
150 mg twice a day. For both drugs, T-max is reached 2 hours (h)
later. Since our aim was to test in vitro the effect of haemostatic
agents on anticoagulant activity, we decided to work at the peak
concentration, i.e. after intake of one dose (7, 8). Therefore, the
healthy volunteers were first randomised to receive either 150 mg
of dabigatran or 20 mg of rivaroxaban in one oral dose. The second
drug was administered after a wash-out period of 15 days for an
identical investigation.

Blood sampling

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Marlu et al. Reversal of dabigatran and rivaroxaban

Table 1: TGT parameters measured at baseline (H0) and 2 hours (H2)

after oral intake of 20 mg rivaroxaban or 150 mg dabigatran. ETP-AUC
denotes endogenous thrombin potential (nM.min); Peak is the maximum
concentration of thrombin (nM); LT is lag time (s); TTP is the time to reach the
maximum concentration of thrombin (s). Data were expressed as means SD
(n = 10).
Rivaroxaban
ETP

Peak

LT

TTP

H0

1076 (223)

216.2 (35.7)

2.29 (0.45)

4.38 (0.65)

H2

839 (147)

73.7 (21.3)

5.59 (1.03)

11.2 (2.9)

< 0.001

< 0.0001

< 0.0001

< 0.0001

Dabigatran
ETP

Peak

LT

TTP

H0

1191 (247)

227.1 (40.2)

2.16 (0.31)

4.23 (0.62)

H2

953 (182)

220.5 (49.2)

3.78 (1.21)

5.4 (1.25)

0.0013

0.5

0.0009

0.002

Results
Anticoagulant effect of rivaroxaban and dabigatran
Classical haemostatic assays allowed us to detect the anticoagulant
effect induced by rivaroxaban, which was responsible for significantly prolonging PT by 1.37-fold (p < 0.0001) and dabigatran,
which was responsible for a significant increase of aPTT by
1.47-fold (p < 0.001).
Figure 1: Effect of non-specific reversal agents on rivaroxaban anti-

Effect of reversal of rivaroxaban on TGT parameters

At 2 h after oral administration, the effect of 20 mg Rivaroxaban on
thrombin generation was particularly marked with the thrombin
peak reduced by a factor of 3, whereas there was only a modest reduction of 22% in the ETP-AUC (Table 1, Fig 1A). The initiation phase of thrombin generation was profoundly altered, as LT
and TTP were more than doubled (Table 1, Fig 1A). This was
followed by a pronounced inhibitory effect on the propagation
phase of thrombin, as shown by the marked effect of rivaroxaban
on the rate index of thrombin generation (16.6 2.9 nM min-1,
vs. 104.6 5.5 at H0, p < 0.0001). There was a good correlation between ETP-AUC and the Peak (rho = 0.68, p < 0.0001), but not for
kinetic parameters. Also we found a strong correlation between LT
and TTP (rho = 0.86, p < 0.0001).
At H2 (Fig. 1B), all reagents but rFVIIa were responsible for a
dose-dependent increase in the ETP-AUC, by 37% for the lowest
dose of PCC (0.25 U/ml, 1,147 232 nM.min) and 50% for the lowest dose of FEIBA (0.25 U/ml, 1,257 240 nM.min) vs. H2 (p <
0.001, Fig 2A). However, only the lower doses of PCC (0.25 and
0.5 U/ml) and FEIBA (0.25 U/ml) reversed the ETP-AUC to near
baseline H0, values (1,313 362 nM.min for PCC 0.5 U/ml, p =
0.07 vs. H0, Fig. 2A). There was over-correction for all other
Schattauer 2012

coagulated plasma. TGT curves were obtained from one representative individual volunteer. A) Anticoagulant effect of rivaroxaban. H0 denotes baseline; H2 denotes 2 hours after 20 mg oral rivaroxaban. B) TGT curves 2 hours
after 20 mg oral rivaroxaban, and after ex vivo addition of non-specific reversal agents at the highest concentration tested.

concentrations of PCC and FEIBA. There was also a significant

dose-dependent correction of the thrombin peak using PCC (96.5
29.5 nM for PCC 0.25 U/ml) or FEIBA (127 27.2 nMfor FEIBA 0.25 U/ml, p < 0.001) vs. H2. Besides the over correction of
ETP-AUC, FEIBA at doses 1 and 2 U/ml corrected the thrombin
peak to close to its H0 value (175.9 77.9 nM for FEIBA 1 U/ml,
p = 0.2) (Fig. 2B).
Regarding LT, PCC was responsible for a slight reduction in LT
(15% for PCC 1 U/ml), compared to FEIBA (47%) (p < 0.0001,
Fig. 1B, 2C). rFVIIa completely reverse the LT increase induced
by rivaroxaban (2.24 s vs. 2.29 s at H0). The effect was similar for
the different doses of rFVIIa or FEIBA. However, only the use of
rFVIIa corrected LT to near baseline H0 (p = 0.6, Fig. 2C). PCC
was without effect on TTP, although rFVIIa and FEIBA significantly decreased TTP by 44% and 30%, respectively, vs. H2 (6.32
1.28 s for rFVIIa; 7.9 1.54 s for FEIBA, p < 0.001), without
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Marlu et al. Reversal of dabigatran and rivaroxaban

reaching baseline H0. None of the different doses of PCC modified

the altered velocity induced by rivaroxaban. In contrast, rFVIIa
and FEIBA increased the rate of thrombin formation to above 25

nM min-1, whatever the tested dose, but without reaching the baseline rate (max observed for FEIBA 2 U/ml, 47.7 7.7 nM.min-1).

Effect of reversal of dabigatran on TGT parameters

At 2 h after receiving 150 mg of dabigatran, the ETP-AUC was
modestly reduced by 20%, but the thrombin peak was unchanged
(Table 1, Fig. 3A). The initiation phase of thrombin was significantly altered, as shown by the LT (Table 1, Fig. 3A). The
index rate of thrombin generation was not inhibited (138.5 10.8
nM.s-1 at H2, vs, 111.9 6.8 at H0). There was very good correlation between the ETP-AUC and the Peak (rho = 0.93, p <
0.0001), and between LT and TTP (rho = 0.94, p < 0.0001).
At H2, PCC and FEIBA increased the ETP-AUC of dabigatrananticoagulated plasma (Fig. 3B) in a concentration-dependent
manner (Fig. 4A). Low doses of PCC or FEIBA reversed ETPAUC results close to baseline H0 (p = 0.02), while there is a dramatic increase in thrombin generated when PCC or FEIBA were
used at regular or half doses. Both the highest dose of rFVIIa (3
U/ml) and the three highest doses of FEIBA (range 0.5 to 2 U/ml)
reduced the LT (Fig. 3B, Fig. 4B), with a more pronounced effect of rFVIIa (2.52 1 s, ratio 0.66 0.13 s vs. H2 values, p < 0.001)
(Fig. 4B). This observed reduction in LT was close to H0 (p =
0.2). For the TTP, similar results were observed for rFVIIa and FEIBA, with, in this case a significant effect of the lowest dose of FEIBA (0.25 U/ml, 4.85 0.52 s, ratio 0.83 0.15 vs. H2, p = 0.03).
Again the observed reversal was close to H0 (p = 0.08). All agents
except rFVIIa were responsible for an increase in the thrombin
peak above the baseline (H0) value (296.2 57.3 nM for PCC
0.25U/ml, 361 28 nM for FEIBA 0.25 U/ml, p < 0.001 vs. H2),
with a maximum observed for PCC at 1 U/ml (462.1 81.9 nM)
and FEIBA at 2 U/mL (551.7 99.5 nM) (data not shown).

Discussion
Due to their ease of use, the novel orally active anticoagulant drugs
will probably be widely used in the near future, including in patients at higher risk of bleeding. Although these treatments have a

Figure 2: Effect of non-specific reversal agents on rivaroxaban anticoagulated plasma. Anticoagulant effect of rivaroxaban was measured by
TGT. Data are mean relative change from H2 (i.e. 2 hours after 20 mg oral rivaroxaban, without reversal agent) SD. A) ETP-AUC denotes endogenous
thrombin potential (nM.min); B) Peak is the maximum concentration of
thrombin (nM); C) LT is lag time (s). Since there is a strong correlation between LT and TTP, only results regarding LT were presented. H0: baseline;
PCC: prothrombin complex concentrates (U/ml); rFVIIa: recombinant FVIIa
(g/ml); FEIBA: activated prothrombin complex concentrate (U/ml). All
reagents are given at their final concentration. * p < 0.001 vs. H2; # was nonsignificant vs. baseline H0.

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Marlu et al. Reversal of dabigatran and rivaroxaban

favourable risk-benefit profile compared to warfarin, major haemorrhages, including intracerebral haemorrhages, have been described in clinical trials evaluating both dabigatran and rivaroxaban (1, 2, 13). Among reversal agents, PCC has been already extensively evaluated to reverse VKA, although rFVIIa was reported
against antiXa-anticoagulant as efficient drug (14). Also FEIBA
was reported to be efficient to reverse VKA (15). These several haemostatic agents have been proposed to reverse the anticoagulant
effect of these new drugs, although the level of evidence is poor in
the absence of available clinical data (35). To improve our knowledge, we tested ex vivo the ability of these non-specific haemostatic drugs to reverse the anticoagulant activity of the new antithrombotics.

Rivaroxaban reversal
In the present study, rivaroxaban significantly affected both the
quantitative and kinetic TGT parameters. As previously described,
the effect of rivaroxaban on the ETP-Peak was more pronounced
than on ETP-AUC (12). As expected, PCC strongly corrected ETPAUC (16) (and more modestly the ETP-Peak), above the baseline
values for the highest doses, whereas rFVIIa only had a relevant effect on kinetic parameters. However, evaluation of potential reversion of rivaroxaban anticoagulant effect should probably not be
based uniquely on analysis of ETP-AUC, since, in a recently published rabbit haemorrhagic model, PCC did not reverse rivaroxaban-induced bleeding, although PCC corrects ETP-AUC by a
factor of 2 (17). Rivaroxaban also altered the initiation (LT) and
propagation phases of thrombin generation. These two parameters have been described as being potentially relevant for determining the antithrombotic activity of rivaroxaban, or its reversion
(18). However, in the present study, the effect of PCC was non-significant on these kinetic parameters. In contrast, rFVIIa, even at its
lowest concentration, showed complete reversion of the rivaroxaban-induced prolonged LT to the baseline level at H0. These results are very close to those already described using rFVIIa to reverse the in vitro anticoagulant effect of the indirect selective FXa
inhibitors, fondaparinux (19), and idraparinux (20). Unfortunately, although rFVIIa had a pronounced effect on lag-time, it
did not reverse rivaroxaban-induced haemorrhage in a rabbit
model (17). Interestingly, FEIBA, which contains FVII, mainly in
the activated form, and FII, FIX and FX, mainly in non-activated
forms, combines the effect of both rFVIIa and PCC, and corrected
all TGT parameters compared to H2, even at the lowest dose. The
observed correction never exceeds baseline (H0) values for the
ETP-Peak, although for kinetics parameters it persisted for longer
than at baseline. Furthermore, only FEIBA corrected the
markedly affected thrombin propagation phase by > 20%, theoretically compatible with a non-bleeding phenotype (21). Finally,
FEIBA at a dose of 50100 U/kg was reported to reduce mesenteric bleeding time in a rat haemorrhagic model (22), and also
bleeding time in baboons (23).

Schattauer 2012

Figure 3: Effect of non-specific reversal agents on dabigatran-anticoagulated plasma. TGT curves were obtained from one representative individual volunteer. A) Anticoagulant effect of dabigatran. H0: baseline; H2: 2
hours after 150 mg oral dabigatran. B) TGT curves 2 hours after 150 mg oral
dabigatran, and after ex vivo addition of specified non-specific reversal
agents at the highest concentration tested.

Dabigatran reversal
Dabigatran has been described as inhibiting thrombin generation
in a concentration-dependent manner (24). The overall generation of thrombin assessed by ETP-AUC has been described as
being moderately decreased. Dabigatran, as a thrombin retardant,
isolated induced alteration of LT, without affecting the velocity of
thrombin generation. Our results are in close agreement with these
previous results. PCC increased ETP-AUC, but had no effect on dabigatran-modified TGT kinetic parameters, as previously described (16). However, PCC has been described as able to reverse
the prolonged bleeding time and blood loss after dabigatran administration (25), and also to prevent intra-cerebral haematoma
expansion in a murine model (26). LT (but not ETP-AUC or Peak)
was described as correlated with circulating concentrations of daThrombosis and Haemostasis 108.2/2012

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Marlu et al. Reversal of dabigatran and rivaroxaban

Figure 4: Effect of non-specific reversal

agents on dabigatran-anticoagulated plasma. Anticoagulant effect of dabigatran was
measured by TGT. Data are mean relative
change from H2 (i.e. 2 hours after 150 mg oral
dabigatran, without reversal agent) SD. A)
ETP-AUC denotes endogenous thrombin potential (nM.min); B) LT is the lag time (s). Since
thrombin peak was unchanged after dabigatran
intake, results with reversal agents are not
shown. Also since there is a strong correlation
between LT and TTP, only results regarding LT
were presented. H0: baseline; PCC: prothrombin complex concentrates (U/ml); rFVIIa: recombinant FVIIa (g/ml); FEIBA: activated prothrombin complex concentrate (U/ml). All
reagents are given at their final concentration.
* p < 0.001 vs. H2; # was non-significant vs.
baseline H0.

bigatran (10). In the present study, rFVIIa at the higher dose, and
FEIBA corrected the altered LT to nearly baseline value. Nevertheless, previous data regarding the effect of rFVIIa on thrombin inhibitors are inconsistent. A single dose of rFVIIa did not reverse the
anticoagulant effect of melagatran in healthy volunteers (27), although it did in another study (28). In a rat model, the addition of
rFVIIa significantly reduced both tail bleeding time and aPTT after
the administration of a high dose of dabigatran (29). Again, in animal models, bleeding caused by a high dose of dabigatran was reduced following the use of FEIBA (29).

Clinical implications
If immediate reversal of the new anticoagulants is required, i.e. in
the event of intra-cerebral haemorrhage, there is currently no clear
evidence that any non-specific reversal agent can counteract the
anticoagulant effect of either rivaroxaban or dabigatran.

FEIBA has the theoretical advantage of combining the effect of

FVIIa and that of PCC. Therefore, a dose of 50 U/kg FEIBA corresponds to 37 g/kg of FVIIa together with 50 U/kg PCC (30). In the
case of rivaroxaban, FEIBA combines the correcting effect on lag
time of rFVIIa and the correcting effect on peak value of PCC.
Higher doses of FEIBA were responsible for an increase in total
thrombin generation, as shown by the increase in ETP-AUC and
Peak. This raises the problem of safety of such a product regarding
thrombotic risk. For this purpose, the incidence of thrombotic adverse events was reported as low, four per 100,000 FEIBA infusions, and a dose-dependent effect on the incidence of thrombosis
was reported (31). This underlines the need to avoid over-reversion, and to better define the potential minimum dose of reversal
agent to use. Regarding TGT, quantitative parameters such as a
Peak higher than 400 to 600 nM were correlated with thrombotic
risk (32, 33). In our study for rivaroxaban, such an increase in
thrombin generation above baseline H0 was not observed with
FEIBA. Moreover, it seems that the lower doses of FEIBA, corresponding to a quarter and a half of the dose usually used, have

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Marlu et al. Reversal of dabigatran and rivaroxaban

comparable reversal profiles to the higher doses, without dramatic

increase in ETP-AUC. Similar results concerning dose effects were
also reported when FEIBA was used to reverse the anticoagulant
effect of fondaparinux.
In view of its wide availability, known safety, and based on recent results (16), PCC has been proposed as a potential option for
the reversal of rivaroxaban (5). In our experiment, PCC does not
correct de lag time enough and appears less efficient than FEIBA.
Also recent data from animal haemorrhagic models (17) do not
completely support the use of PCC. Finally, although rFVIIa corrected Rivaroxaban-induced prolongation of LT, animal models
do not support the use of rFVIIa to reverse rivaroxaban (17).
Therefore, although a complete correction was not obtained,
FEIBA seems thus theoretically to represent the most appropriate
non-specific haemostatic agent to reverse the rivaroxaban effects
on the TG curve.
In the case of dabigatran, rFVIIa is the only drug able to correct
the kinetic of the TG curve. rFVIIa was reported to reduce bleeding
time in animal models treated with dabigatran, and recently in
human to manage dabigatran-associated post-surgery bleeding
(34). However, in this case, the use of rFVIIa, together with haemodialysis, makes difficult to assess the real efficacy of the haemostatic
drug. Moreover, in our experiment, the effect on TGT was obtain
with the higher dose of rFVIIa, and also raises the problem of
safety, even if rFVIIa did not dramatically increases the amount of
thrombin generated. Indeed, as for FEIBA, a dose-dependent effect
on the incidence of thrombotic complications was reported. For
dabigtran, the minimal efficient dose of FEIBA was not clearly determined, as the dose of 0.5 U/ml, but not 0.25 U/ml corrected LT
close to baseline, but the dose of 0.5U/ml overcorrected the ETPAUC. However, the increase Peak of thrombin generation above
baseline H0 remains below 400 nM for FEIBA at 0.25 to 0.5 U/ml.
Therefore, it is probably necessary to refine the dose of FEIBA for a
potential use in case of dabigatran. Nevertheless, based on our results, and also on animal models (26, 29), PCC but especially
FEIBA seem to be most reasonable (in the lower normally used
dose) approach to reverse dabigatran.
Finally, interpretation of TGT tests remains speculative, because of inconsistent experimental data, and also by the fact that
we actually do not have enough clinical data. However, these experimental data can be used to design prospective trials to assess
the efficacy and safety of such drugs to reverse effect of new anticoagulants.

What is known about this topic?

New anticoagulants such as dabigatran and rivaroxaban can be responsible for haemorrhagic complications.
As promising drugs, it is expected that these treatments will be
widely used in long-term indications such as atrial fibrillation.
The management of any bleeding event or emergency surgery, as
for any anticoagulant, is challenging.

What does this paper add?

This study tested ex vivo, the ability of all putative non-specific haemostatic agents currently available, at different dosages, to reverse
the anticoagulant activity of both dabigatran and rivaroxaban.
For both drugs, PCC corrected the ETP-AUC, whereas rFVIIa only
modified kinetic parameters. Higher doses of PCC and FEIBA were
responsible for an over correction of ETP-AUC and could be harmful. Lower doses of FEIBA, corresponding to a quarter to half the
dose usually used, have potential reversal profile of interest.
Prospective validation in clinical trials is urgently needed.

life of product such as rFVIIa, and we did not measure the duration
of the response. Third, the doses of anticoagulant tested, 150 mg
for dabigatran and 20 mg for rivaroxaban in one oral administration are not really comparable, as in clinical practice dabigatran
is usually prescribed twice a day, and is potentially accumulated at
steady state. Moreover, we did not test overdose conditions, such as
the high concentrations potentially observed in patients who have
kidney failure. Fourth, we performed TGT in platelet-poor plasma,
at standard concentration of tissue factor. Therefore, the effect of
drugs such as rFVIIa is likely to be different in vivo when there may
be massive exposure of TF. Fifth, we did not use a haemorrhagic
model, and our conclusions remain a theoretical extrapolation
from in vitro to in vivo. Finally, Dabigatran increased aPTT, TT and
ECT (35), and prolongation of TT or ECT was proposed to reflect
a high concentration of circulating dabigatran and to help monitor
surgery. Rivaroxaban significantly increased PT, as well as diluted
PT (36), and anti-FXa activity was suggested as a better indicator of
anti-FXa drugs plasma concentration (37). Unfortunately, we did
not explore, for the different concentration of haemostatic agents,
these tests, available in clinical practice, to judge the potential reversal effect.

Study limitations
Conclusion
They are several limitations in our study. First, our study was an ex
vivo study, by contrast to already published studies in which the reversing agent has been administered and in vivo observations were
made (16). However, in the absence of clear available data regarding the better choice to reverse anticoagulant effect, this was the
only way to test several agents at several doses. Second, the ex vivo
design has the disadvantage to not take into account the short half Schattauer 2012

In case of severe haemorrhage, i.e. intracerebral haemorrhage

needing rapid reversal of anticoagulant and, in the absence of specific antidotes, alternatives such as one of the non-specific haemostatic agents must be considered. However, clinical evaluation in
haemorrhagic situations and a meticulous risk-benefit appraisal of
the use of these treatments is urgently needed.
Thrombosis and Haemostasis 108.2/2012

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Marlu et al. Reversal of dabigatran and rivaroxaban

Acknowledgments

This study was sponsored by the Clinical Research and Innovation

Department (Dlgation la Recherche Clinique et Innovation) of
Grenoble University Hospital This study was also supported by a
grant from Diagnostica STAGO, France. All reagents for in vitro
analysis were graciously given by Diagnostica-STAGO, Asnires,
France. Novoseven, FEIBA and Kanokad were graciously given
by NovoNordisk, Baxter and Laboratoire Franais des Biotechnologies, respectively. The authors thank Alison Foote (Grenoble
Clinical Research Center) for help in preparing the English version
of the manuscript.
Conflicts of interest

G. Pernod was investigator for the EINSTEIN program using Rivaroxaban for venous thromboembolic disease. None of the
other authors declares any conflict of interest.

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