1. Effect of non-specific reversal agents on anticoagulant activity
of dabigatran and rivaroxaban
Raphael Marlu, Enkelejda Hodaj, Adeline Paris, Pierre Albaladejo, Jean Luc Crackowski, Gilles Pernod
Thrombosis and Haemostasis 108.2/2012
Journal Club presented by: Megan Handley December 5, 2014
Background and Overview
Background New anticoagulant medications have recently been approved for the treatment of venous thromboembolic
disease (VTE) and for prevention of systemic embolism in non-valvular atrial fibrillation. They do not require
frequent monitoring, and have little interactions with foods and other medications. However, unlike warfarin,
these medications have no antidote. In the case of major bleeding or emergency surgery, the effects of these
medications would need to be reversed immediately. In this study, the effect of all putative hemostatic agents on
rivaroxaban (direct factor Xa inhibitor) and dabigatran (direct thrombin inhibitor) were tested via thrombin
generation tests to determine which anticoagulation reversal strategy was most effective for each
anticoagulation agent.
Study Objective To test the ability of all purative non-specific hemostatic agents currently available, at various doses, to reverse
the anticoagulation activity of rivaroxaban and dabigatran using thrombin generation tests.
Methods
Study Design Randomized, crossover ex vivo study in healthy volunteers.
Subject
Selection
Inclusion Criteria Exclusion Criteria
• Age 18-45 years old
• BMI between 18 and 30 kg/m2
• White males
• Personal or family history of bleeding disorders.
• Renal or liver impairment
Study
Protocol
• 10 healthy, white, male subjects between the ages of 18 and 45 were included in the study between November
2010 and March 2011. They all gave written informed consent, and received financial compensation for their
participation.
• Volunteers were randomized to receive either 150mg of dabigatran or 20mg of rivaroxaban in one oral dose.
• A 15-day washout period preceded the administration of the second drug for an identical investigation.
• Venous blood samples were collected just before drug administration and 2 hours after. Following a double
centrifugation, platelet poor plasma was collected, quick-frozen, and stored at -80°C until the analyses were
performed.
• The three reversal agents were recombinant factor VIIa (rFVIIa), activated prothrombin complex concentrate
(FEIBA), and the four-factor prothrombin complex concentrate (PCC).
• Thrombin generation was measured using Calibration Automated Thrombinography (CAT) reagents. Quantitative
parameters included: endogenous thrombin potential (ETP-AUC) and the maximum concentration of thrombin
(peak). Kinetic parameters included: lag time (LT) and time to reach the maximum concentration of thrombin
(time to Peak, TTP).
• Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were also determined.
Outcome
Measures
• Endogenous thrombin potential (ETP-AUC)
• Thrombin peak
• Lag-time (LT)
• Time to peak (TTP)
Statistical
Analysis
• Quantitative data were expressed as means ± standard deviation (SD)
• Paired t-tests were used to analyze the reversal effect for each quantitative parameter.
• Normality assumption was checked with a Shapiro-Wilk’s test and the Wilcoxon signed-ranks test was used if the
normality assumption was violated.
• Spearman’s coefficient of rank correlation was used to assess the correction between the different parameters
of the thrombin generation test
• Two-sided significance tests were used throughout the study.
• Stata software was used for all analyses
• P-value < 0.05 was considered statistically significant.
Results

2. Medication
characteristics
(prior to reversal)
Table 1: Thrombin generation test parameters measured at baseline (H0) and 2 hours (H2) after oral intake of
20 mg rivaroxaban or 150 mg dabigatran. ETP-AUC (nM.min); Peak is the maximum concentration of thrombin
(nM); LT (seconds); TTP (seconds). 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)
p <0.001 <0.0001 <0.0001 <0.0001
• Rivaroxaban significantly prolonged PT by 1.37-fold (p<0.0001)
• 2 hours after administration, the effect of 20mg rivaroxaban was marked by the reduction of
thrombin peak by a factor of 3, representing the effect of rivaroxaban on thrombin generation.
• Modest reduction (22%) in the ETP-AUC
• The initiation phase of thrombin generation was profoundly altered, as LT and TTP were more than
doubled.
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)
p 0.0013 0.5 0.0009 0.002
• Dabigatran significantly increased aPTT by 1.47-fold (p<0.0001)
• After receiving 150mg dabigatran, ETP-AUC was modestly reduced by 20%
• Thrombin peak unchanged
• Initiation phase of thrombin was significantly altered, as shown by the LT increase.
• The index rate of thrombin generation was not inhibited
• 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)
Outcomes RIVAROXABAN

3. ETP-AUC
• At H2, all reagents but rFVIIa resulted in a dose-dependent increase in the ETP-AUC
o Lowest dose of PCC (0.25 U/mL) resulted in 37% decrease vs. H2
o Lowest dose of FEIBA (0.25 U/mL) resulted in 50% decrease vs. H2
• 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 (p=0.07)

4. • Over-correction occurred for all other PCC and FEIBA concentrations
Thrombin Peak
• Significant dose-dependent correction of thrombin peak using PCC or FEIBA vs. H2
• FEIBA at doses 1 and 2 U/mL corrected the thrombin peak close to its H0 value (p=0.2); however, these
doses resulted in over correction of ETP-AUC
Lag-time
• PCC resulted in slight reduction in LT (15% for 1U/mL)
• FEIBA resulted in 47% decrease in LT (p<0.0001)
• rFVIIa completely reversed the LT increase induced by rivaroxaban (2.24 seconds at H2 vs. 2.29 seconds at
H0). This was the only agent that corrected LT to near baseline H0 (p=0.6)
Time to reach maximum concentration of thrombin (TTP)
• PCC did not effect TTP
• rFVIIa and FEIBA significantly decreased TTP by 44% and 30% respectively vs. H2 (p<0.001).
DABIGATRAN

5. ETP-AUC
• At H2, PCC and FEIBA increased the EUC-AUC of dabigatran-anticoagulated plasma in a concentration-
dependent manner
• Low doses of PCC or FEIBA reversed the ETP-AUC results close to baseline H0 (p=0.02)
Thrombin Peak
• Dramatic increase in thrombin generated occurred when PCC or FEIBA were used at regular or half doses.
• All agents except rFVIIa were responsible for an increase in the thrombin peak above baseline H0 value
(p<0.0001)
• Maximum increase in thrombin peak with PCC at 1 U/mL and FEIBA at 2 U/mL
Lag-time
• Both the highest dose of rFVIIa (3U/mL) and the three highest doses of FEIBA (range 0.5-2 U/mL) reduced
the LT (p<0.001)
Time to reach maximum concentration of thrombin (TTP)
• Similar results observed for rFVIIa and FEIBA, with a significant effect of the lowest dose of FEIBA (0.25
U/mL, p=0.03)
Author’s Conclusion
RIVAROXABAN REVERSAL
The effect of rivaroxaban on the ETP-Peak was more pronounced than on the ETP-AUC. PCC strongly corrected ETP-AUC above the
baseline values for the highest doses. rFVIIa only had a relevant effect on kinetic parameters. Rivaroxaban altered the LT and TTP,
which have been found to be potentially relevant for determining the antithrombotic effect of rivaroxaban, or its reversal. rFVIIa, at
all studied doses, showed complete reversion of the prolonged LT caused by rivaroxaban to the baseline H0 level. FEIBA corrected all
TGT parameters compared to H2 at all studied doses. Only FEIBA corrected the markedly affected TPP by >20%.
DABIGATRAN REVERSAL
The overall generation of thrombin determined by ETP-AUC is moderately decreased with dabigatran therapy. LT was increased,
without affecting the velocity of thrombin generation. PCC increased ETP-AUC. LT was described as correlated with circulating
concentrations of dabigatran. rFVIIa at a higher dose and FEIBA corrected the LT to nearly baseline value.

6. CLINICAL IMPLICATIONS
• FEIBA combines the rFVIIa effect on LT and the PCC correcting-effect on peak value. Higher doses of FEIBA resulted in an
increase in ETP-AUC and peak, indicating an increase in total thrombin formation. The combined effects of FEIBA make
thrombotic risk a safety concern. Ehlrich et al. reported the incidence of thrombotic adverse events as four per 100,000
FEIBA infusions. This incidence was dose-dependent. In the rivaroxaban studies, FEIBA did not increase thrombin generation
above baseline. PCC has been proposed as a possible reversal agent for rivaroxaban. However in the study, PCC does not
appear to correct the LT enough, and appears less efficient than FEIBA. Based on the study, FEIBA seems to be the most
appropriate non-specific hemostatic agent to reverse rivaroxaban effects on the TGT.
• In the case of dabigatran, rFVIIa is the only drug able to correct the thrombin generation curve. This effect was seen in the
experiment with the higher dose of rFVIIa. This presents a safety concern for over-reversion and thrombotic risk. PCC
increased ETP-AUC, but had no effect on TGT parameters. FEIBA dosed at 0.5U/mL corrected LT close to baseline, unlike the
0.25U/mL dose. The increase peak of thrombin generation above baseline remains below 400nM for FEIBA at 0.25 and
0.5U/mL. However, the ETP-AUC was overcorrected with the 0.5U/mL FEIBA dose. Therefore, PCC and especially FEIBA (at
the lower normally used dose) seem to be reasonable agents to reverse the effects of dabigatran.
Critique & Discussion
Weaknesses • Ex vivo observations were used, which are inferior to in vivo observations
• Ex vivo design also prevents the duration of the responses seen in the study.
• The effects of renal insufficiency leading to potential overdose of the anticoagulation agents were not
studied due to the ex vivo design and the exclusion of those with renal insufficiency.
• Hemorrhagic conditions, which is the indication for anticoagulation reversal, were not studied due to the
ex vivo design.
• The population used was 18-45 year old healthy, white males, which does not match the patient
population of anticoagulation therapy.
• Dabigatran was dosed as a single 150mg dose, when in reality it is usually prescribed twice daily, and
accumulates at steady state.
• TGT were conducted in platelet-poor plasma, at a standard concentration of tissue factor. The effect of
drugs such as rFVIIa is likely to be different in vivo where there is extensive exposure to tissue factor.
• The study also did not address the relevance of PT and plasma concentrations of the anticoagulation
medications to judge the reversal effect.
• Small amount of participants.
• Not a prospective, randomized, controlled trial (optimal study design)
Strengths • Evaluating the various hemostatic agents in one trial was an effective way of comparing the effects of
the agents on reversal of the two anticoagulation agents.
• The use of various doses of the reversal agents was also a strength of the study. It highlighted the dose
limitations and risks, as well as the dose-dependent discrepancies.
• The crossover design creates consistency in response for the two agents since the study subject is the
same.
Relevant
Studies
 Eerenberg et al. studied the reversal of rivaroxaban and dabigatran by PCC and found PCC effective in
reversing rivaroxaban, but not dabigatran. PCC only resulted in slight correction of the thrombin
generation tests in the Marlu et al. study. However, PCC was not compared to FEIBA in the Eerenberg
trial. (2011)
 Zhou et al. established a murine model of intracranial hemorrhage associated with rivaroxaban and
examined the effectiveness of PCC, rFVIIa, and FFP in preventing intracerebral hematoma expansion. All
agents were proven effective in prevention. The clinical symptom of bleeding was studied. All the tested
agents were effective. Since bleeding was not observed in the Marlu study, results cannot be compared.
(2013)
 Zhou et al. conducted a similar study as above with dabigatran reversal. The evidence shows PCC, and
less consistently, FFP prevent hematoma expansion associated with dabigatran. This study supports the
result of PCC being somewhat effective in reversal, however FFP was not studied in the Marlu trial.
(2011)
 Lu et al. designed a modified form of factor Xa as an antidote for factor Xa inhibitors. The recombinant
protein (r-Antidote) restored hemostasis in rabbits treated with rivaroxaban. (2013)
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