Reversal of dabigatran anticoagulation ex vivo: Porcine study comparing prothrombin complex concentrates and idarucizumab.

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Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Reversal of dabigatran anticoagulation ex vivo: Porcine study comparing prothrombin complex concentrates and idarucizumab Markus Honickel1; Stefanie Treutler1; Joanne van Ryn2; Sabine Tillmann1; Rolf Rossaint1; Oliver Grottke1 1Department

of Anaesthesiology, RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co. KG, Biberach,

Germany

Summary Urgent surgery or life-threatening bleeding requires prompt reversal of the anticoagulant effects of dabigatran. This study assessed the ability of three- and four-factor prothrombin complex concentrate (PCC) and idarucizumab (specific antidote for dabigatran) to reverse the anticoagulant effects of dabigatran in a porcine model of trauma. Twelve animals were given dabigatran etexilate (DE) orally and dabigatran intravenously, before infliction of trauma. Six animals received tranexamic acid plus fibrinogen concentrate 12 minutes post-injury. Six PCCs (each 30 and 60 U/kg) and idarucizumab (30 and 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by several coagulation assays. All coagulation parameters were altered after dabigatran infusion (plasma level: 442 ± 138 ng/ml). Both threeand four-factor PCCs mostly or completely reversed the effects of dabigatran on thromboelastometry variables and PT but not on aPTT. Idarucizumab neutralised plasma concentrations of dabigatran, and reversed the effects of the drug on coagulation variables. Thrombin

Correspondence to: Oliver Grottke, MD PhD Department of Anaesthesiology RWTH Aachen University Hospital Pauwelsstrasse 30, 52074 Aachen, Germany Tel.: +49 241 8080972, Fax: +49 241 8082406 E-mail: [email protected]

generation showed dose-dependent over-correction following the addition of PCC, implying that elevated levels of thrombin are required to overcome dabigatran-induced coagulopathy. In contrast, treatment with idarucizumab returned thrombin generation to baseline levels. Following trauma, therapy with tranexamic acid plus fibrinogen improved correction of coagulation parameters by PCC, and thromboelastometry parameters by idarucizumab. All investigated PCCs improved dabigatran- and trauma-induced coagulopathy to a similar degree. In conclusion, this study shows that three- and four-factor PCCs are similarly effective for dabigatran reversal. Idarucizumab also reversed the effects of dabigatran and, unlike PCCs, was not associated with over-correction of thrombin generation.

Keywords Dabigatran, idarucizumab, prothrombin complex concentrate, trauma, thrombin generation

Received: August 29, 2014 Accepted after major revision: November 19, 2014 Epub ahead of print: January 8, 2015 http://dx.doi.org/10.1160/TH14-08-0712 Thromb Haemost 2015; 113: ■■■

Note: This study was performed at the RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany.

Introduction Dabigatran is a new oral anticoagulant approved for the prevention and treatment of venous thromboembolic events and for the prevention of stroke and systemic embolism in adult patients with non-valvular atrial fibrillation (1, 2). As a direct thrombin inhibitor, dabigatran functions by binding both free and clot-bound thrombin, thereby impeding the conversion of fibrinogen to fibrin and preventing thrombus fomation. It is administered orally as the prodrug dabigatran etexilate (DE) and is characterised by rapid onset of action, few drug or food interactions, no requirement for routine coagulation monitoring and a short half-life of 12–14 hours (h) (2). Based on a large phase III trial (RE-LY), the overall bleeding risk is similar to warfarin with improved stroke prevention with the higher 150 mg dose (3). Thus, appropriate prescription of dabigatran and consideration of contraindications, includ-

ing renal insufficiency (clearance rate < 30 ml/kg), should result in low rates of bleeding complications. In the case of life-threatening bleeding (e. g. following injury or intra-cranial haemorrhage), immediate and prompt reversal of anticoagulation is required (4). The management of bleeding is complicated by the lack of coagulation assays to provide timely, quantitative information on the degree of anticoagulation (i. e. point-of-care assays), and pre-clinical studies reported confliciting data regarding the reversal of dabigatran. In haemodynamically stable patients, dabigatran may be removed from the circulation by dialysis (5). This approach is especially effective in patients with end-stage renal disease or overdosing, but the approach is not immediate (i. e. takes 4–6 h) and may not be feasible in haemodynamically unstable patients. Current European Society of Cardiology guidelines suggest prothrombin complex concentrate (PCC) or activated PCC (aPCC) for life-threatening bleeding under dabi-

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Honickel et al. Reversal of dabigatran

gatran anticoagulation (6), based on positive results from preclinical studies (7–9). PCCs are derived from human plasma and their principal constituents are the vitamin K–dependent coagulation factors II, IX and X, with or without significant levels of factor VII. They also contain varying amounts of heparin and the endogenous coagulation inhibitors, protein C and protein S. PCCs with low levels of factor VII are known as three-factor PCCs, while those with higher levels of factor VII are four-factor PCCs. They are all standardised and administered according to the quantity of factor IX, but there are considerable differences between PCCs in the quantities of coagulation factors II, VII, and X, relative to factor IX (10). In a phase III clinical trial, administration of a four-factor PCC reversed the effect of vitamin K antagonists on the international normalised ratio (INR) in 62 % of patients within 1 h (11). Several case reports and a limited number of pre-clinical studies have shown that PCC may be an effective treatment for coagulopathic bleeding outside vitamin-K antagonist reversal (12). The possibility of using PCC for reversal of novel oral anticoagulants is also being investigated (12, 13). Using an experimental trauma model with high plasma concentrations of dabigatran, our group has shown that a four-factor PCC effectively reverses dabigatran-induced coagulopathy (14). Additional animal studies have shown that four-factor PCC can reverse dabigatran-induced bleeding, and that with highdose PCC (300 IU/kg) the presence of dabigatran may inhibit or prevent thrombosis (15, 16). However, due to the substantial differences between quantities of coagulation factors and inhibitors within PCCs, it is difficult to predict the impact of these products on overall coagulation status and thrombin generation in patients taking dabigatran. In trauma-induced coagulopathy, early use of tranexamic acid is associated with improved survival (17). Additionally, low levels of fibrinogen are an important contributor to trauma-induced coagulopathy: fibrinogen supplementation has the potential to correct coagulopathy and improve outcomes such as blood loss and survival rates (18–20). No studies have so far investigated the clinical impact of combined treatment with tranexamic acid and fibrinogen in severely bleeding patients under dabigatran anticoagulation. This ex vivo study was performed to evaluate a number of commercially available three- and four-factor PCCs, and a specific dabigatran antidote (idarucizumab) to reverse coagulopathy in a dabigatran anticoagulation/trauma model in pigs. Additional post-injury therapy with tranexamic acid plus fibrinogen concentrate was also investigated, alone and in combination with PCCs.

Materials and methods Ethics All experiments were performed in accordance with German legislation governing animal studies following The Principles of Laboratory Animal Care (21). Ethical approval was obtained from the regional governmental animal care and use office (No. 84–02.04.2013.A083). Twelve pigs were housed in ventilated rooms and acclimatised to their surroundings for at least seven

days. Animals were fasted overnight before surgical procedures, with unrestricted access to water.

Dabigatran administration and infliction of trauma DE (Pradaxa®, Boehringer Ingelheim, Biberach, Germany) was administered orally twice daily for three days (30 mg/kg bid). Animals were then prepared for surgery and anaesthetised as previously described, with maintenance anesthesia for the duration of the procedure (14). On the 4th day, after surgical preparation dabigatran (active substance) was infused at a rate of 0.77 mg/kg/h for 30 min and 0.2 mg/kg/h for 60 min. This approach enabled consistent, supratherapeutic plasma concentrations to be achieved, together with complete distribution of the drug into the body tissues. Standardised polytrauma comprised bilateral femur fractures and a blunt liver injury. The fractures were induced with a captive bolt gun (Karl Schermer, Ettlingen, Germany) placed vertically against the femur and fired while the leg was extended (22). Liver injury was induced by clamping once through the parenchyma of the right middle liver lobe using a custom-made instrument (23). Five minutes after injury, all animals received Ringer’s solution as a fluid bolus over 5 minutes (min) at 5 ml/kg/min, followed by continuous infusion at a rate of 40 ml/kg/h. The animals were then allocated to one of two groups. At 12 min after injury, animals in group 1 (n=6) received placebo as Ringer’s solution; animals from group 2 (n=6) received an intravenous bolus of tranexamic acid (20 mg/kg, Cyclocapron, Pfizer, New York, NY, USA) plus fibrinogen concentrate (80 mg/kg, Haemocomplettan P, CSL Behring, Marburg, Germany). At 60 min after trauma, the abdomen was reopened and any blood was collected to determine total blood loss post-injury. A summary of the study is shown in ▶ Figure 1.

Ex vivo addition of PCCs and idarucizumab The following haemostatic agents were tested: four four-factor PCCs (Beriplex P/N 500 [US brand-name Kcentra] [CSL Behring, Marburg, Germany], lot 80970111A; Cofact 500 [Sanquin, Amsterdam, The Netherlands], lot VNP5L003; Prothromplex NF 600 [Baxter, Deerfield, IL, USA], lot VNP5N010; Octaplex 500 [Octapharma, Vienna, Austria], lot B342A264A) and two three-factor PCCs (Bebulin [Baxter], lot VNP6L005; Profilnine [Grifols, Los Angeles, CA, USA], lot B2PB138001). Idarucizumab (Boehringer Ingelheim, Biberach, Germany; lot 6001325) was obtained in Tween 20 buffer (25 mM acetate, 220 mM sorbitol and 0.2 % polysorbate 20) at a concentration of 44 mg/ml. Aliquots were stored at –80˚C and thawed at 37˚C for 10 min prior to application.

Blood collection and ex vivo addition of haemostatic agents Blood samples were collected into sodium citrate (Sarsted, Nuembrecht, Germany) at the following four time-points: baseline (before administration of DE), 12 h after the last oral dose of DE representing the trough level of dabigatran (“low dabigatran”), after dabigatran infusion representing the peak level of dabigatran

Thrombosis and Haemostasis 113.4/2015

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Figure 1: Study overview. Flowchart summarising the main steps of the

study.

(“high dabigatran”) and 60 min post-injury (“post-trauma”; 60 min after stopping the dabigatran infusion). Placebo (saline), four-factor PCCs, three-factor PCCs, or idarucizumab were added ex vivo to each citrated whole-blood sample at each timepoint. The amounts of PCC were designed to reflect plasma concentrations achieved with doses of 30 U/kg and 60 U/kg. Idarucizumab was added to achieve levels equivalent to doses of 30 or 60 mg/kg (14).

Thrombin generation in plasma was measured using the Calibrated Automated Thrombogram (CAT; Thrombinoscope, Maastricht, The Netherlands) as described previously, using final concentrations of 5 pM tissue factor and 4 µM phospholipids (24). As heparin can also inhibit thrombin generation, heparinase (Dade Hepzyme, Siemens, Marburg, Germany; lot B4240–10; amount for neutralising 1 USP/mL of heparin) was added to all plasma samples after PCC addition and prior to performing the assay. The in vitro effect of the heparin component in each PCC tested is shown in a representative thrombin generation measurement with and without addition of heparinase (Suppl. Figure 1, available online at www.thrombosis-online.com).

Analytical methods including coagulation assays Haemoglobin concentrations were measured with a blood gas analyser (ABL720, Radiometer, Copenhagen, Denmark). Prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration were determined by standard laboratory tests (all reagents from Dade Behring, Marburg, Germany) on a coagulometer (MC 4 plus, Merlin Medical, Lemgo, Germany). The aPTT test is very sensitive to heparin concentrations and PCC preparations contain differing amounts of heparin. To eliminate this effect, heparinase (Dade Hepzyme, Siemens, Marburg, Germany; lot B4240–10; amount for neutralising 1 USP/ml of heparin) was added to all plasma samples after PCC addition. Plasma concentrations of dabigatran were determined using the diluted thrombin time (Hemoclot, HyphenBiomed, Neuville sur-Oise, France).

Thromboelastometry and thrombin generation Coagulation was assessed in whole blood using thromboelastometry (ROTEM, Tem International, Munich, Germany) and the EXTEM assay was performed according to the manufacturer’s instructions.

Statistical analysis Statistical analysis was performed using SPSS 22 (SPSS, Chicago, IL, USA). For graphical purposes, GraphPad Prism (Version 6, GraphPad Software, San Diego, CA, USA) was used. A priori ranking of laboratory parameters would have been highly speculative, so the primary outcome parameters were not defined and the study was carried out using an exploratory approach. Based on a previous study with comparable experimental design, a sample size of six per group was considered to be sufficient (14). Differences between the control and intervention groups were analysed with a one-way analysis of variance (ANOVA), with the Tukey post-hoc test for multiple comparisons. These analyses were performed two-tailed and p-values < 0.05 were considered as statistically significant. ‘Non-measurable’ was entered for clot formation time (CFT) when the required clot amplitude of 20 mm was not reached within 4,000 seconds (sec), and for lag time in CAT if 10 sec was exceeded. Data are presented as mean ± standard devi-

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ation (SD). Baseline values are shown by a grey dotted line in all figures.

Results Twelve male German land-race pigs weighing between 37 and 43 kg were included in this ex vivo study.

Effects of dabigatran administration and infliction of trauma At baseline, all coagulation parameters were within reference ranges. After oral administration of DE, the mean plasma concentration of dabigatran was 160 ± 110 ng/ml (“low dabigatran” in ▶ Table 1). Coagulation parameters were prolonged at this time compared with baseline: PT 11.9 ± 2.2 vs 8.7 ± 0.6 sec; aPTT 15.4 ± 3.5 vs 11.3 ± 2.5 sec (values after oral administration of DE are shown as “control” in ▶ Figure 2 A, D). Consistent with this, the thromboelastometry variables CT and CFT were also prolonged after oral administration of DE (“control” in ▶ Figure 3 A, B). Another thromboelastometry variable which indicates the speed of clot formation, maximum velocity, was reduced from a baseline value of 44.0 ± 5.9 to 31.8 ± 7.1 mm/min (▶ Figure 3 D). However, oral DE administration did not appreciably affect clot strength (MCF; “control” in ▶ Figure 3 C), or levels of haemoglobin or fibrinogen; ▶ Table 1). Following the 90-min infusion of dabigatran, the mean plasma concentration of dabigatran increased to 442 ± 138 ng/ml (▶ Table 1). This supratherapeutic level was associated with further prolongation of PT, aPTT, CT and CFT (▶ Figure 2 B, E and ▶ Figure 4 A, B). These changes in coagulation parameters were compounded by blood loss following trauma (total blood loss at 60 min, group 1: 2,367 ± 253 ml; tranexamic acid plus fibrinogen concentrate, group 2: 2,004 ± 283 ml; between-group difference not statistically significant) and by dilution following the infusion of crystalloids. At 60 min after trauma, 4/6 animals in group 1 and 2/6 animals in group 2 had no measurable clot formation (EXTEM CFT ≥ 4,000 sec). Also at this time-point, clot strength (EXTEM MCF) and maximum velocity were reduced (▶ Figure 5). In addition, the

mean plasma fibrinogen concentration had decreased to 36 ± 10 mg/dl, the haemoglobin level had dropped to 3.8 ± 0.6 g/dl (group 1 animals; ▶ Table 1), and further prolongations of PT and aPTT were observed (▶ Figure 2 C, F). Dabigatran appeared to affect all three thrombin generation parameters in a concentration-dependent manner. Prolonged lag time, reduced peak thrombin concentration and reduced ETP were observed (▶ Figure 6).

Measurements after haemostatic therapy ex vivo Ex vivo treatment with PCCs The effects of dabigatran anticoagulation and trauma-induced coagulopathy were reduced by all six PCCs (for time point “posttrauma” the impact on PCC30 on thromboelastometry, PT and aPTT are not shown). This was evident from significant decreases versus control in PT, CT and CFT (▶ Figures 2–5). A similar pattern was observed following trauma: PT, CT and CFT were significantly shortened by all PCCs, and clot strength and maximum velocity substantially increased without reaching baseline values (▶ Figure 2 C and ▶ Figure 5 A–D). Despite ~80–90 % reversal of these parameters, PCC treatment without prior fibrinogen supplementation had no effect on aPTT at any time-point. When dabigatran was present at therapeutic peak concentrations (“low dabigatran”), addition of PCCs dose-dependently and significantly increased the ETP versus control and also vs baseline (▶ Figure 6). At supratherapeutic dabigatran concentrations (“high dabigatran”) and after trauma, ETP and peak thrombin generation showed that thrombin generation was increased significantly above baseline by adding PCC. The addition of PCCs had no effect on dabigatran concentrations (data not shown). Additional treatment with tranexamic acid plus fibrinogen concentrate (group 2) significantly enhanced plasma fibrinogen concentration (107 ± 22 mg/dl) (▶ Table 1). PCC reversal of coagulopathy was enhanced, as shown by significant reductions in PT, aPTT and clot formation time (▶ Figure 2 C, F and ▶ Figure 5 B). In addition, clot strength and maximum velocity with PCC were significantly increased by tranexamic acid plus fibrinogen concentrate (▶ Figure 5 C, D). However, the additional treatment had no

Table 1: Haematological parameters and plasma fibrinogen concentration during the study. Plasma concentration (activity, measured by diluted thrombin time) of dabigatran (ng/ml) during the study. At 12 min after trauma, animals from group 1 (controls) received placebo (saline), and animals from group 2 received tranexamic acid (20 mg/kg) plus fibrinogen concentrate (80 mg/kg).

Baseline (n=12)

Low dabigatran (n=12)

High dabigatran (n=12)

Post-trauma Group 1 (n=6)

Group 2 (n=6)

Fibrinogen [mg/dl]

149 ± 20

146 ± 26

136 ± 25

36 ± 10

107 ± 22*

Platelet count [x 103µl]

478 ± 71

411 ± 63

348 ± 41

166 ± 35

189 ± 43

Haemoglobin [g/dl]

10.5 ± 0.9

10.5 ± 0.52

8.8 ± 0.25

3.8 ± 0.6

4.2 ± 0.6

Dabigatran [ng/ml]

NA

160 ± 110

442 ± 138

338 ± 119

293 ± 102

Plasma concentrations of dabigatran were not measured at baseline since DE had not yet been administered. *P < 0.05 vs Group 1. Data are shown as mean ± SD. NA: not assessed.


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Figure 2: Prothrombin time and aPTT at low dabigatran concentration, high dabigatran concentration and 60 min after trauma. Prothrombin time (A-C) and aPTT (D-F) determined the effect of ex vivo haemostatic therapy at various time points. For clarity at 60 min post-trauma, only

significant impact on PCC effects on clotting time (Figure ▶ Figure 5 A) or on thrombin generation (▶ Figure 6). Plasma levels of dabigatran were also unaffected by tranexamic acid plus fibrinogen concentrate.

the effects of the high concentration of haemostatic therapy are shown. Grey dotted lines indicate baseline mean values. Data (mean ± SD) were analysed by ANOVA with Tukey as a post-hoc test. *P < 0.05 vs control; §P < 0.05 vs PCC of the same group; #P < 0.05 vs all PCCs.

Ex vivo treatment with idarucizumab Addition of idarucizumab restored dabigatran-prolonged coagulation parameters to baseline values (▶ Figure 2). This was ob-

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Figure 3: Thromboelastometry (ROTEM) parameters after oral administration of dabigatran etexilate. A) The clotting time (CT) represents the initiation of clot formation and corresponds to the reaction time. B) Clot formation time (CFT) reflects coagulation time until 20 mm of amplitude are reached. C) The maximum clot firmness (MCF) reflects the strength of a resulting clot. D) The maximum velocity (V) of clot formation is shown. Grey dotted lines indicate baseline mean values. Data (mean ± SD) were analysed by ANOVA with Tukey as a post-hoc test. *P < 0.05 vs control; §P < 0.05 vs PCC of the same group; #P < 0.05 vs all PCCs.


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Figure 4: Thromboelastometry parameters after intravenous administration of dabigatran. EXTEM values obtained after 90 min of intravenous infusion of dabigatran, “high dabigatran”. Grey dotted lines indicate baseline mean values. Data (mean ± SD) were analysed by ANOVA with Tukey as a post-hoc test. *P < 0.05 vs control; §P < 0.05 vs PCC of the same group; #P < 0.05 vs all PCCs.

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Figure 5: Thromboelastometry parameters 60 min after trauma. EXTEM measurements 60 min after stopping the dabigatran infusion and the infliction of trauma to measure the impact of the different PCC or idarucizumab preparations on dabigatran reversal. For clarity at 60 min post-trauma, only effects of high concentration (60 U/kg) of haemostatic therapy and idarucizumab (60 mg/kg) are shown. Grey dotted lines indicate baseline mean values. Data (mean ± SD) were analysed by ANOVA with Tukey as a post-hoc test. *P < 0.05 vs control; §P < 0.05 vs PCC of the same group; #P < 0.05 vs PCC.


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served with low and high levels of dabigatran, as well as post-trauma. The restoration of aPTT to baseline values (▶ Figure 2 D–F) distinguished idarucizumab from substitution with PCC; a significant difference between idarucizumab and PCC was also observed in relation to PT at the “high dabigatran” time-point. Small differences vs baseline in thromboelastometry parameters remained with idarucizumab (e. g. MCF and maximum velocity post-trauma; ▶ Figure 5). On the other hand, thrombin generation data showed that both ETP and peak height were returned to baseline by idarucizumab (▶ Figure 6), without the overcompensation seen with PCC addition. No significant differences between the two concentrations of idarucizumab were detected with any of the tests. Treatment with tranexamic acid plus fibrinogen concentrate post-trauma improved idarucizumab reversal of PT, CFT, MCF and maximum velocity. Mean plasma concentrations of dabigatran were below the limit of detection (< 30 ng/ml; data not shown) after the addition of both concentrations of idarucizumab (14).

Discussion This pre-clinical trial is the first to show that three- and four-factor PCCs are equally effective for the reversal of dabigatran-induced coagulopathy in a model of anticoagulation and polytrauma. In contrast to other parameters, thrombin generation showed a clear dose-dependent effect with an overcorrection of dabigatran-induced impairment after the addition of PCC. The antibody fragment idarucizumab corrected all coagulation parameters to baseline values; in contrast to PCCs, there was no overcorrection of any coagulation parameter with idarucizumab. The impact of posttrauma therapy with tranexamic acid plus fibrinogen concentrate was evident by greater enhancement of clot strength and greater improvements in clot formation after the ex vivo addition of PCC or idarucizumab. Addition of tranexamic acid and fibrinogen concentrate (in the absence of PCC or idarucizumab) had no significant effect on bleeding after 60 min. The mechanism(s) by which PCCs reverse the effects of dabigatran are not well understood. One possible explanation is that excess thrombin molecules, produced from the prothrombin in PCCs, are in such excess that dabigatran molecules are saturated with thrombin and enough unbound thrombin still remains to stimulate coagulation without dabigatran inhibition. In vivo this may not be a systemic phenomenon, but happen locally at an injury site where coagulation is activated. This is supported by the observation that sufficient concentrations of PCC are needed to overcome dabigatran-induced anticoagulation (25). Monotherapy with 25 U/kg PCC appears insufficient to terminate severe bleeding under supratherapeutic anticoagulation with dabigatran. Similarly, van Ryn showed a dose-dependent effect with a four-factor PCC in a rat-tail bleeding model (26). Further evidence from preclinical studies supports a potential role for using PCCs to reverse the effects of dabigatran (16, 27, 28). Unfortunately, there are only limited data relating to the use of PCCs in dabigatran-anticoagulated humans. In two studies, PCC

increased thrombin generation in dabigatran-anticoagulated volunteers (29, 30). Several case reports have also shown that PCC therapy is effective in reducing dabigatran-associated bleeding (31). One study failed to show any effect of PCC (Cofact) on aPTT after dabigatran anticoagulation (32). However, as shown by the results of the present study, this lack of effect may be attributable to the choice of coagulation assay. A clear effect of PCCs including Cofact was observed in our study on a variety of parameters, including PT, thromboelastometry variables and thrombin generation; however, aPTT was not altered. A substantial shortcoming of PCC use is the potential risk of thromboembolic complications (8). We have shown in an experimental animal model of liver injury that high levels (50 U/kg) of PCC may increase the risk for disseminated intravascular coagulation, due to an imbalance of pro- and anti-coagulant proteins. These data are in line with another observational study reporting treatment with a four-factor PCC in severely injured patients (33). Although no thromboembolic complications were observed, ETP was increased for several days, which presumably increased the patients’ prothrombotic risk. Despite high plasma concentrations of dabigatran, thrombin generation data indicate an overcorrection of thrombin generation after application of low concentrations (30 U/kg) of PCC. Thus, clinically it seems reasonable to titrate PCCs for emergency reversal as opposed to employing high doses initially. The lack of a routinely used point-of-care measurement for detecting dabigatran complicates treatment in bleeding patients requiring urgent interventions. In emergency surgery and/or trauma-related coagulopathy, delays in the detection of coagulopathy may influence outcome. Conventional coagulation tests such as aPTT or diluted thrombin time usually have slow turnaround times (e. g. 60 min), whereas thromboelastometry allows a rapid assessment of the patient’s coagulopathy. Thromboelastometry may, therefore, prove useful for guiding PCC therapy in dabigatran-treated patients with traumatic coagulopathy (14). The EXTEM assay is similar to PT in that it assesses tissue factor-initiated extrinsic coagulation, making it the most suitable thromboelastometric assay for guiding PCC reversal of dabigatran (34). There has been little investigation of the effects of dabigatran on viscoelastic coagulation parameters but, consistent with the present findings, prolonged activated clotting time with rapid thromboelastography has been reported in several patients taking dabigatran (35). We found that PCC and idarucizumab reversed the effects of dabigatran on CT and CFT. Although thrombin generation may provide the best insight into the anticoagulant effect of dabigatran and their reversal by PCC, routine and timely assessment of thrombin generation parameters is not currently practical in the clinical setting (9). The results of the present in vitro study show that the specific antidote to dabigatran, idarucizumab, has a greater impact on coagulation variables than PCC. Reversal of dabigatran even at supratherapeutic concentrations has been reported previously in a rabbit model of anticoagulation (16). Similarly, data from a phase I study including 145 healthy volunteers showed that dabigtran anticoagulation can be reversed by idarucizumab (36). Idarucizumab

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was infused at three fixed doses (1, 2 or 4 g) and the effects were dose-dependent: immediate reversal of plasma concentrations of dabigatran was observed, with the two higher doses of idarucizumab completely reversing dabigatran anticoagulation. No adverse reactions were observed. In our study, in contrast to PCC, idarucizumab returned thrombin generation to baseline values. This observation underlines the principal difference between PCC and idarucizumab for dabigatran reversal. It should be recognised that pro-haemostatic agents are not specific antidotes to dabigatran and do not remove its effect from plasma. Instead, they reduce the ongoing inhibitory effect of the drug by elevating concentrations of its substrate, thrombin (precursor prothrombin). For treatment of coagulopathic bleeding, coagulation factor concentrates may be useful in reducing the need to administer allogeneic blood products (4, 37). Early use of antifibrinolytic therapy with tranexamic acid as first-line therapy has been shown to reduce blood loss in severely injured patients (17). Fibrinogen supplementation is currently being considered as a primary therapeutic option for haemostatic management of trauma-related bleeding. Fibrinogen concentrate can be effective for the treatment of dilutional coagulopathy; it may reduce transfusion of allogeneic blood products and improve clinical outcomes (20, 38, 39). The beneficial effects of fibrinogen concentrate are attributed mainly to its role in forming fibrin as the basis of the clot and increased clot strength through binding of GPIIb/IIIa receptors on platelets (40). We have shown that the administration of tranexamic acid plus fibrinogen concentrate had no effect on bleeding in the presence of trauma- and dabigatran-induced coagulopathy. This could be related to the fact that dabigatran inhibits thrombin and, since thrombin is required to convert fibrinogen to fibrin, the addition of excess fibrinogen and an antifibrinolytic agent may have been a useless intervention. However, once sufficient thrombin was made available through the addition of PCC or idarucizumab, conversion of fibrinogen to fibrin was restored meaning that the effect of tranexamic acid plus fibrinogen concentrate could also be restored. This could explain the greater improvements in some coagulation/thromboelastometry parameters with PCC and idarucizumab in the group receiving tranexamic acid plus fibrinogen concentrate. These outcomes are consistent with previous studies of fibrinogen concentrate in porcine models (41, 42). There are some limitations of our study that need to be acknowledged. PCCs and idarucizumab were supplemented ex vivo. This approach was taken mainly for ethical reasons: it enabled investigation of a variety of haemostatic agents with a small number of animals Figure 6: Thrombin generation (CAT) measurements showing the effects of PCCs on increasing concentrations of dabigatran, 60 min after trauma. Thrombin generation in plasma was assessed using 5 pM tissue factor and 4 µM phospholipids. Changes over time in lag time (A, D & G), peak height (B, E & H) and endogenous thrombin potential (ETP, C, F & I). Grey dotted lines indicate baseline mean values. Data (mean ± SD) were analysed by ANOVA with Tukey as a post-hoc test. *P < 0.05 vs control; §P < 0.05 vs 30 U/kg dose of the same PCC; #P < 0.05 vs PCC; †P < 0.05 vs all PCCs.

(43). As a consequence, however, the effect of PCC or idarucizumab treatment on blood loss could not be measured in this study and it is uncertain to what extent our observations may be predictive of clinical dabigatran reversal. Lastly, there remains a possibility of species differences in aspects such as the tissue factor/rFVIIa complex, and the aPTT/PT responses to dabigatran (44).

Conclusion In summary, the results of this pre-clinical study support the use of either a three- or four-factor PCC to reverse the anticoagulant effects of dabigatran, at plasma levels similar to or higher than those achieved clinically. PCC reversal of dabigatran was improved posttrauma by prior administration of tranexamic acid plus fibrinogen concentrate, most prominently with thromboelastometry parameters. PCCs increased thrombin generation measurements above baseline in a concentration-dependent manner, while treatment with idarucizumab returned thrombin generation to baseline by removing active dabigatran from plasma. This implies for PCCs that elevated levels of thrombin are required to overcome dabigatran-induced coagulopathy. This study indicates that treatment with either PCC or idarucizumab could potentially be considered a therapeutic option to control life-threatening bleeding among patients under treatment with dabigatran. However, effectiveness and appropriate doses of both of these treatments need to be studied and established in bleeding human patients.

What is known about this topic? • Prothrombin complex concentrate (PCC) is effective and well tolerated for emergency reversal of vitamin K antagonist therapy. • Limited clinical data indicate that PCC is effective for the reversal of dabigatran-induced anticoagulation. • Tranexamic acid and fibrinogen supplementation are essential parts of the treatment of trauma-induced coagulopathy. However, the impact of these agents in trauma-associated coagulopathy under anticoagulation with dabigatran is not known. What does this paper add? • Three- and four-factor PCCs are equally effective to reverse dabigatran-induced coagulopathy in an anticoagulation/polytrauma pig model. • Thrombin generation data indicate that the mechanisms of dabigatran reversal with PCC and idarucizumab are different. • Additional treatment with tranexamic acid and fibrinogen concentrate mainly affect the kinetics of clot formation and clot strength without influencing thrombin generation. Combined therapy with coagulation factors (PCC and tranexamic acid and fibrinogen concentrate) yields additional improvements in coagulation. • Thromboelastometry parameters, rapidly available via point-ofcare testing, may provide the best means of guiding and monitoring PCC therapy for reversing the effects of high concentrations of dabigatran.

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Abbreviations ANOVA, analysis of variance; aPCC, activated prothrombin complex concentrate; aPTT, activated partial thromboplastin time; CAT, Calibrated Automated Thrombogram; CFT, clot formation time; CT, clotting time; DE, dabigatran etexilate; ECG, electrocardiography; ETP, endogenous thrombin potential; INR, International normalised ratio; MaxV, maximum velocity; MCF, maximum clot firmness; PCC, prothrombin complex concentrate; PT, prothrombin time; SD, standard deviation; U, international unit.

Acknowledgements

The authors thank Renate Nadenau (Department of Anaesthesiology, RWTH Aachen University Hospital) and Johanna Schurer for excellent laboratory assistance. Dabigatran and idarucizumab were provided by Boehringer Ingelheim, Germany. The sponsor of the study had no influence on its design or the interpretation of results. Author contributions

M. H. conducted the experimental laboratory work and helped to draft the manuscript. J.vR. performed CAT analysis. S.T. helped to perform the experimental laboratory. R.R. helped to draft the manuscript. O.G. designed the study, conducted the experimental laboratory work and wrote the manuscript. All authors read and approved the final manuscript. Conflicts of interest

M. H. has received travel support from Boehringer Ingelheim. R.R. has received honoraria for lectures and consultancy from CSL Behring and Novo Nordisk. J.vR. is an employee of Boehringer Ingelheim Pharma GmbH & Co., Germany. O.G. has received research funding from Novo Nordisk, Biotest, CSL Behring, Nycomed and Boehringer Ingelheim. He has also received honoraria for consultancy and/or travel support from Bayer Healthcare, Boehringer Ingelheim, CSL Behring and Portola.

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