Reversal of Trauma-Induced Bleeding and Anticoagulation with a Dabigatran-Specific Antidote (idarucizumab) As Assessed By Shed and Washed Blood Tests in a Pig Model of Supratherapeutic Anticoagulation and Trauma
Authors: Grottke Oliver , Honickel Markus , Schurer Johanna , van Ryn Joanne 
Published: October 2019

Abstract
Background: The most frequent side effect associated with anticoagulant therapy is bleeding. Testing reversal strategies in patients is difficult due to the unpredictability, rarity and heterogeneity of these events. Currently methods to reverse this bleeding are being tested in a variety of clinical settings in volunteers by using reversal of anticoagulation as a marker of reversal of bleeding in patients. However, the limitation of volunteer studies is the lack of understanding between the assay being used to measure anticoagulation and if its reversal has any relevance for bleeding reversal in patients. A marker of bleeding that was safe to use in volunteers and also relevant for predicting reversal of bleeding in a patient would be advantageous.
Objective: The feasibility of two markers of bleeding - fibrin formation (fibrinopeptide A, FPA) in blood as it exits a wound and blood loss measured via a washed blood method, both at a superficial wound site - were tested in pigs under high dabigatran anticoagulation. Reversal of dabigatran anticoagulation and bleeding following trauma injury were evaluated with idarucizumab and outcomes compared to the markers of bleeding from superficial wound.
Methods: After ethical approval dabigatran etexilate (30 mg/kg p.o. twice daily, n=24) or placebo (n=6, no dabigatran anticoagulation) was administered to male pigs for 3 days. On Day 4, the pigs were anesthetized and given a 90 min infusion of active dabigatran. A standardized blunt liver injury (0 min) was performed and animals underwent hemorrhagic shock, 15 min later were randomized to receive idarucizumab (30, 60 or 120 mg/kg) or vehicle. Blood loss was measured at defined time points over 4 hrs post injury. At the same time points a standardized cut was made on the inner side of the ear using an adult Surgicutt® device, one cut for each method. Shed blood was collected from the wound site as it emerged over 4 min, placed in protease inhibitor solution and processed for FPA measurement by ELISA. For washed blood method, blood as it exited the wound in 10 sec intervals was diluted in saline into 96 wells and measured photometrically. AUC over 15 min was recorded as blood loss. Blood samples were collected over time to measure active dabigatran by diluted thrombin time (dTT). Data shown as mean ± SE.
Results: Cumulative blood loss was 630 ± 56 mL in the placebo group and 2977 ± 129 mL in the dabigatran-treated group. There was a dose-dependent reduction in blood loss by 47% (1586 ± 253 mL), 64% (1064 ± 40 mL) and 62% (1140 ± 44 mL) following treatment with 30, 60 and 120 mg/kg idarucizumab (p<0.001). Dabigatran plasma levels were 1228 ± 320 ng/mL prior to injury with no significant differences between groups. Dabigatran activity decreased within 5 min of idarucizumab administration, with a dose-dependent reduction over 240 min and was completely inhibited with 120 mg/kg.
FPA in blood from the wound in dabigatran-treated groups was decreased ~74% vs non-anticoagulated group (211.2 ± 86 vs 53.7 ± 13 ng/mL). FPA increased by 2.6-fold (136.3 ± 96 ng/mL), 1.4-fold (103.9 ± 59 ng/mL) and 6.2-fold (251.9 ± 139 ng/mL) over baseline following 30, 60 and 120 mg/kg idarucizumab, respectively. At 240 min, FPA levels were 12%, 25% and 52% of non-anticoagulated animals with increasing idarucizumab doses and correlated with levels of dabigatran at 240 min.
Washed blood loss (AUC) in dabigatran-treated animals was increased ~7-fold prior to injury vs non-anticoagulated groups (2110 ± 420 vs 309.1 ± 11.2 OD*sec). There was a dose-dependent reduction in washed blood loss by 2.5% (1682 ± 688 OD*sec), 48% (889 ± 304 OD*sec), and 79% (370 ± 97 OD*sec) 30 min following 30, 60 and 120 mg/kg idarucizumab vs dabigatran-treatment (1724 ± 298 OD*sec). Washed blood loss was comparable to the non-anticoagulated group (186 ± 59 vs 281 ± 201 OD*sec) with 120 mg/kg idarucizumab after 240 min, also correlating with lack of dabigatran anticoagulant activity.
Conclusions: This study shows that bleeding and its reversal with a dabigatran-specific antidote can be evaluated using alternative methods that measure FPA levels in shed blood and washed blood loss at non-trauma superficial wound sites. The reversal of blood loss and FPA generation correlated with reversal of bleeding from a trauma wound. These methods may serve as potential markers in experimental bleeding and trauma models to sensitively assess blood loss and to also monitor reversal agents.

Disclosures
Grottke: Boehringer Ingelheim : Consultancy, Research Funding; CSL Behring: Research Funding. Honickel:Boehringer Ingelheim : Travel support Other. Schurer:Boehringer Ingelheim Pharma GmbH & Co KG: Employment. van Ryn:Boehringer Ingelheim Pharma: Employment.