Neutralization Of Hemorrhage Induced By Direct Factor Xa and Thrombin Inhibitors In a Rat Model
Authors: Jeske Walter , Escalante Vicki , McGuire Brian , Walenga Jeanine M. , Fareed Jawed , Bakhos Mamdouh 
Published: October 2019

Abstract

Introduction
Direct factor Xa and thrombin inhibitors (apixaban, Bristol-Myers Squibb, Princeton, NJ; rivaroxaban, Bayer Healthcare, Leverkusen, Germany; dabigatran, Boehringer Ingelheim, Ridgefield, CT) have shown favorable efficacy profiles in comparison to standard therapy in a variety of clinical conditions including the prevention of stroke in patients with non-valvular atrial fibrillation. Although clinical trials have shown that the safety of these new drugs in terms of the incidence of major hemorrhage is similar or better than with conventional therapies, there remains concern over the ability to reverse their anticoagulant effects in cases of overdose or medical emergency. Prothrombin complex concentrates (PCCs) have been used to reverse the effect of warfarin and in cases of significant bleeding in patients with coagulopathy. PCCs may be useful in modulating hemorrhage caused by the new oral anticoagulant drugs. This study tested the ability of three PCCs to reverse bleeding induced by apixaban, rivaroxaban or dabigatran in a standardized rat model.

Methods
Following anesthesia with an IM injection of ketamine and xylazine, male Sprague-Dawley rats were anticoagulated by an IV injection of apixaban (100 or 300 µg/kg), rivaroxaban (100 or 300 µg/kg) or dabigatran (50 or 100 µg/kg). After 5 minutes, rats were treated with vehicle, a 3-factor PCC (Profilnine, Grifols Biologicals, Los Angeles, CA), a 4-factor PCC (Beriplex, CSL Behring, Marburg, Germany) or an activated 4-factor PCC (FEIBA, Baxter, Deerfield, IL). Five minutes after PCC administration, a standardized tail snip was performed and the time for bleeding to stop was measured. Upon cessation of bleeding, a blood sample was collected by cardiac puncture and the rat was humanely euthanized. Platelet poor plasma was prepared from the blood samples and was analyzed using PT, aPTT and PiCT assays. Results for individual treatment groups were compared using one-way ANOVA (SigmaPlot 12, Systat, San Jose, CA).

Results
While at a dose of 100 µg/kg, apixaban did not increase the bleeding time vs. saline (6.25 ± 0.8 vs. 6.0 ± 1.7 min), a dose of 300 µg/kg apixaban significantly prolonged the bleeding time (17.1 ± 5.6; p=0.024). Administration of Beriplex at doses up to 10 U/kg did not shorten the bleeding time. 10 U/kg Profilnine shortened bleeding time, but not completely to baseline (9.8 ± 4.2 min). The administration of FEIBA dose-dependently prolonged bleeding beyond that seen with apixaban alone (22.6 ± 11.4 and 37.5 ± 5.2 min for doses of 5 and 10 U/kg, respectively; p<0.001 apixaban + 10 U/kg FEIBA vs. apixaban alone). Doses of 100 and 300 µg/kg rivaroxaban prolonged bleeding time (20.8 ± 2.5, p=0.002 vs. saline; 25.0 ± 10.0 min, p<0.001 vs. saline, respectively). While 10 U/kg Beriplex or Profilnine shortened the bleeding time, Profilnine was more effective (9.7 ± 3.2 min; p=0.508 vs. saline). FEIBA at a dose of 5 U/kg did not reverse rivaroxaban-induced bleeding. Dabigatran caused a dose-dependent increase in bleeding time which could be prevented by the administration of Beriplex or Profilnine (10 U/kg). As with apixaban, the administration of FEIBA enhanced the bleeding seen with dabigatran (50.3 ± 8.3 vs. 15.9 ± 1.2 min; p<0.001). As it was hypothesized that the increased bleeding observed following FEIBA administration may be due to a Protein C mediated facilitation of fibrinolysis, additional groups of rats were anticoagulated with apixaban or rivaroxaban and then treated with FEIBA + 10 mg/kg epsilon aminocaproic acid. In these rats, the bleeding times were comparable to those in non-anticoagulated rats (6.7 ± 5.0 min with apixaban; 7.7 ± 6.4 min with rivaroxaban).

Discussion
PCCs appear useful for neutralizing bleeding induced by direct factor Xa and thrombin inhibitors. Owing to their different compositions, each PCC exhibits a distinct neutralization profile. Co-administration of a fibrinolytic inhibitor with a PCC may enhance the effectiveness of hemorrhage reversal.

Disclosures:
Jeske: Bristol-Myers Squibb: Research Funding.