γT-S195A thrombin reduces the anticoagulant effects of dabigatran in vitro and in vivo

Abstract

Background: Dabigatran etexilate (DE) is an oral direct thrombin inhibitor used to prevent strokes in patients with atrial fibrillation. No licensed DE antidote is currently available. We hypothesized that active site-mutated S195A thrombin (S195A-IIa) and/or its trypsinized derivative (γT-S195A-IIa) would sequester dabigatran, the active form of DE, and reduce its anticoagulant effects. Objective: To assess active site-mutated S195A or γT-S195A-IIa as dabigatran reversal agents in vitro and in vivo. Methods: Diluted thrombin time (dTT) assays were performed using human or murine plasma containing dabigatran, combined with S195A-IIa, γT-S195A-IIa or FPR-chloromethyl ketone-treated thrombin (FPR-IIa). Bleeding times were determined in anesthetized DE-treated mice also receiving γT-S195A-IIa or vehicle 15 min prior to tail transection. The time to occlusion of carotid arteries of DE-treated mice also receiving S195A-IIa, γT-S195A-IIa, prothrombin complex concentrate (PCC) or vehicle, 15 min prior to topical FeCl3, was determined using Doppler ultrasound. Results: γT-S195A-IIa reduced dTT values of dabigatran-containing human and murine plasma more effectively than S195-IIa; FPR-IIa had no effect. A dose of 13 mg kg-1 DE abrogated occlusive thrombus formation in the carotid arteries of FeCl3-treated mice; γT-S195A-IIa (6 mg kg-1) or PCC (14.3 IU kg-1), but not saline vehicle or S195A-IIa (6 mg kg-1), was equally effective in restoring thrombus formation. Bleeding times of mice treated with 60 mg kg-1 DE and γT-S195A-IIa (6 mg kg-1) or saline vehicle did not differ. Conclusions: Our data suggest that γT-S195A-IIa decreases the anticoagulant effects of dabigatran in vitro and is partially effective at restoring hemostasis-related thrombus formation in DE-treated mice in vivo. © 2014 International Society on Thrombosis and Haemostasis.