γ T ‐ S 195 A thrombin reduces the anticoagulant effects of dabigatran in vitro and in vivo

Summary 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 S 195 A thrombin ( S 195 A ‐ II a) and/or its trypsinized derivative (γ T ‐ S 195 A ‐ II a) would sequester dabigatran, the active form of DE , and reduce its anticoagulant effects. Objective To assess active site‐mutated S 195 A or γ T ‐ S 195 A ‐ II a as dabigatran reversal agents in vitro and in vivo . Methods Diluted t hrombin t ime (d TT ) assays were performed using human or murine plasma containing dabigatran, combined with S 195 A ‐ II a, γ T ‐ S 195 A ‐ II a or FPR ‐chloromethyl ketone‐treated thrombin ( FPR ‐ II a). Bleeding times were determined in anesthetized DE ‐treated mice also receiving γ T ‐ S 195 A ‐ II a or vehicle 15 min prior to tail transection. The time to occlusion of carotid arteries of DE ‐treated mice also receiving S 195 A ‐ II a, γ T ‐ S 195 A ‐ II a, prothrombin complex concentrate ( PCC ) or vehicle, 15 min prior to topical F e C l 3 , was determined using D oppler ultrasound. Results γ T ‐ S 195 A ‐ II a reduced d TT values of dabigatran‐containing human and murine plasma more effectively than S 195‐ II a; FPR ‐ II a had no effect. A dose of 13 mg kg −1 DE abrogated occlusive thrombus formation in the carotid arteries of F e C l 3 ‐treated mice; γ T ‐ S 195 A ‐ II a (6 mg kg −1 ) or PCC (14.3  IU  kg −1 ), but not saline vehicle or S 195 A ‐ II a (6 mg kg −1 ), was equally effective in restoring thrombus formation. Bleeding times of mice treated with 60 mg kg −1 DE and γ T ‐ S 195 A ‐ II a (6 mg kg −1 ) or saline vehicle did not differ. Conclusions Our data suggest that γ T ‐ S 195 A ‐ II a decreases the anticoagulant effects of dabigatran in vitro and is partially effective at restoring hemostasis‐related thrombus formation in DE ‐treated mice in vivo .