A DE tablet (110mg, Pradaxa, Boehringer Ingelheim, Ingelheim, Germany) was dissolved with 1% dimethylsulfoxide (DMSO) in saline solution. Solutions with three different DE concentrations were prepared: 10mg/ml, 20mg/ml, and 30mg/ml. DE mice were fed three times orally using a gastric tube with intervals of eight hours. Each feeding consisted of 0.15mL of the respective solution resulting in a dose of 37.5mg/kg, 75mg/kg or 112.5mg/kg body weight per feeding for a 40g mouse. Oral gavages of comparable dosages of DE were previously shown to cause significant activated partial thromboplastin time (aPTT) prolongation in rats.16 (link) Control mice were fed three times with 0.15mL saline following the same modus of application. Determination of coagulation parameters or ICH induction by collagenase injection and laser-induced vessel rupture, respectively, was performed 0.5 hours after the last oral gavage of either DE or saline. In a subset of mice, multiple coagulation parameter analyses over time were performed, in order to determine the kinetics of DE anticoagulation. Furthermore, the effect of the solvent DMSO alone on coagulation parameters and ICH volume was determined.
Warfarin was applied via drinking water following a previously established protocol.4 (link), 15 (link) For mice with a body weight of 40g, a daily water consumption of 15mL/100g provided an estimated warfarin intake of 0.1mg (2.5mg/kg) within a 30 hour feeding period. Determination of coagulation parameters or ICH induction, respectively, was done at the end of the warfarin feeding period.
In addition, we treated mice with lepirudin (1.5mg/kg), heparin (80IU/kg), or fondaparinux (0.1mg/kg). Heparin inhibits thrombin indirectly by catalyzing its inactivation by antithrombin III. In addition, it inhibits factor Xa in complex with antithrombin III. As a selective factor Xa inhibitor, fondaparinux does not have direct effects on thrombin.17 (link) Each agent was dissolved in saline (total volume 0.1mL) and administered by a single retro-orbital i.v. injection, immediately followed by a single subcutaneous injection of the same dose and volume. These doses were chosen according to the literature.18 (link)-20 (link) Determination of coagulation parameters or ICH induction by collagenase injection, respectively, was done 0.5 hours after the subcutaneous application.
Warfarin was applied via drinking water following a previously established protocol.4 (link), 15 (link) For mice with a body weight of 40g, a daily water consumption of 15mL/100g provided an estimated warfarin intake of 0.1mg (2.5mg/kg) within a 30 hour feeding period. Determination of coagulation parameters or ICH induction, respectively, was done at the end of the warfarin feeding period.
In addition, we treated mice with lepirudin (1.5mg/kg), heparin (80IU/kg), or fondaparinux (0.1mg/kg). Heparin inhibits thrombin indirectly by catalyzing its inactivation by antithrombin III. In addition, it inhibits factor Xa in complex with antithrombin III. As a selective factor Xa inhibitor, fondaparinux does not have direct effects on thrombin.17 (link) Each agent was dissolved in saline (total volume 0.1mL) and administered by a single retro-orbital i.v. injection, immediately followed by a single subcutaneous injection of the same dose and volume. These doses were chosen according to the literature.18 (link)-20 (link) Determination of coagulation parameters or ICH induction by collagenase injection, respectively, was done 0.5 hours after the subcutaneous application.
