Dabigatran

Dabigatran (1 nM; Selleck Chemicals) or thrombin (5 U/ml; Sigma-Aldrich; Merck KGaA) with/without Dabigatran were used to treat the induced cells, or the induced cells were treated with thrombin (5 U/ml) with/without 3-MA (Sigma-Aldrich; Merck KGaA).
The H/R induced cells were treated with thrombin (5 U/ml) and transfected with small interfering (si)-SPRED2 or si-negative control (NC; 5 nM; all synthesized and purchased from Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The Lipofectamine^® 2000-si-SPRED2 complex was formed by mixing for 20 min at room temperature. Then, Lipofectamine^® 2000-si-SPRED2 complex was added to the cells and the cells were cultured in serum-free Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C for 4 h, followed by incubation in serum-containing medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) at 37˚C for 72 h. After 48 h, the transfection efficiency was evaluated by reverse transcription-quantitative (RT-qPCR). The sequences were as follows: si-SPRED2, 5'-AUAGCAUUCCAAUACAACCAG-3' and si-NC, 5'-AAUUGACCCAACAAUCACAU-3'.

Serine protease activity was quantified in colonic washes from rats with BOC-val-pro-arg-amino-4-methylcoumarin [VPR-AMC] hydrochloride [200 μM, Sigma-Aldrich B9395] as substrate in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH = 8.3. Initial velocity was calculated by the change in fluorescence [excitation: 355 nm, emission: 460 nm], measured over 15 min at 37°C using a Varioskan Flash microplate reader [Thermo Fisher Scientific]. Thrombin specific proteolytic activity was accounted for in the VPR-AMC assay, by pre-incubating samples with dabigatran [1 µM, Sigma] for 10 min at 37°C before starting enzymatic reaction.