Dabigatran

LC-MS analysis was performed using the liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled with a triple quadrupole 6500 tandem mass spectrometer (AB Sciex, Foster City, CA, USA) according to the previously published method [8 (link),9 (link)]. Dabigatran, rivaroxaban, and stable isotopically labelled D3-Dabigatran (Toronto Research Chemicals Inc, Toronto, ON, Canada) and apixaban (Pfizer Inc., New York, NY, USA) were dissolved in methanol (LC-MS quality, Sigma, St. Louis, MO, USA) at a final concentration of 1 mg/mL expressed as free substances. These stock solutions were used for the preparation of all other standards. For quantification, a series of calibration standards in methanol were prepared (concentrations 0, 10, 50, 100, and 500 ng/mL of DABI, API, and RIVA). The calibration standards were prepared in addition to drug-free plasma from healthy volunteers. All solutions were stored at −20 °C.

The cover and capillary chips used in the flow chamber system (Fig. S1A) were manufactured by Richell Corp. (Toyama, Japan). The following materials were obtained from commercial sources: porcine type I collagen (Nitta Gelatin, Inc., Osaka, Japan), tissue thromboplastin (Sysmex, Hyogo, Japan), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD41 immunoglobulin G (IgG), and FITC-conjugated mouse IgG (Beckman Coulter, Miami, FL, USA), rabbit anti-human fibrinogen IgG (Dako, Tokyo, Japan), normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Alexa594 (Invitrogen, Carlsbad, CA, USA).
Dabigatran and rivaroxaban were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). AR-C66096, a specific P₂Y₁₂-receptor antagonist, was obtained from Tocris Bioscience (Bristol, UK). For the TG assay, PPP-Reagent (with phospholipids), PRP-Reagent (without phospholipids), and FluCa-reagent, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ). Recombinant TF (r-TF) was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan). Corn trypsin inhibitor (CTI) was prepared as reported previously [13] (link).

Bovine trypsin (catalog number LS003707) was purchased from Worthington Biochemical Corporation and was active-site titrated with p-nitrophenyl p’-guanidino-benzoate (catalog number N-8010, MilliporeSigma) (59 (link)). Ecotin was produced and purified as reported previously (60 (link)). The concentration of ecotin was determined by titration against active-site titrated bovine trypsin. Recombinant human pro-enterokinase (catalog number 1585-SE-010) was purchased from R&D Systems and was activated with human cationic trypsin. The trypsin substrate GPR-pNA (catalog number 4000768.0100) was purchased from Bachem Americas. Benzamidine hydrochloride (code number 401790050) was obtained from Acros Organics (through Thermo Fisher Scientific) and dissolved in distilled water to prepare a 100 mM stock solution, which was further diluted with distilled water to a 10 mM working solution. dabigatran etexilate mesylate (catalog number D100150) and dabigatran (catalog number D100090) were purchased from Toronto Research Chemicals. dabigatran used for biochemical studies was dissolved in 0.1N HCl first to prepare an 800 μM stock solution, which was further diluted with distilled water to a 10 μM working solution.