Ticagrelor

Fifty µl of bacterial broth containing 10⁵ CFU was added to microtiter plate wells containing eight serially double-diluted ticagrelor concentrations to make the final volume 100 µL and the final ticagrelor (Sigma-Aldrich) concentrations 50 μg/ml to 0.75 μg/ml followed by incubation for 24 h at 37°C. Antibacterial activity was measured by determining the OD at 600 nm. The minimum bactericidal concentration (MBC) was determined by quantifying bacteria from wells with no visible growth, using the drop dilution method (Naghili et al., 2013 (link)). The minimum ticagrelor concentration that reduced bacterial concentration by more than 99.9% was taken as MBC. To determine the antibiofilm activity of ticagrelor, we used previously reported biofilm assay procedures with a slight modification (Singh et al., 2019 (link)). The culture supernatant was discarded and the residual biofilm was fixed with 2% sodium acetate for 10 min. Then the biofilm was stained overnight with crystal violet followed by rinsing with tap water and air drying. The crystal violet retained was then reconstituted with absolute ethanol, and OD values were measured at 570 nm. The experiments were performed in triplicates. S. aureus growth in ticagrelor diluent dimethylformamide (DMF) (4.15%) was used as a positive control while the sterile DMF was used as a negative control.

Dimethyl sulfoxide (DMSO), ristocetin (ristomycin monosulfate), ethylenediaminetetraacetic acid disodium salt (EDTA), platelet-activating factor-16 (PAF), acetylsalicylic acid (ASA), 4-methyl-1,2-benzenediol (4-methylcatechol, 4-MC), 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619) and ticagrelor were purchased from Sigma (St. Louis, MO, USA). Arachidonic acid (AA), adenosine-5’-diphosphate (ADP) and thrombin receptor agonist peptide-6 (TRAP) were obtained from Roche (Basel, Switzerland). The physiological solution was purchased from B. Braun (Melsungen, Germany), and collagen from Diagnostica, a.s. (Prague, Czech Republic). Vorapaxar was purchased from Selleck Chemicals GmbH (Munich, Germany).

Apyrase, ADP, ATP, gemcitabine and ticagrelor were obtained from Sigma-Aldrich, (St. Louis, MO, USA). Inhibitor of the transforming growth factor-β (TGF-β) type I receptor, SB431542 was from Tocris Bioscience, Bristol, UK. AsPC-1 and BxPC-3 cell lines were obtained from ATCC and tested negative for mycoplasma (tissue culture facility routine testing). Cells were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 1 mM Sodium Pyruvate, 1 mM non-essential amino acids (all from Gibco^® Life Technologies Australia Pty Ltd., Mulgrave, Australia), and 10% Foetal Bovine Serum (FBS, from Bovogen Biologicals, Keilor East, Australia).