Apixaban

Apixaban (SelleckChem, Houston, TX, USA), D-phenylalanyl-prolyl-arginyl chloromethylketone (PPACK; Essex Junction, VT, USA), adenosine 5’-diphosphate sodium salt (ADP), prostaglandin E₁ (PGE₁), citrate concentrated solution, and apyrase (Sigma-Aldrich, St. Louis, MO, USA), the GPVI agonist convulxin (Cayman Chemical, Ann Arbor, MI, USA), the TP agonist U46619, the IP agonist iloprost (Tocris Bioscience, Bristol, UK), the PAR4 agonist peptide H-Ala-Tyr-Pro-Gly-Lys-Phe-NH₂ trifluoroacetate salt (AYPGKF) and the PAR1 agonist peptide H-Ser-Phe-Leu-Leu-Arg-Asn-OH trifluoroacetate salt (SFLLRN) (Bachem, Torrance, CA, USA), Fluo-4 NW calcium dye and probenecid (Invitrogen, Carlsbad, CA, USA), and FITC mouse anti-human PAC-1, PE mouse anti-human CD62P, and Cy5 Annexin V fluorescent antibodies (BD Biosciences, San Jose, CA, USA) were used and stored according to manufacturers’ instructions. HEPES-buffered saline (HBS, pH 7.4) was prepared with 20 mM N-2-hydroxyethylpiperazine-N’−2-ethanesulfonic acid (HEPES, Fisher Scientific, Fair Lawn, NJ, USA) and 150 mM NaCl (Fisher); Tyrode’s buffer (pH 7.4) was prepared with 10 mM HEPES, 127.2 mM NaCl, 11.9 mM NaHCO₃ (Fisher), 5 mM KCl (Fisher), 0.4 mM NaH₂PO₄ (Fisher), 1 mM MgCl₂ · 6 H₂O (Sigma), and 5 mM D-glucose (Sigma). Where specified, Ca²⁺ HBS was prepared by dissolving CaCl₂ · 2 H₂O in HBS to achieve a calcium concentation of 2 mM.

Whole blood (4 μg/mL) CTI was separated into 8 separate 100 μL aliquots. 1 μL samples were drawn at predefined time points (0–35 min) from a single aliquot and added to a well containing 30 nM Apixaban (Selleck Chemicals, Houston, TX) and 100 μM PPACK (Haematologic Technologies) in 100 μL of HBS. An aliquot was briefly mixed before the sample was drawn and discarded immediately after. To measure platelet activation, 20 μg/mL fluorescent fibrinogen (Life Technologies, Grand Island, NY), 1:100 fluorescently labeled anti-P-selectin (Becton Dickson), and 1:100 fluorescently labeled Annexin V (Life Technologies) were added to the diluted whole blood. Samples were incubated for 10 min with the fluorescent labels before reading with the flow cytometer (Accuri C6, Becton Dickson). Positive controls were activated with 5 nM convulxin (Centerchem, Norwalk, CT).