Fitc mouse anti human pac 1

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.