Adenosine diphosphate (adp)

Collagen (native Collagen fibrils (type I) from equine tendons) and ADP were purchased from Chrono-Log (Havertown, PA, USA). An ATP assay kit was purchased from Biomedical Research Service Centre (Buffalo, NY, USA), and TRIZOL solution was obtained from Invitrogen (Carlsbad, CA, USA). Primers against SREBP-2, ACAT-1, ACAT-2, HMG-CoA, and GAPDH were obtained from Bioneer (Daejeon, Republic of Korea). Primer sequences have been given in Table 1. Water was obtained from J. T. Baker (Phillipsburg, NJ, USA). All chemicals were of reagent grade.

Platelet aggregation induced by either ADP (10 μM final concentration, Chrono-Log Corporation; Havertown, PA.) or Thrombin (1 IU/ml final concentration, Baxter Healthcare Corporation; Deerfield, IL) was assessed in citrated whole blood on both clopidogrel and control donors. A Model 560VS Dual Channel Whole Blood Lumi-Aggregometer (Chrono-log; Havertown, PA) using an impedance technique was used for testing each sample in duplicate per agonist, with the duplicates being averaged to obtain a final result.

Flow cytometry reagents included CD61 PerCP (RUU-PL7F12, cat. No 3473408), PAC-1 FITC (Mouse BALB/c IgM, κ cat. No 34507), CD62-PE (P-selectin, Mouse BALB/c IgG1 κ, cat. No 550888), CellFIX (10 concentrated, cat no 340181), which were acquired from Becton Dickinson (Franklin Lakes, NJ, USA). The PL-chip (cat. No 18002), reservoir kit for PL-chip (cat. No 18003) and BAPA tubes (3 mL) and other equipment needed for the T-TAS were purchased from Bionicum (Warsaw, Poland). Collagen and ADP were obtained from Chrono-Log Corporation (Havertown, PA, USA). Dimethylsulfoxide (DMSO), isorhamnetin, Triton X-100 and p-nitrophenylphosphate were purchased from Sigma-Aldrich (Saint Louis, MO, USA). A stock solution of Aronox (Aronia melanocarpa berry extract, Agropharm Ltd., Warsaw, Poland) was prepared in water. The remaining reagents, including NaCl, Tris, NaOH and glucose were obtained from POCh (Gliwice, Poland).

LC–MS grade, acetonitrile (MeCN), methanol (MeOH), tetrahydrofuran (THF), dimethyl sulfoxide, MS grade formic acid (HCOOH) and all the analytical standards of testosterone, dihydrotestosterone, α-estradiol, β-estradiol, methyltestosterone, as well as the hormone preparations used for in vitro measurements of platelet activation/reactivity were obtained from Sigma (St. Louis, MO, USA) and had a minimum purity specification of 99%. Nitric acid (HNO₃) was purchased from POCH (Gliwice, Poland). Dichloromethane (DCHM, HPLC grade) was provided by VWR International (Radnor, PA, USA).
PBS was from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Arachidonate, equine tendon collagen and ADP were from Chrono-Log Corp. (Havertown, PA, USA). Fluorolabelled monoclonal antibodies (moAbs): anti-CD61/PerCP, antiCD62/PE, PAC-1/FITC, isotype controls IgG/PE and IgM/FITC, as well as CellFix (1% formaldehyde in PBS) were from Becton Dickinson (San Diego, CA, USA). Ultrapure water was obtained from Milli-Q purification system (Millipore, Bedford, MA, USA). Nitrogen (NM32LA Nitrogen Generator, Peak Scientific Instruments, Billerica, MA, USA) was used as a drying gas.

ADP was obtained from Chrono-Log Corporation (Havertown, PA), divided into small 500 μmol/L stock aliquots and stored at 2–8°C until use (for adhesion and aggregation). Collagen type I, bovine serum albumin (BSA), and bicinchoninic acid (BCA) solution were delivered from Sigma (St. Louis, MO, USA). Thrombin was purchased from BioMed (Lublin). Fibrinogen was prepared from citrated human plasma, by the combination of cold and ethanol precipitations technique by Doolittles' method [18 (link)].