Cd62 pe

Adenosine receptor agonists were purchased from Sigma (St. Louis, MO, USA) (NECA (CAS № 35920-39-9)), and Cayman (Ann Arbor, MI, USA) (regadenoson (CAS № 313348-27-5)). LUF5835 (2-amino-6-(1H-imidazol-2-ylmethylsulfanyl)-4-(3-hydroxy-phenyl) pyridine-3,5dicarbonitrile) was synthesized at Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. Cangrelor (AR-C69931MX) was from Cayman Chemical (Ann Arbor, MI, USA). Prasugrel metabolite (R-138727) was obtained from BOC Sciences (Shirley, NY, USA). Calcein AM was obtained from Molecular Probes (Eugene, OR, USA). Antibodies anti-human CD61/PErCP, CD61/PE, CD62/PE, PAC-1/FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype control, Cellfix, buffered sodium citrate was purchased from Becton-Dickinson (San Diego, CA, USA). Phosphate buffered saline pH 7.4 (PBS) was obtained from Corning (New York, NY, USA). Dimethyl sulfoxide (DMSO), adenosine diphosphate (ADP), and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

PSB 0777 (ammonium salt) was purchased from Tocris Bioscience (Bristol, United Kingdom) and cangrelor (sodium salt, AR-C69931MX) was from Cayman Chemical (Ann Arbor, MI, United States). Human serum albumin (HSA, purity 96–99%), warfarin sodium salt, ibuprofen sodium salt, and adenosine 5′-diphosphate (ADP, purity ≥95%), prostaglandin E₁ (PGE₁) were obtained from Sigma (St. Louis, MO, United States). Monoclonal antibodies anti-human CD61/PerCP (Cat# 347408, RRID: AB_2811174), CD62/PE (Cat# 348107, RRID: AB_2184974), mouse IgG1/PE isotype control (Cat# 340013, RRID: AB_399997) and Cellfix were obtained from Becton Dickinson (San Diego, CA, United States). Biacore sensor chips (CM5), Biacore amine coupling kit and BIAmaintenance kit were from GE Healthcare (Little Chalfont, United Kingdom). The BCA Protein Assay Kit was purchased from Thermo Scientific (Waltham, MA, United States). Phosphate buffered saline (PBS) was obtained from Biomed-Lublin (Lublin, Poland). All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials (Gliwice, Poland). All reagents were of analytical grade. Water used for solution preparation and glassware washing was passed through an Easy Pure UF water purification system (Thermolyne Barnstead, IA, United States).

Fluorescently labelled CD62-PE, CD61-perCP antibodies and CellFix reagent were purchased from Becton Dickinson (Franklin Lakes, NJ, USA), glutaraldehyde, absolute ethyl alcohol, phosphate buffered saline, pH 7.4 (PBS), adenosine diphosphate (ADP) and sodium citrate were purchased from Sigma Aldrich (Saint Louis, MI, USA).

Surface P-selectin was analyzed by flow cytometry as we performed in a previous study (Csongrádi et al., 2011 (link)). Briefly, fixed platelets were incubated with saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to GPIX-receptor (CD42a) and phycoerythrin (PE)-labeled anti-P-selectin (CD62-PE, Becton Dickinson) for 20 min in the dark at RT to investigate the level of platelet activation. As a control for immunolabeling with anti-CD62 antibody, platelets were incubated with PE-coupled non-immune mouse IgG₁ antibody. A total of 10,000 dual-color labeled platelet events were acquired on an FC-500 flow cytometer (Beckman Coulter, Pasadena, CA, United States). Results were expressed as the percentage of double-positive platelets.
The number of PMPs was quantified by a standardized method we set earlier (Csongrádi et al., 2011 (link)) with some minor modifications. PMP count was calculated based on the event count from the bead tube collected for the same time (60 s). PMPs were gated into a restricted area by forward scatter (FSC) and side scatter (SSC) parameters and then identified by Annexin V-FITC and CD41a-PeCy5 positivity.