Anti p selectin pe

PRP, pre‐incubated with PAM or vehicle, as described earlier, was incubated with PGI₂, DEA/NONOate or vehicle and then activated with TRAP‐6 (25 μm) with gentle mixing at 37 °C. After 2 min, the reaction was stopped by dilution with a 10‐fold excess of cold saline. Platelets were immediately stained with anti‐CD61‐allophycocyanin (CD61‐APC, eBioscience, Hatfield, UK), PAC‐1‐FITC (BD Bioscience, Oxford, UK), and anti–P‐selectin‐PE (eBioscience) for 15 min at 4 °C and then fixed in 2% (v/v) formalin (Sigma). PAC‐1‐FITC and anti‐P‐selectin‐PE immunoreactivity was measured by flow cytometry using a FACSCalibur instrument (Becton Dickinson, Oxford, UK). Representative histograms are shown in Supplementary Figure 5A–D.

After anesthesia, mouse blood was collected from the inferior vena cava with anticoagulant acid-citrate dextrose (ACD, 65 mmol/L trisodium citrate, 70 mmol/L citric acid, 100 mmol/L dextrose, pH 6.5). For washed platelets, the whole blood was centrifuged at 100 g for 15 min to obtain platelet-rich plasma, which was then centrifuged at 1300 g for 5 min in the presence of 1 μmol/L prostaglandin E1. Platelets were resuspended in modified Tyrode’s buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO₃, 0.4 mmol/L NaH₂PO₄, 5 mmol/L HEPES, 5.6 mmol/L glucose, 0.35% bovine serum albumin, pH 7.4) at a density of 2 × 10⁸ platelets/ml to test their reactivity as we previously reported [17 (link)]. Briefly, the washed platelets were stimulated with thrombin (0.05 U/ml) in the presence of 1 mmol/L CaCl₂, and stained with anti P-selectin-PE (eBioscience) or anti-activated integrin GPIIb/IIIa (JON/A-PE; Emfret Analytics, Eibelstadt, Germany) and anti-CD41-APC (eBioscience) for 15 min at room temperature (25 ± 5) °C. The reactions were stopped by the addition of 400 μl modified Tyrode’s buffer and analyzed using a FACSverse flow cytometer within 30 min. Platelets were selected based on forward scatter (FSC) and side scatter characteristics and CD41⁺ platelets were selected for analysis. The antibodies used were listed in Additional file 1: Table S1.