Platelet granule secretion and calcium mobilization measurements

P‐selectin exposure was measured by flow cytometry. Washed platelets (2 × 10⁸ cells mL⁻¹) were diluted in HEPES‐buffered saline (HBS) (3.2 mm NaCl, 0.148 mm KCl, 0.054 mm Na₂HPO₄:2H₂O, 0.4 mm glucose, 2 mm HEPES, pH7.0) containing anti‐human‐P‐selectin antibody (1 : 500 dilution) and either anti‐ERp72 or control antibody added for 5 min prior to stimulation. Platelets were stimulated with CRP‐XL (0.5 μg mL⁻¹) for 20 min and then fixed by dilution in paraformaldehyde (0.2% v/v). Mouse platelets were stained using anti‐mouse CD62P and JON/A antibodies (both at a dilution of 1 : 500) and stimulated with thrombin (0.1U mL⁻¹). Data for 10 000 (human platelets) or 5000 (mouse platelets) gated events were recorded using an Accuri C6 flow cytometer (Beckton Dickinson, Oxford, UK).

ATP secretion from dense granules was measured using lumi‐aggregometry. Washed platelets (4 × 10⁸ cells mL⁻¹) were pre‐incubated with anti‐ERp72 or control antibody for 5 min, Chronolume reagent (50 μL) was added 2 min prior to stimulation with collagen (1 μg mL⁻¹) and secretion recorded for 180 s.

For calcium mobilization assays, washed human platelets (4 × 10⁸ cells mL⁻¹) were loaded with FURA‐2AM calcium sensitive dye (2 μm) and then incubated with either anti‐ERp72 or control antibody for 5 min prior to stimulation with CRP‐XL (0.5 μg mL⁻¹). Fluorescence measurements with excitation at 340 and 380 nm and emission at 510 nm were recorded using a NOVOstar plate reader (BMG Labtech, Aylesbury, UK) 23, 24.