Chrono lume luciferin luciferase reagent

Adenosine triphosphate (ATP) release was measured from washed platelets (245 μl at 3 × 10⁸ platelets/ml) as a surrogate for dense granule secretion, as previously described.²² Prior to activation, washed platelets were incubated with 5 μl of Chrono‐Lume luciferin‐luciferase reagent (CHRONO‐LOG), an ATP‐sensitive dye, protected from light at 37°C for 1 min. Platelets were stimulated with PAR1‐AP (0.5, 1, 2.5, and 5 μM), PAR4‐AP (10, 25, 50, and 75 μM), thrombin (0.1, 0.25, 0.5, and 1 nM), or collagen (0.125, 0.25, 0.5, and 1 μg/ml) under stirring conditions. Fluorescence was measured in real‐time using a Lumi‐Aggregometer (CHRONO‐LOG Model 700D).

Cerium oxide nano powder (#544841), phalloidin-FITC, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), purified fibrinogen from human plasma, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), prostaglandin E-1 (PGE-1) and thrombin were purchased from Sigma. Collagen and Chrono-lume luciferin luciferase reagent were products of Chrono-log. Mitotracker red, MitoSOX red, Calcein-AM, Fluo-4-AM and Alexa Fluor 488-conjugated human fibrinogen were from Invitrogen. FITC-conjugated annexin V, PAC1 and anti-CD62P antibodies were from BD BioSciences. Hydrogen peroxide was from Thermo Fisher and trypan blue was from SRL laboratories. All other reagents used were of analytical grade. Type I deionized water (18.2 MΩ⋅cm, Millipore) has been used throughout the experiments.