Journal: British Journal of Pharmacology

Article Title: Zafirlukast is a broad‐spectrum thiol isomerase inhibitor that inhibits thrombosis without altering bleeding times

doi: 10.1111/bph.15291

Figure Lengend Snippet: Platelet granule secretion and calcium flux in human and mouse platelets is diminished by zafirlukast (ZFL). Washed human platelets (4 × 10 8 cells·ml −1 ) were incubated with ZFL (0.1–10 μM) or vehicle for 5 min prior to stimulation with collagen (1 μg·ml −1 ). Chronolume reagent was added for 2 min prior to stimulation, and luminescence recorded. (a) Representative traces of ATP secretion, and (b) is log dense granule secretion values, n = 5. (c) For α‐granule secretion, human platelet P‐selectin exposure was measured by flow cytometry. Platelets (2 × 10 8 cells·ml −1 ) were incubated with vehicle or zafirlukast (0.1–20 μM) for 5 min prior to stimulation with 0.25 μg·ml −1 CRP‐XL. Anti‐human P‐selectin PE conjugate was added for 20 min (1:500 dilution), and samples were fixed with 0.2% (v/v) paraformaldehyde, n = 6. (d) Following incubation with vehicle or zafirlukast (0.6–20 μM), mouse platelets were stained with anti‐mouse P‐selectin FITC conjugate and stimulated with thrombin (0.1 U·ml −1 ), n = 12. For Ca 2+ mobilisation assays, platelets (4 × 10 8 cells·ml −1 ) were loaded with Fura‐2 AM and incubated with either vehicle or zafirlukast (2.5–40 μM) for 5 min before addition of CRP‐XL (0.5 μg·ml −1 ), (e) mean Ca 2+ trace. (f) peak Ca 2+ concentration normalised to vehicle values, n = 6. Graphs represent mean ± SEM; data were analysed by one‐way ANOVA, * P < 0.05

Article Snippet: Chronolume ATP standard reagents were from Chronolog (Havertown, PA, USA).

Techniques: Incubation, Flow Cytometry, Staining, Concentration Assay