Proteasome inhibitor inhibits platelet dense, alpha granule, PS exposure and release of intracellular calcium.

<p>(A–D) Platelets were incubated with proteasome inhibitor (MG132; 10 μM) for 3 min, 12.5 μL of luciferase luciferin was added and stimulated with thrombin (0.025 or 0.05 U/mL) or collagen (2.5 or 5 μg/mL) for 3 min. Release of ATP (for dense granule release) as a luminescence was measured by aggregometer. Each experiment was repeated at least 3 times, with blood obtained from three separate donors. (E) Washed platelets were incubated in the presence or absence of MG132 (10 μM) for 3 minutes and then stimulated with thrombin (0.025 U/mL) for 3 minutes. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. Platelets were incubated with FITC-conjugated anti—P-selectin antibody (for alpha granule); ***P < 0.001, Mann-Whitney test and (F) FITC-conjugated Annexin V antibody, the fluorescent intensities were measured by flow cytometry, and the data were plotted as bar diagram; ***P < 0.001, Mann-Whitney test. (G) Platelets were loaded with Fura-2/AM to measure intracellular [Ca²⁺]_i in presence or absence of proteasome inhibitor (MG132, 10 μM) upon activation with thrombin (0.025 U/mL). Bar graph representing [Ca^{2 +}]_i values from platelets treated with or without MG132 (10 μM) in the presence of 7 mM extracellular Ca²⁺, expressed as mean [Ca²⁺]_i ± SEM (<i>n</i> = 3); ***P < 0.001, Student’s <i>t</i>-test.