Fig. 2

BML-111 inhibits platelet aggregation, integrin αIIbβ3 activation and secretion. Human isolated platelets (4 × 10⁸ cells/ml) were incubated with modified-Tyrode’s HEPES buffer or different concentrations of BML-111 (3.125, 6.25, 12.5, 25, and 50 µM) for 5 min and then stimulated with CRP-XL (0.25 µg/ml) and thrombin (0.05 U/ml). The change in light transmission was monitored and recorded for 300 s. (a, c) Representative aggregation traces from three separate donors. (b, d) Cumulative data from three different donors. The percentage of aggregation measured at 300 s with a vehicle control was taken as 100%. Human PRP was incubated with different concentrations of BML-111 (3.125, 6.25, 12.5 and 50 µM) or vehicle control (modified-Tyrode’s HEPES buffer) for 5 min prior to stimulating with (e) CRP-XL (0.25 µg/ml) or (f) thrombin (0.05U/ml) for 20 min followed by flow cytometry analysis. Integrin αIIbβ3 activation (inside-out signaling) was determined by measuring the level of fibrinogen binding to the platelets. The bar charts represent the percentage of fibrinogen binding compared with the positive control, which is defined as 100%. The level of P-selection exposure on the platelet surface was measured using anti-human CD62P antibodies by flow cytometry. The bar charts represent the percentage of P-selectin exposure compared with the positive control, which is defined as 100% (g, h). The level of ATP release was observed by lumi-aggregometery using a luciferin-luciferase detection system. (i, k) Representative traces for ATP release. (j, l) ATP secretion was calculated as a percentage of the area under the curve, where 100% was expressed as the level of ATP release achieved with a vehicle control at 300 s. Data represent mean ± SEM (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 values were as calculated by One-way ANOVA.