oxLDL stimulates ROS production in platelets. (A) Washed human platelets (1 × 108 platelets/mL) were incubated with the superoxide-detection probe for 30 minutes at 37°C and then treated with either nLDL or oxLDL (1-50 µg/mL) for 15 minutes, and fluorescence was measured at 650 nm (n = 4). *P < .05 compared with basal. (B) As in panel A, except platelets were incubated with DHE (5 µmol/L) for 30 minutes followed by LDL (50 µg/mL) or thrombin (0.01 U/mL) for 15 minutes, and DHE oxidation product for superoxide anion was measured by LC-MS. The data are presented as % increase in superoxide anion above basal (n = 4). *P < .05 compared with basal. (C) Washed human platelets (5 × 107 platelets/mL) were preincubated with a superoxide-detection probe for 30 minutes at 37°C and adhered to slides coated with oxLDL or nLDL (50 µg/mL) or human fibrinogen (100 µg/mL). Platelet fluorescence emission was then captured over time. Representative images of 5 separate experiments are shown. Scale bar, 20 µm. (D) Human platelets were stained with superoxide-detection probe, treated with nLDL or oxLDL (50 µg/mL), reconstituted with autologous RBCs, and perfused through fibrinogen-coated capillary tubes at arterial shear (1000 s−1). (i) Images were then captured under bright-field or fluorescence microscopy (n = 4 separate experiments with different blood donors). (ii) The number of platelets in each field is presented as mean ± SEM (n = 4).
