Figure 2
Figure 2. oxLDL stimulates platelet ROS production in a CD36- and NOX2-dependent manner. (A) Suspended human platelets (1 × 108 platelets/mL) were incubated with the superoxide-detection probe for 30 minutes at 37°C, then treated with oxLDL (50 µg/mL) for 15 minutes, in the presence or absence of SSO (100 µmol/L) or fucoidan (50 µg/mL). Fluorescence was measured at 650 nm (n = 4). *P < .05 compared with oxLDL alone. (B) As in panel A, except platelets were treated with oxPCCD36 or PAPC (5 µmol/L) (*P < .05, PAPC compared with oxPCCD36). (C) Suspended human platelets (1 × 108 platelets/mL) were incubated with DHE (5 µmol/L) for 30 minutes followed by oxPCCD36 (5 µmol/L) or thrombin (0.1 U/mL) for 15 minutes, and the DHE oxidation product for superoxide anion was measured by LC-MS. The data are presented as % increase in superoxide above basal (n = 4). *P < .05 compared with basal. (D) WT and CD36−/− murine platelets were stained with superoxide-detection probe and then treated with oxPCCD36 (5 µmol/L). Reconstituted blood was perfused through fibrinogen-coated capillary tubes, and images of adherent platelets were then taken under bright-field or fluorescence microscopy (n = 4 separate experiments with different mice in each group). (i) Representative bright-field and fluorescence images are shown. (ii) The number of platelets in each field is presented as mean ± SEM (n = 4). (E) As in panel B, except WT and CD36−/− murine platelets were used (n = 4). *P < .05, basal compared with oxPCCD36. (F) Human platelets were treated with nLDL or oxLDL platelets in the presence of TEMPOL (1 mmol/L), MnTMPyP (100 µmol/L), gp91ds-tat, or sc-gp91ds-tat (2 µmol/L), or left untreated (control). Representative images of adherent platelets taken under bright-field or fluorescence microscopy are shown (n = 4). (G) Suspended murine platelets (1 × 108 platelets/mL), NOX2−/− or WT, were incubated with the superoxide-detection probe for 30 minutes at 37°C and then treated with oxPCCD36 (5 µmol/L) for 15 minutes at 37°C, and fluorescence was measured at 650 nm (n = 4 individual mice). *P < .05 compared with basal. NS, not significant.

oxLDL stimulates platelet ROS production in a CD36- and NOX2-dependent manner. (A) Suspended human platelets (1 × 108 platelets/mL) were incubated with the superoxide-detection probe for 30 minutes at 37°C, then treated with oxLDL (50 µg/mL) for 15 minutes, in the presence or absence of SSO (100 µmol/L) or fucoidan (50 µg/mL). Fluorescence was measured at 650 nm (n = 4). *P < .05 compared with oxLDL alone. (B) As in panel A, except platelets were treated with oxPCCD36 or PAPC (5 µmol/L) (*P < .05, PAPC compared with oxPCCD36). (C) Suspended human platelets (1 × 108 platelets/mL) were incubated with DHE (5 µmol/L) for 30 minutes followed by oxPCCD36 (5 µmol/L) or thrombin (0.1 U/mL) for 15 minutes, and the DHE oxidation product for superoxide anion was measured by LC-MS. The data are presented as % increase in superoxide above basal (n = 4). *P < .05 compared with basal. (D) WT and CD36−/− murine platelets were stained with superoxide-detection probe and then treated with oxPCCD36 (5 µmol/L). Reconstituted blood was perfused through fibrinogen-coated capillary tubes, and images of adherent platelets were then taken under bright-field or fluorescence microscopy (n = 4 separate experiments with different mice in each group). (i) Representative bright-field and fluorescence images are shown. (ii) The number of platelets in each field is presented as mean ± SEM (n = 4). (E) As in panel B, except WT and CD36−/− murine platelets were used (n = 4). *P < .05, basal compared with oxPCCD36. (F) Human platelets were treated with nLDL or oxLDL platelets in the presence of TEMPOL (1 mmol/L), MnTMPyP (100 µmol/L), gp91ds-tat, or sc-gp91ds-tat (2 µmol/L), or left untreated (control). Representative images of adherent platelets taken under bright-field or fluorescence microscopy are shown (n = 4). (G) Suspended murine platelets (1 × 108 platelets/mL), NOX2−/− or WT, were incubated with the superoxide-detection probe for 30 minutes at 37°C and then treated with oxPCCD36 (5 µmol/L) for 15 minutes at 37°C, and fluorescence was measured at 650 nm (n = 4 individual mice). *P < .05 compared with basal. NS, not significant.

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