OxLDL-induced platelet activation requires the FcRγ. (A) Washed platelets (5 × 108/mL) from WT and FcRγ−/− mice were either unstimulated or stimulated with oxLDL (50 μg/mL) for 15 seconds and then lysed. Lysates were immunoblotted for pSrc-tyr416, pSyk-tyr352, pSLP-76-tyr128, and β-tubulin. Representative blots (i) and densitometric analysis (ii-iv) of 4 independent experiments. ∗P < .05. (B) Washed platelets (2.5 × 108/mL) from WT, FcRγ−/−, and CD36−/− mice were incubated with a combination of apyrase (2 U/mL), indomethacin (10 μM), and EGTA (1 mM) for 15 minutes and then stimulated with oxLDL (50 μg/mL) for 2 minutes. Representative traces of 4 independent experiments. (C) Washed platelets (2.5 × 108/mL) from WT, FcRγ−/−, or CD36−/− mice were either incubated with oxLDL (50 μg/mL; red line) or nLDL (50 μg/mL; black line) for 30 seconds followed by stimulation with thrombin (0.02 U/mL) and aggregation was recorded for 4 minutes. Representative aggregation traces of 3 independent experiments (i) and aggregation (percent) (ii) expressed mean ± standard error of the mean (SEM; n = 3). ∗P < .05. (D) Whole blood from WT and FcRγ−/− mice was incubated with oxLDL (100 μg/mL) or vehicle for 1 minute and then perfused at arterial shear 1000 s−1 for 2 minutes over immobilized fibrinogen (100 μg/mL). Images of adherent platelets were taken by fluorescence microscopy. Representative images of arterial flow experiments (left panels) and surface coverage (percent) presented as a function of time (right panels). Data are expressed mean ± SEM (n = 5). ∗P < .05. Fgn, fibrinogen; pSLP-76-tyr128, phospho–SLP-76-tyr128; pSrc-tyr416, phospho–Src-tyr416; pSyk-tyr352, phospho–Syk-tyr352; T, thrombin.
