Figure 4
Figure 4. OxLDL induces the activation of Src kinases, which leads to platelet shape change and the phosphorylation of MLC. (A) Platelets (2.5 × 108/mL) were treated with PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL). Traces were recorded for 2 minutes. Shown are representative traces of 6 independent experiments. (B) Platelets (5 × 107/mL) were adhered to nLDL or oxLDL (50 μg/mL) slides in the presence or absence of PP2 or PP3 (20 μM) for 30 minutes and were viewed by fluorescence microscopy. Representative images of 3 independent experiments were taken under ×60 magnification. Bar = 20 µm. (C) Platelets (5 × 108/mL) were stimulated with either oxLDL (50 µg/mL) or nLDL (50 µg/mL) for 15 seconds. Samples were then lysed, separated by SDS-PAGE, and immunoblotted for phospho-SrcTyr416 followed by reprobing for β-tubulin. (Ci) Representative blots. (Cii) Densitometric analysis of 4 independent experiments. *P < .05. (D) Samples were processed as in (C), except that platelets were pretreated with SSO (50 μM) or fucoidan (5 μg/mL). (Di) Representative blots. (Dii) Densitometric analysis of 3 independent experiments. *P < .05. (E) Platelets (3 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then processed as in (C) and were immunoblotted for phospho-MLCSer19 followed by reprobing for β-tubulin. (Ei) Representative blots. (Eii) Densitometric analysis of 4 independent experiments. *P < .05. (F) Platelets (5 × 108/mL) were stimulated with (Fi) varying concentrations of oxLDL (10-200 µg/mL) or nLDL (50 µg/mL) for 15 seconds. (Fii) Platelets (5 × 108/mL) were incubated with 50 µg/mL of oxLDL for various time points (15-300 seconds) before lysis. Representative blots of 4 independent experiments are shown. (G) Platelets (5 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples (F-G) were then processed as in (B) and were immunoblotted for phosphotyrosine, followed by reprobing for β-tubulin. All experiments were carried out in the presence of apyrase (2 U/mL), indomethacin (10 µM), and EGTA (1 mM), and are representative of 3 independent experiments.

OxLDL induces the activation of Src kinases, which leads to platelet shape change and the phosphorylation of MLC. (A) Platelets (2.5 × 108/mL) were treated with PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL). Traces were recorded for 2 minutes. Shown are representative traces of 6 independent experiments. (B) Platelets (5 × 107/mL) were adhered to nLDL or oxLDL (50 μg/mL) slides in the presence or absence of PP2 or PP3 (20 μM) for 30 minutes and were viewed by fluorescence microscopy. Representative images of 3 independent experiments were taken under ×60 magnification. Bar = 20 µm. (C) Platelets (5 × 108/mL) were stimulated with either oxLDL (50 µg/mL) or nLDL (50 µg/mL) for 15 seconds. Samples were then lysed, separated by SDS-PAGE, and immunoblotted for phospho-SrcTyr416 followed by reprobing for β-tubulin. (Ci) Representative blots. (Cii) Densitometric analysis of 4 independent experiments. *P < .05. (D) Samples were processed as in (C), except that platelets were pretreated with SSO (50 μM) or fucoidan (5 μg/mL). (Di) Representative blots. (Dii) Densitometric analysis of 3 independent experiments. *P < .05. (E) Platelets (3 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then processed as in (C) and were immunoblotted for phospho-MLCSer19 followed by reprobing for β-tubulin. (Ei) Representative blots. (Eii) Densitometric analysis of 4 independent experiments. *P < .05. (F) Platelets (5 × 108/mL) were stimulated with (Fi) varying concentrations of oxLDL (10-200 µg/mL) or nLDL (50 µg/mL) for 15 seconds. (Fii) Platelets (5 × 108/mL) were incubated with 50 µg/mL of oxLDL for various time points (15-300 seconds) before lysis. Representative blots of 4 independent experiments are shown. (G) Platelets (5 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples (F-G) were then processed as in (B) and were immunoblotted for phosphotyrosine, followed by reprobing for β-tubulin. All experiments were carried out in the presence of apyrase (2 U/mL), indomethacin (10 µM), and EGTA (1 mM), and are representative of 3 independent experiments.

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