Sulforaphane attenuates platelet granule secretion through down-regulating glycoprotein VI-mediated p38 MAPK/cPLA₂ signaling pathway

Figures & data

Figure 1. SFN attenuates platelet granule secretion and aggregation. Human gel-filtered platelets were pre-incubated with SFN (1, 5, or 10 µm) or the vehicle control (0.05% DMSO) for 40 min. (a-b) Platelet surface expression of CD62P was analyzed by flow cytometry after stimulation with 2 μg/mL collagen for 5 min. (c-d) the secretion of platelet PF4 (c), CCL5 (d), ATP (e) and levels of intracellular calcium concentration ([Ca²⁺]_i) (f) were determined after stimulation with 2 μg/mL collagen for 5 min. (g-h) Same as (a-b), except platelet surface expression of CD63 was analyzed. (i-j) Platelet aggregation stimulated by 2 μg/mL collagen was performed. *P < .05, **P < .01 and ***P < .001 vs. the vehicle control as assessed by a one-way ANOVA followed by Dunnett’s t-test (n = 3).

Figure 2. SFN attenuates platelet granule secretion involving in reducing TxA₂ generation. Human gel-filtered platelets were pre-incubated with various concentrations of SFN or the vehicle control for 40 min. The levels of TxB₂ generation were measured in platelets stimulated with 2 μg/mL collagen (a) or 50 ng/mL convulxin (b) for 5 min (n = 5). (c-e) Human gel-filtered platelets were incubated with SFN (10 µm) or the vehicle control for 40 min in the absence or presence of 100 µm ASA. The levels of platelet PF4 (c), CCL5 (d) and ATP (e) secretion were determined after stimulation with 2 μg/mL collagen for 5 min (n = 3). (f-g) Human gel-filtered platelets were incubated with various concentrations of SFN or the vehicle control for 40 min. The levels of platelet PF4 (f) and CCL5 (g) secretion were determined after stimulation with 5 µm U46619 for 5 min (n = 3). Data were assessed by a one-way ANOVA followed by the Newman–Keuls test in (c-e) and by Dunnett’s t-test in a, b, f and g. *P < .05 and ***P < .001 vs. the vehicle control, and ^#P < .05, ^##P < .01; NS, not significant difference.

Figure 3. SFN attenuates platelet TxA₂ generation and granule secretion via down-regulating cPLA₂ phosphorylation. (a) Human gel-filtered platelets were incubated with various concentrations of SFN or the vehicle control for 40 min, followed by the stimulation of 2 μg/mL collagen for 5 min. Western blotting was performed to assess total and phosphorylation of cPLA₂ at Ser⁵⁰⁵. (b-d) Human gel-filtered platelets were incubated with SFN (10 µm) or the vehicle control for 40 min in the absence or presence of pyrrophenone (1 µm), then the levels of TxB₂ generation (b), and PF4 (c) and CCL5 (d) release were measured after stimulation with 2 μg/mL collagen for 5 min. Data were assessed by a one-way ANOVA followed by Dunnett’s t-test in a and by the Newman–Keuls test in b-d (n = 3). *P < .05, **P < .01 and ***P < .001 vs. the vehicle control; NS, not significant difference.

Figure 4. SFN regulates platelet TxA₂ generation and granule secretion by down-regulating p38 MAPK/cPLA₂ signaling pathway. Human gel-filtered platelets were incubated with SFN (10 µm) or the vehicle control for 40 min in the absence or presence of 5 µm U0126 (a, e), 10 µm SP600125 (b, f), or 20 µm SB203580 (c, g) followed by the stimulation of 2 μg/mL collagen. The levels of TxB₂ generation (a-c) and PF4 secretion (e-g) were measured. (d) same as (C), except cPLA₂ (Ser⁵⁰⁵) phosphorylation was assessed by Western blotting. (h) same as (E), except platelets were incubated with SFN or the vehicle control in the absence or presence of 740 Y-P (25 µg/mL). (i-k) Human gel-filtered platelets were incubated with SFN (10 µm) or the vehicle control for 40 min in the absence or presence of H89 (10 µm), followed by the stimulation of 2 μg/mL collagen. The levels of platelet surface CD62P expression (i), PF4 (j) and CCL5 (k) release were measured. *P < .05, **P < .01 and ***P < .001 vs. the vehicle control using a one-way ANOVA followed by the Newman–Keuls test (n = 3). #P < .05 and ^##P < .01; NS, not significant difference.