Fig 1.
Effects of nifedipine on the membrane CD40L expression and sCD40L release.
Washed platelets were preincubated with various doses of nifedipine or nifedipine combined with GSK0660 (5 μ M) or GW9662 (5 μ M) for 3 min, followed by addition of collagen (10 μ g/ml) for 6 min. The platelet surface CD40L expression (A) and the levels of sCD40L (B) were determined. Data were presented as means ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the collagen alone group; +p < 0.05, ++p < 0.01, +++p < 0.001 compared with respective collagen+nifedipine-treated group.
Fig 2.
Effects of PPAR-β/-γ antagonists on nifedipine-mediated NO/cyclic-GMP formation and CD40L/sCD40L cascade.
Platelets were incubated with various drugs, followed by addition of collagen. The levels of NOx (A) cyclic GMP formation (B) platelet surface CD40L expression (C) and sCD40L concentrations (D) were determined. Date were expressed as means ± SEM (n = 5). **p < 0.01, ***p < 0.001 compared with collagen alone group; +p < 0.05, ++p < 0.01, +++p < 0.001 compared with respective collagen+nifedipine-treated group.
Fig 3.
Effects of PPAR-β/-γ antagonists on nifedipine-mediated phosphorylation of P38MAPK, ERK1/2 and HSP27.
Platelets were pretreated with 5 μ M nifedipine or nifedipine combined with various drugs for 3 min followed by addition of collagen for 6 min. The expression of phospho-P38MAPK and total-P38MAPK (A), phospho-ERK1/2 and total ERK1/2 (B), phospho-HSP27 and total-HSP27 (C) were determined. Date were expressed as means ± SEM (n = 5). ***p < 0.001 compared with collagen alone group; ++p < 0.01, +++p < 0.001 compared with respective collagen+nifedipine-treated group.
Fig 4.
Effects of PPAR-β/-γ antagonists on nifedipine-mediated MMP2 activity.
Platelets were treated with nifedipine (5 μ M), or nifedipine combined with various drugs for 3 min, followed by addition of collagen for 6 min. The MMP-2 expression (A) and MMP-2 activity (B) were determined. Date were expressed as means ± SEM (n = 5). ***p < 0.001 compared with collagen alone group; ++p < 0.01, +++p < 0.001 compared with respective collagen+nifedipine-treated group.
Fig 5.
Effects of PPAR-β/-γ antagonists on nifedipine-mediated ROS production.
Platelets were preincubated with nifedipine or nifedipine combined with GSK0660 or GW9662 for 3 min; then NBT or DCFH-DA was added, followed by collagen to measure superoxide (A) and hydrogen peroxide (B) formation respectively. Data were expressed as means ± SEM (n = 5). ***p < 0.001 compared with collagen alone group; ++p < 0.01 compared with respective collagen+nifedipine treated group.
Fig 6.
Hypothetical model of the signaling pathways of nifedipine-mediated reduction of sCD40L release from collagen-stimulated platelets.
Nifedipine initially activates PPAR-β/-γ followed by increased formation of NO/cyclic GMP and down-regulation of p38MAPK/ERK1/2/HSP27 signaling as well as ROS generation, which then attenuates MMP-2 activity and ultimately inhibits sCD40L release from activated platelets.