PLTs induce apoptosis in a dose-dependent manner in in vitro grown neuroblastoma cells. (A) Isolated human PLTs were stimulated with ADP, fixed with PFA to exclude PLT-derived signal, and incubated with SH-SY5Y neuroblastoma target cells. As positive control, cells were treated with 1 µM STS. After 6 hours, caspase activity was measured and calculated as mean ± SEM (n = 6). *P < .05 vs control. Results are expressed as percentage of buffer-treated control. (B) LDH activity was measured in the supernatant of cultured SH-SY5Y cells after application of various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of isolated membrane protein and soluble fractions of buffer- (−) or ADP-stimulated (+) human PLTs for 6 hours. Data were normalized to the total LDH level from the same amount of untreated and lysed cells. Data represent mean ± SEM (n = 9) and are shown as percentage of total cellular LDH levels of untreated cells. *P < .05 vs corresponding soluble fraction or membrane fractions of resting PLTs. (C) Caspase 3/7 activity kinetics were measured in SH-SY5Y cells incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of the isolated membrane protein and soluble fractions of ADP-stimulated human PLTs for 6 hours. Data were normalized to the corresponding samples from buffer-stimulated PLTs (control). Data represent mean ± SEM (n = 12) and are shown as percentage of control. *P < .05 vs corresponding soluble fraction. (D) Primary murine neuronal cells were incubated with buffer (ctrl) or with membrane proteins of ADP-activated murine PLTs (0.5 × 108 or 2.5 × 108 PLTs). To analyze cell death, cultures were co-stained for NeuN and active caspase 3. For quantification, staining was analyzed in a blinded fashion by fluorescence microscopy. Data are mean ± SEM (n = 5) and are shown as percentage of total cell number per section. *P < .05 vs control. Scale bar, 20 µm.
