FIGURE 3.

Effects of PP2 on AYPGKF-elicited Ca²⁺ mobilization and A23187-induced aggregation and TXA₂ generation. A--C, washed platelets from P2Y₁₂ deficient platelets were labeled with 12.5 μm Fura-2/AM, 0.2% Pluronic F-127 and resuspended in Tyrode's solution. A and B, platelets were then preincubated with PP2 or dimethyl sulfoxide (DMSO) for 5 min and stimulated with AYPGKF (500 μm) (A) or collagen (5 μg/ml) (B). C, changes in the intracellular free calcium level were measured every 2 s and expressed as a ratio of fluorescence (FL) detected at 509-nm emission with excitation wavelengths of 340 and 380 nm. Summarized data from three experiments are shown. D, washed platelets from C57BL/6 mice were preincubated with PP2 or dimethyl sulfoxide for 5 min and stimulated with A23187 (100 nm) in a lumi-aggregometer at 37 °C. Real time ATP secretion and platelet aggregation were simultaneously recorded. E, washed platelets from TP^+/+ or TP^−/− mice were stimulated with A23187 in a lumi-aggregometer at 37 °C. Real time ATP secretion and platelet aggregation were simultaneously recorded. F, washed platelets from P2Y₁₂ knock-out mice or wild-type controls were preincubated with PP2 or dimethyl sulfoxide for 5 min and stimulated with various concentrations of A23187 for 5 min at 37 °C with stirring. The reactions were stopped by adding 10 mm of EDTA (final concentration). Because TXA₂ has a half-life of 37 s and is rapidly converted to the stable product TXB₂, TXA₂ was measured as TXB₂ with TXB₂ EIA kits. TXB₂ production data were obtained from three tests. Statistical differences were examined by Student t test. The data are the means ± S.D. *, p < 0.001 versus dimethyl sulfoxide in the presence of same concentration of AYPGKF.