Cytoplasmic calcium and 1,4,5-inositol triphosphate (IP₃) levels following agonist stimulation. (A) FURA-2am–loaded washed platelets were stimulated with the indicated agonists (ADP, 20 μM; PAR4 activating peptide [AYPGFK], 250 μM; U46619, 100 μM; convulxin, 1 nM) and cytoplasmic-free calcium was determined by measuring the fluorescence emission spectra following excitation by 340-nm and 380-nm wavelength UV light. Data are representative of 3 independent experiments and are presented as a 340:380 ratio. (B) Intracellular IP₃ levels are presented for wild-type (white bars) and caspase-12^–/– (dotted bars) platelets at 3 and 6 seconds following stimulation with thrombin. Data presented are the means ± SEM from 3 independent experiments. Wild-type platelets are assigned a value of 100% for each experiment. (C) Western blots for expression of PLCβ1, β2, and β3, isoforms, for PLCβ3 phosphoserine 537 and 1105 before and following activation with PAR4 receptor-activating peptide, and for total PLCγ2 are presented. All show similar expression levels in wild-type (WT) and caspase-12^–/– (C12^–/–) platelets. Each membrane was reprobed for the indicated loading control.