Cytoplasmic calcium, IP₃ levels, and ¹²⁵FAK phosphorylation following agonist stimulation with either CRP, thrombin, or PAR-4. (A) Fura-2 am loaded washed wild-type or PECAM-1^–/– platelets were stimulated with the indicated agonists ([i,ii] PAR-4 agonist peptide at 125 and 250 μM, respectively; [iii,iv] thrombin at 0.5 and 1.0 U/mL, respectively; and [v,vi] CRP at 2.5 and 5 μg/mL, respectively) and cytoplasmic-free calcium was determined by measuring fluorescence emission spectra following excitation at 340 and 380 nm. Data are presented as 340:380 nm ratio. Arrow indicates addition of agonist. (B) Intracellular IP₃ levels are presented for wild-type (▪) and PECAM-1^–/– (□) platelets at 5 and 10 seconds following stimulation with thrombin. Data are presented as the mean ± SEM from 3 independent experiments. Wild-type platelets are assigned a value of 100% for each experiment. (C) Tyrosine phosphorylation of ¹²⁵FAK was induced following stimulation of wild-type or PECAM-1^–/– platelets with thrombin (1 U/mL) and stirring at different time points in the platelet aggregometer (0-1 minute). Platelet suspensions were lysed and immunoprecipitated with anti-FAK antibody. Immune complexes were separated by 10% SDS-PAGE and transferred by Western blotting. Top row: 4G10; bottom row: total FAK antigen.