CD151^–/– platelets show normal inside-out integrin α_IIbβ₃-mediated signaling. (A) Flow cytometric analysis of FITC-conjugated fibrinogen binding to platelets stimulated with thrombin (1 U/mL), PMA (20 μM), ADP (10 μM), and ADP (10 μM) + Epinephrine (20 μM) or unstimulated (control). Triplicate samples were analyzed. Graph shows compilation of mean fluorescence intensity ± SEM from wild-type and CD151^–/– mice. Data are representative of 3 independent runs tested. (B) Flow cytometric analysis of JON/A-PE mAb binding to platelets stimulated with 0 to 1.0 U/mL thrombin and 100 to 500 μM PAR-4 agonist peptide or unstimulated (control). Triplicate samples were analyzed. Graph shows compilation of mean fluorescence intensity ± SEM from wild-type and CD151^–/– mice. Data are representative of 3 independent runs tested. (C-E) Fura-2–loaded washed wild-type or CD151^–/– platelets were stimulated with the indicated agonists (thrombin, 1 U/mL; PAR-4 agonist peptide [AYPGFK], 250 μM; and collagen-related peptide [CRP], 20 μg/mL), and cytoplasmic-free calcium was determined by measuring fluorescence emission spectra following excitation by 340- and 380-nm wavelengths. Arrow indicates addition of agonist. Data are representative of 3 independent experiments and are presented as a 340:380-nm ratio. (F) Intracellular IP₃ levels are presented for wild-type (□) and CD151^–/– (▪) platelets at 5 and 10 seconds following stimulation with thrombin. Data are presented as the mean ± SEM from 3 independent experiments.