The effect of an inhibitor of 14-3-3–GPIb interaction on thrombin-induced cellular responses in GPIb-expressing CHO cells and platelets. (A) CHO cells or CHO cells expressing GPIb-IX were loaded with a calcium-sensitive dye (FLIPR Calcium 5) and then treated with MPαC or scrambled control peptide prior to stimulation with 3 nM thrombin. Thrombin-induced calcium responses were determined as in Figure 1. (B) Quantification of data shown in A (mean ± SEM, 3 experiments; **P < .01, 1-way ANOVA). (C) Thrombin-stimulated aggregation of human platelets pretreated with the MCsC (control), MPαC micellar peptide, or buffer alone (no peptide treatment). (D) Quantitative data of 4 aggregation experiments as shown in C (mean ± SEM). (E) Thrombin-stimulated ATP release (mean ± SEM, 4 experiments; *P < .05 and **P < .01, t test). (F) Thrombin-induced calcium mobilization in human platelets pretreated with MCsC, MPαC, or buffer alone. (G) Quantification of the area under the curves (AUC) as shown in F (mean ± SEM, 3 experiments; **P < .01, and ***P < .001, 1-way ANOVA). (H) Stacked fluorescence histograms of 3 nM S195A thrombin binding to washed human platelets pretreated with 10 μM MPαC or control peptide MCsC as analyzed with flow cytometry using a goat antithrombin antibody and an Alexafluor 488–labeled anti-goat IgG antibody. Prior to thrombin addition, samples were preincubated with IgG or SZ2 to verify GPIb-dependent thrombin binding. Thr, 3 nM S195A thrombin. (I) Quantification of specific binding of thrombin to MCsC or MPαC-treated platelets (mean ± SEM, 3 experiments; P = .6847). Specific binding is calculated by subtracting thrombin binding mean fluorescence intensity of SZ-2–treated platelets from IgG-treated platelets.