Platelets expressing talin 1(R35E) exhibit normal GPIIb-IIIa integrin activation. (A) Generation of mice expressing talin 1(R35E) mutant. (B) Sequencing chromatogram of the mutated region of Tln1(R35E) gene. (C) Expression of talin 1(R35E) mutant in platelets was assayed by western blotting. Results are representative of 3 independent experiments, n = 3 mice each time. (D-F) Normal GPIIb-IIIa integrin activation in Tln1R35E/R35E platelets. (D) Real-time GPIIb-IIIa activation assay. Jon/A-PE binding to washed platelets was recorded continuously for 10 minutes by flow cytometry. Jon/A antibody was added at the time of stimulation with 75 or 500 μM PAR4-AP (indicated by the arrow). Flow cytometry assay to measure binding of GPIX-labeled platelets in whole blood to Jon/A antibody (E) or washed platelets to fibrinogen (F) in response to agonist stimulation. Alexa Fluor 488–coupled fibrinogen and Jon/A-PE were added simultaneously with agonists for 10 minutes. Bar graphs represent MFI ± standard error of the mean (n = 4 mice, representative of ≥4 independent experiments).