Graduated αIIbβ3 activation response in Tln1-mR35E, Tln1-mR118E, Tln1-mR35E,R118E, and Tln1-mKO platelets. (A) Flow cytometry assay to measure binding of GPIX-labeled platelets in whole blood to Jon/A-PE antibody in response to PAR4-AP stimulation. Bar graph represents mean fluorescence intensity (MFI) ± standard error of the mean (n = 6 mice, representative of ≥3 independent experiments). Two-way analysis of variance with Tukey posttest. (B-C) Real-time αIIbβ3 activation assay. JonA/PE binding to washed platelets was recorded continuously for 9 minutes by using flow cytometry in response to PAR4-AP (B) or convulxin (C) stimulation. Arrows indicate addition of agonists. (D) Representative aggregation responses of Tln1-mR118E and Tln1-mR35E,R118E platelets stimulated with various concentrations of agonists. Arrows indicate addition of agonists. *P < .05; ***P < .001. ns, not significant.