Blockade of Rap1 binding to both F0 and F1 domains in talin-1 prevents integrin activation in platelets in Tln1-mR35E,R118E mice. (A) Expression of talin-1 mutant in Tln1-mR35E,R118E platelets was assayed by using western blotting. Results are representative of 3 independent experiments, n = 3 mice each time. (B) Surface expression of αIIb, β3, α2, α5, and β1 integrins in Tln1-mR35E,R118E platelets was measured by using flow cytometry. Bar graph represents mean fluorescence intensity (MFI) ± standard error of the mean (n = 6 mice). Two-tailed Student t test. (C-D) Impaired integrin activation in Tln1-mR35E,R118E. Flow cytometry assay to measure binding of GPIX-labeled platelets in whole blood to JonA/PE antibody (C) or Alexa Fluor 488–coupled 9EG7 antibody (D) in response to agonist stimulation. Bar graphs represent MFI ± standard error of the mean (n = 6 mice, representative of ≥3 independent experiments). Two-way analysis of variance with Tukey posttest. (E) Representative aggregation responses of Tln1-mR35E,R118E platelets stimulated with various concentrations of agonists (indicated by arrows). (F) Intravital microscopy studies to monitor hemostatic plug formation after laser injury to the saphenous vein in Tln1-mR118E and Tln1-mR35E,R118E mice. The experiment was terminated at the end of 10 minutes to avoid excessive loss of blood. Individual bleeding times with median were plotted for 3 experimental groups. Data depicted are determinations in 3 control mice (17 injury sites), 4 Tln1-mR118E mice (42 injury sites), and 5 Tln1-mR35E,R118E mice (39 injury sites). Statistical significance was assayed by a one-way analysis of variance with Tukey posttest. ***P < .001. ns, not significant.