Rap1 binding to talin-1 F1 domain contributes to integrin activation in platelets. (A) Generation of mice harboring Tln1 R118E mutation. Two guide RNAs were used to target Tln1 exon 3. A silent mutation corresponding to the PAM sequence of gRNA2 was inserted into the donor single-stranded DNA oligo to prevent re-editing. (B) Sequencing chromatogram of mutated region of Tln1(R118E) gene. (C) Expression of talin-1 mutant in Tln1-R118E platelets was assayed by using western blotting. Results are representative of 3 independent experiments, n = 3 mice each time. (D) Surface expression of αIIb, β3, α2, α5, and β1 integrins in Tln1-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. (E-G) Impaired integrin activation in Tln1-R118E platelets. Flow cytometry assay to measure binding of GPIX-labeled platelets in whole blood to the JonA/PE antibody (E-F) or the Alexa Fluor 488–coupled 9EG7 antibody (G) 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. *P < .05; ***P < .001. ns, not significant.