Tln1-mR35E,R118E mice exhibit milder defects in hemostasis compared with Rap1a/b-mKO mice, along with preserved capacity of platelets to secrete α-granules and expose surface PS. (A-B) Intravital microscopy studies to monitor hemostatic plug formation after laser injury to the saphenous vein in Tln1-mR35E,R118E and Rap1a/b-mKO mice. Before laser injury, animals were injected with Alexa Fluor 488–conjugated antibodies to GPIX to label circulating platelets and Alexa Fluor 647–conjugated antibodies to fibrin. The experiment was terminated at the end of 10 minutes to avoid excessive loss of blood. (A) Representative images taken 30, 60, and 300 seconds after laser injury. Scale bar, 50 μm. (B) Percentage of bleeding injury sites was plotted over time for each experimental group. Seventeen injury sites in 3 control mice, 39 injury sites in 5 Tln1-mR35E,R118E mice, and 8 injury sites in 2 Rap1a/b-mKO mice were tested. Statistical significance was assayed by using the Gehan-Breslow-Wilcoxon test with Bonferroni correction. (C-D) Comparisons of the 3 pairs of groups were all statistically significant using a family-wise significance level of 5%. Flow cytometry analysis of P-selectin (CD62P) surface expression onto GPIX-labeled platelets in whole blood in response to PAR4-AP (C) or convulxin (D) stimulation. Bar graphs represent Δ mean fluorescence intensity (MFI) ± standard error of the mean (n = 6 mice). Two-way analysis of variance with Tukey post-test. (E) Determination according to flow cytometry of PS exposure onto GPIX-labeled platelets in whole blood in response to dual stimulation with 100 ng/mL of convulxin and 500 μM of PAR4-AP. Bar graph represents the percent mean ± standard error of the mean (n = 6 mice). Two-way analysis of variance with Tukey posttest. **P < .01; ***P < .001. ns, not significant.