Genetic ablation of Nix impairs platelet activation. (A-B) Characterization of Nix knockout (Nix^−/−) mice by polymerase chain reaction (A) and western blotting (B). (C) Platelet counts from Nix^−/− mice and WT controls (WT) were illustrated. Student t test. (D-F) Platelet membrane receptors GPIb (D), GPVI (E), and GPIIbIIIa (F) were analyzed by flow cytometry using specific antibodies. Student t test. (G-H) Platelet aggregation induced by thrombin, collagen, ADP, and U46619 was monitored. (G) Baseline of platelet aggregation trace was indicated with arrowhead. (H) The aggregation amplitudes were analyzed (n = 11). Student t test. (I-J) P-selectin surface expression of platelets treated with 0.05 U/mL α-thrombin was examined by flow cytometry using anti–P-selectin antibody, and negative controls (IgG control), resting platelet controls, and positive controls (A23187-treated platelets) have been included (I); statistical analysis of the data is shown in panel J. Student t test. (K-L) Platelet activation of platelets treated with 0.05 U/mL α-thrombin was detected by flow cytometry using an antibody against the active α_IIbβ₃, and negative controls (IgG control), resting platelet controls, and positive controls (A23187-treated platelets) have been included (K); statistical analysis of the data is demonstrated in panel L. Student t test. (M) Comparison of tail-bleeding time in Nix^−/− mice and WT controls. Nonparametric test. (N-O) The occlusion times in the FeCl₃-induced thrombus models in Nix^−/− mice and WT controls (N); statistical analysis of the data using the nonparametric test is shown in panel O. *P < .05; **P < .01. n = 9 for each group if it is not stated in the figures. MFI, mean fluorescence intensity; mRNA, messenger RNA; NS, not significant.