CSPα^−/− platelets have defects in signaling pathways and defective hemostasis. (A) Washed platelets (200 x 10³/µL) from CSPα^+/+ and CSPα^−/− mice were kept in a resting state (R) or stimulated with 0.1 U/mL thrombin (T) or 100 ng/mL convulxin (C) for 10 minutes and were probed by western blotting to look at phospho-Akt and phospho- myosin light chain (MLC) levels. (B) Quantification of protein levels was performed using ImageLab and data were plotted as either the ratio of phospho-Akt to Akt, or phospho-MLC to β-tubulin. (C) Washed platelets (200 x 10³/µL) from CSPα^+/+ and CSPα^−/− mice were kept in a resting state (R) or stimulated with 0.1 U/mL thrombin (T) or 100 ng/mL convulxin (C) for 10 minutes and were probed by western blotting to determine phosphorylation of PKC substrates, which was interpreted as PKC activity. (D) Quantification of protein levels was performed using ImageLab and data were plotted as the ratio of PKC activity to β-tubulin. (E) Tail bleeding assay was performed to analyze thrombus formation in vivo. CSPα^+/+ (n = 27; male = 16, and female = 11), CSPα^+/− (n = 83; male = 40, and female = 43), and CSPα^−/− (n = 9; male = 4, and female = 5) mice were used to perform the experiment. Statistical analyses were performed using the Kaplan-Meier method using the log-rank test. Akt, phosphatidylinositol 3-kinase (PI3K)/protein kinase B signaling pathway; phosphor, phosphorylated.