PS exposure and Ca²⁺ influx in platelets and MKs. (A) Membrane PS on platelets from SMS1-KO or WT mice (n = 3 per group) was stained with FITC-conjugated annexin V (FITC–annexin V) and detected by using flow cytometry. WT mouse platelets were pretreated with calcium ionophore A23187 for 10 minutes before PS staining. The percentages of FITC-annexin V–positive platelets (CD41⁺) are presented. (B) MKs were stained with FITC–annexin V and anti–CD41-phosphatidylethanolamine (PE). The nuclei were then counterstained with 4,6-diamidino-2-phenylindole (DAPI), and images were obtained with confocal microscopy. The fluorescent intensity of FITC–annexin V was quantified with ImageJ software and is presented in arbitrary units (AU). Scale bar, 20 µm. Values show the mean ± standard deviation. (C) Sections of mouse spleen were stained with Alexa Fluor 488–conjugated anti-CD68 antibody and anti–CD41-PE, and then observed with fluorescent microscopy. Scale bar, 50 µm. (D) Splenectomy was performed in WT and SMS1-KO mice (n = 3-5 each). Blood was then collected from the tail vein, and platelet numbers were counted on the indicated day after surgery. Values show the mean ± standard deviation. (E) WT (n = 5) and SMS1-KO (n = 3) mice were intravenously injected with N-hydroxysuccinimide ester (NHS)-conjugated biotin. Platelets were then isolated from whole blood collected at the indicated time points and stained with FITC-conjugated anti-CD41 antibody and allophycocyanin-conjugated streptavidin. The biotinylated platelets were analyzed with flow cytometry. t_1/2 represents the half-life of platelets in hours. Values show the mean ± standard deviation. (F) Platelets from WT (n = 4) and SMS1-KO (n = 4) mice were stained with Furo-4 AM and anti–CD41-PE and then analyzed with flow cytometry. The intracellular Ca²⁺ levels in platelets were detected by measuring the Furo-4 AM fluorescence intensity and are presented as mean fluorescent intensity (MFI). Values show the mean ± standard deviation. *P < .05; **P < .005.