Defective Zn²⁺homeostasis in platelets from Tmem163-KO mice and HPS mouse models of BLOC-1, BLOC-2 and AP-3. (A-B) MEG-01 cells were transfected with Cherry-VMAT2 (A) or Cherry-TMEM163 (B) for 48 hours then incubated with 2 μM Zn²⁺ indicator (FluoZin-3) for 30 minutes at 37°C. Pictures are representative confocal images. Outline of the cell is indicated by the white line. Insets show 5× magnified images of the boxed region. Scale bars, 10 μm. (C) MEG-01 cells were transfected with Cherry-TMEM163 for 48 hours and then incubated with 2 μM Zn²⁺ indicator (FluoZin-3) for 30 minutes at 37°C. Picture (left) is representative maximum intensity projection image in X-Y, X-Z, and Y-Z planes. Pictures (right) are Z-stack images in different dimensions. The arrows represent the classical colocalization of Cherrry-TMEM163 with FluoZin-3. (D-I) Washed mutant and control mice platelets were incubated with 2.5 µM FluoZin-3 and stimulated with 0.01 U/mL thrombin, and the kinetic curve of fluorescence changes was recorded by flow cytometry (appending the first 50 seconds [resting stage] to 400 seconds [activated stage]). Data shown are the quantitative results (D) of normalized FluoZin3 mean fluorescence intensity relative to each control at resting conditions and representative kinetic curves of Zn²⁺ release after thrombin treatment (E-I). B6 vs Tmem163-KO, n = 11, 11; sdy^+/− vs sdy, n = 10, 10; pa^+/− vs pa, n = 12; 12, ru^+/− vs ru, n = 12, 12; and pe^+/− vs pe, n = 12, 12, respectively. MFI, mean fluorescence intensity. Mean ± SEM, **P < .01; ***P < .001.