Figure 4
Loss of Bak does not prevent thrombocytopaenia in BCL-2tg mice. (a) Blood platelet counts in WT (white), Bak−/− (light grey), BCL-2tg (dark grey) and Bak−/−BCL-2tg (black) mice at 6–8 weeks (n=4–7 per genotype), 12 weeks (n=9–14) and 24 weeks (n=15–20). Bars represent mean±S.E.M. Statistical significance is shown only for 12-week-old mice; ***P<0.001, Student’s t-test. (b) Platelet clearance. Peripheral blood samples were taken daily following injection of NHS-biotin to determine the proportion of biotin-labelled platelets. Data represent mean±S.E.M.; n=6. (c) Megakaryocyte frequency is normal in the bone marrow of BCL-2tg and Bak−/− mice and somewhat increased in Bak−/−BCL-2tg mice. Megakaryocytes were counted manually at × 200 magnification in sternum sections stained with haematoxylin and eosin (>10 fields scored per section). Data represent overall mean per field±S.E.M.; n=6–10 male mice; *P<0.05, **P<0.01, Student’s t-test. (d) Platelet maturation in BCL-2tg mice. The percentage of reticulated platelets was determined by flow cytometry following staining with thiazole orange and APC-conjugated CD41 antibody, then converted to absolute platelet numbers using the ADVIA platelet count. Data represent mean±S.E.M.; n=5–7 male mice at 6 weeks. ***P<0.001, Student’s t-test. (e) Overexpression of Bcl-2 does not impair proplatelet formation in vitro. Fetal liver cells were cultured with thrombopoietin and the frequency of large, mature BSA gradient-purified megakaryocytes displaying proplatelet formation assessed. Data are representative of two independent experiments; n=4 technical replicates. (f) Recovery from anti-platelet serum (APS)-induced thrombocytopaenia is impaired in BCL-2tg mice. Mice were treated with a single dose of APS and blood samples taken by tail pricks daily. Platelet counts were determined by flow cytometry. Data represent mean±S.E.M., n=6. *P<0.05, ***P<0.001, Student’s t-test
