Fig. 7: Tropomyosin 4 is required for the final steps in platelet generation in the pulmonary vasculature.

Megakaryocytes (MKs) from Tropomyosin4^−/− (Tpm4^−/−) mice labelled with FITC-conjugated anti-CD41 antibody, were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. For controls, wild-type (WT) MKs stained either with FITC-conjugated anti-CD41 or isotype antibodies, were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. Lungs were ventilated with air throughout. a Representative FACS dot plot images are shown for generated platelets (CD41-FITC positive events are within the red square). b Numbers of generated platelets per Tpm4^−/− MKs, or control WT MKs, in perfusates after different passage numbers through WT lung, were quantified by FACS. Tpm4^−/− platelets were consistently undetectable after up to 18 passages, in the perfusate. Data shown are platelets generated per MK from either Tpm4^−/− MKs or control WT MKs and displayed as mean ± SEM. N = 5 independent experiments for each group. Two-way ANOVA with Šídák’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. c Cells were imaged from samples of perfusates after passage numbers 0, 1, 2, 3, 6 and 9 through murine lung vasculature ex vivo. Cells were morphologically classified as 5 subgroups: intact MKs (as per Fig. 1b) and MK derivatives (shown in Fig. 6a) and quantified as a percentage of total number of cells. Quantification was from at least 150 fields of view, counting at least 170 cells in total for each of the subgroups. N = 4 independent experiments and data are mean ± SEM. Numbers above each column indicate significant difference to other passages within each group. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Dollar sign ($) above columns represents significant difference to corresponding wild-type column in Fig. 6b. Two-tailed unpaired t test (passages 1, 2, 6 and 9) or Mann–Whitney test (passage 3), *p < 0.05, **p < 0.01. d Abundant fluorescent objects, ~10 µm diameter, were visible in sections of mouse lung after 18 passages of stained Tpm4^−/− MKs, as shown in extended focus stacks of 20 continuous two-photon planes of lung. Images shown are representative of n = 3 independent experiments. Scale bar: 20 µm. Source data are provided in the Source Data file.