Main

Platelets are anucleate blood cells that play essential roles in haemostasis, wound healing and a range of other processes, including inflammation and immunity.1 They are produced by megakaryocytes, large polyploid cells that develop primarily in the bone marrow, spleen and foetal liver.2 Recent work has demonstrated that the survival of megakaryocytes and platelets is governed by the BCL-2 family proteins.3 Both cell types possess a classical BAK/BAX-mediated intrinsic apoptosis pathway that must be restrained in order for them to develop and survive.

In platelets, BCL-XL is the critical pro-survival BCL-2 family member required to keep BAK and BAX in check. The first evidence of this came from Wagner et al.,4 who reported severe thrombocytopaenia in mice after MMTV-Cre-mediated deletion of Bclx in the haematopoietic system, skin and various secretory tissues. It has since been shown that megakaryocyte-restricted deletion of Bclx in mice reduces platelet lifespan from ~5 days to ~5 h, with a concomitant decrease in platelet counts to ~2% of wild-type levels.5, 6 Pharmacological inhibition of BCL-XL with the BH3 mimetics ABT-7377 or Navitoclax (ABT-263)8 (which both also inhibit BCL-2 and BCL-W) triggers BAK/BAX-mediated platelet apoptosis.9, 10, 11 As a result, these drugs cause dose-dependent thrombocytopaenia in mice, dogs and humans.9, 11, 12, 13, 14 Indeed, thrombocytopaenia is the dose-limiting toxicity for Navitoclax.12, 13, 14 This fact provided additional impetus for the development of agents that specifically target BCL-2, beginning with ABT-199,15 a BCL-2-selective antagonist currently in clinical trials for the treatment of a range of haematological malignancies including chronic lymphocytic leukaemia, non-Hodgkin’s lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma and acute myeloid leukaemia. ABT-199 has already shown very promising anti-tumour activity, with little to no impact on platelet counts.15, 16 These data suggest that BCL-2 is dispensable for the development and survival of platelets.

In megakaryocytes, BCL-XL is also critical for survival. Although not absolutely required for their growth and maturation, BCL-XL is essential for megakaryocytes to proceed safely through pro-platelet formation and platelet shedding.5 In addition to BCL-XL, megakaryocytes also depend on the pro-survival activity of MCL-1. Conditional deletion of Mcl1 alone has no effect on this lineage. In contrast, combined megakaryocyte-specific loss of Bclx and Mcl1 results in the failure of megakaryopoiesis, systemic haemorrhage and embryonic lethality.5, 17, 18 These defects are rescued by deletion of Bak and Bax.18

Consistent with the genetic studies, administration of ABT-737 to Mcl1Pf4Δ/Pf4Δ mice, which lack MCL-1 in megakaryocytes and platelets, induces acute, fulminant BAK/BAX-dependent megakaryocyte apoptosis. Given that, in addition to BCL-XL, ABT-737 also targets BCL-2,7 these data suggested that BCL-2 might also contribute to the development and survival of the megakaryocyte lineage. This is supported by recent studies demonstrating that neonatal human platelets contain increased levels of BCL-2 relative to adult counterparts,19 and that platelet lifespan is extended in transgenic mice expressing BCL-2 under the control of the pan-haematopoietic Vav promoter.20 In light of these observations, and intense ongoing activity surrounding the development of novel BH3 mimetics,21 we set out to elucidate the role of BCL-2 in megakaryocytes and platelets. Mice with a megakaryocyte-specific deletion of Bcl2, either alone or in combination with deletion of Mcl1 or Bclx, were generated. The effect of these mutations, and of BCL-2 or BCL-XL-selective BH3 mimetics, on the megakaryocyte lineage was assessed.

Results

Platelet production and platelet lifespan are normal in the absence of BCL-2

Mice lacking BCL-2 in the megakaryocytic lineage were generated by crossing animals carrying a floxed allele of Bcl222 with Pf4-Cre transgenic animals.23 Bcl2Pf4Δ/Pf4Δ mice were born at the expected Mendelian ratios, and were outwardly healthy. Deletion of BCL-2 in bone marrow-derived megakaryocytes and washed platelets was confirmed by western blotting (Figure 1a). Peripheral blood platelet counts (Figure 1b) and platelet survival (Figure 1c) in adult Bcl2Pf4Δ/Pf4Δ mice were comparable with control animals. Megakaryocyte numbers and ploidy in bone marrow of Bcl2Pf4Δ/Pf4Δ mice were normal (Figures 1d and e). Additionally, we assessed platelet and megakaryocyte counts in young (1–5-week old) mice with a constitutive deletion of Bcl2.24 Despite their various phenotypic abnormalities, including kidney polycystic disease, growth retardation and lymphopaenia, blood platelet counts (Figure 1f) and bone marrow and spleen megakaryocyte numbers (Figure 1g) in Bcl2−/− mice were comparable with those of wild-type controls. Together, these results indicated that BCL-2 is dispensable for steady state platelet production. To establish whether this is also the case under conditions of stress, we induced transient thrombocytopaenia by injecting anti-platelet serum. This typically leads to platelet depletion in wild-type mice within 24 h, followed by recovery and rebound thrombocytosis at ~5 days post injection. Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl mice responded similarly to anti-platelet serum treatment (Figure 1h), indicating that even under conditions of stress thrombopoiesis, BCL-2 is dispensable for the development and survival of megakaryocytes and platelets.

Figure 1
figure 1

Loss of BCL-2 does not affect platelet survival or platelet production. (a) Western blot analysis of protein lysates from platelets and bone marrow-derived megakaryocytes from Bcl2fl/fl and Bcl2Pf4Δ/Pf4Δ mice. Bone marrow progenitor cells were cultured in thrombopoietin (TPO), and mature megakaryocytes were purified on a BSA gradient. Probing for actin was used as a control for protein loading. Each lane represents platelets from an individual mouse. (b) Platelet counts in Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl control mice at 7–10 weeks of age. Each symbol represents an individual mouse. (c) Platelet survival curves in Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl control mice. Platelets were labelled via i.v. injection of a DyLight 488-conjugated anti-CD42c (GPIbβ) Ab. n=3 Bcl2Pf4Δ/Pf4Δ and n=5 Bcl2fl/fl mice. Time 0 (100%) was set at 1 h post injection. (d) Morphologically recognisable bone marrow megakaryocytes in H&E-stained sternum sections from Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl control mice. Field of view (FOV). n=22 Bcl2fl/fl; n=11 Bcl2Pf4Δ/Pf4Δ mice. (e) Ploidy distribution profile of CD41+ bone marrow cells in Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl control mice, as determined by flow cytometry. n=3 Bcl2fl/fl; n=4 Bcl2Pf4Δ/Pf4Δ mice. (f) Platelet counts in Bcl2-/- and Bcl2+/+ mice at 2–5 weeks of age. Each symbol represents an individual mouse. (g) Morphologically recognisable bone marrow (BM) and spleen megakaryocytes in H&E-stained sections from Bcl2−/− and Bcl2+/+ mice at 1–4 weeks of age. n=5 Bcl2+/+, n=6 Bcl2-/-mice. (h) Platelet counts in mice treated with anti-platelet serum. n=5–6 Bcl2fl/fl, n=4 Bcl2Pf4Δ/Pf4Δ mice per time point (except 96 h, n=2). Data are presented as mean±S.D.

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Combined loss of BCL-2 and MCL-1 does not affect platelet production or platelet survival

We and others have previously shown that platelet production and platelet counts are normal in Mcl1Pf4Δ/Pf4Δ mice, whereas combined deletion of Mcl1 and Bclx in megakaryocytes results in haemorrhage and embryonic lethality.17, 18 To examine any potential functional redundancy between BCL-2 and MCL-1 in the megakaryocyte lineage, we conditionally deleted both of the genes encoding these proteins. We began by measuring body weight, platelet counts and the proportion of reticulated platelets in newborn mice 5–10 days after birth. Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ double knockout animals gained weight at a rate comparable with control littermates (Supplementary Figure 1a). Platelet counts significantly increased from day 7 to 10 in all mice with no differences between genotypic classes (Figure 2a). This correlated with a reduction in the proportion of reticulated (thiazole orange-stained) platelets (Figure 2b). In adult Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ mice, platelet counts and platelet survival were equivalent to those of control littermates (Figures 2c and d). We confirmed efficient deletion of BCL-2 and MCL-1 in bone marrow-derived BSA-gradient-purified megakaryocytes by western blotting (Supplementary Figure 1b). Bone marrow megakaryocyte numbers and ploidy were normal in Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ mice, and these animals exhibited a wild-type response to anti-platelet serum-induced thrombocytopaenia (Figures 2e and f, Supplementary Figure 1c). Consistent with these observations, administration of a single dose of the BCL-2-selective antagonist ABT-199 (100 mg/kg) to Mcl1Pf4Δ/Pf4Δ and Mcl1fl/fl mice did not markedly affect blood platelet or bone marrow megakaryocyte numbers 6 and 24 h post injection (Figures 2g and h). Although a statistically significant reduction in platelet counts at 24 h post treatment was observed in Mcl1Pf4Δ/Pf4Δ mice, relative to Mcl1fl/fl counterparts (Figure 2g), the numbers were in the normal range (987.6±235.1 × 103/μl) and not significantly different from vehicle-treated Mcl1Pf4Δ/Pf4Δ animals. Collectively, these results demonstrated that the combined functions of BCL-2 and MCL-1 are dispensable for platelet production and platelet survival.

Figure 2
figure 2

Combined deletion of BCL-2 and MCL-1 does not affect platelet production. (a) Platelet counts and (b) % reticulated thiazole orange (TO)-positive platelets in floxed control, Bcl2Pf4Δ/Pf4Δ and Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ neonatal mice on perinatal day (P) 5, 7 and 10. Each symbol represents an individual mouse. (c) Platelet counts in adult Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ and Bcl2fl/fl Mcl1fl/fl control mice at 7–10 weeks of age. n=6 Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ; n=20 Bcl2fl/fl Mcl1fl/fl. (d) Platelet survival curves in Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4 and floxed control mice. Platelets were labelled via i.v. injection of a Dylight 488-conjugated anti-CD42c mAb. n=6 mice per genotype. Time 0 (100%) was set at 8 h post injection. (e) Numbers of morphologically recognisable megakaryocytes in H&E-stained bone marrow sections. n=4 Bcl2fl/fl Mcl1fl/fl; n=7 Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4 mice. (f) Ploidy distribution profile of CD41+ bone marrow cells, as determined by flow cytometry. n=4 Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ, n=2 Bcl2fl/fl Mcl1fl/fl. Platelet (g) and bone marrow megakaryocyte counts (h) 6 and 24 h post oral administration of the BCL-2 selective BH3 mimetic ABT-199 (A-199) 100 mg/kg or vehicle control. n=3–5 mice per group, except Mcl1fl/fl 6 h vehicle where n=2. *P<0.05; **P<0.005; ***P<0.001. Data are presented as mean±S.D.

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BCL-XL antagonism in MCL-1/BCL-2-deficient mice

The recent development of BCL-XL-specific BH3 mimetic compounds25, 26, 27 enabled us to determine the combined effect of BCL-XL, BCL-2 and MCL-1 inhibition on the megakaryocyte lineage in vivo. Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ double knockout and control littermates were treated with a single dose of the BCL-XL-selective antagonist A-1155463.7 (A-463, 5 mg/kg). In Bcl2fl/fl Mcl1fl/fl mice, A-463 induced rapid thrombocytopaenia, but had no effect on bone marrow megakaryocyte numbers at 2 and 24 h post injection (Figures 3a, b and d). In contrast, in Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ animals, acute thrombocytopaenia was observed (Figure 3a). Additionally, apoptotic megakaryocytes with pyknotic nuclei were apparent in the bone marrow and spleen 2 h post administration (Figures 3c and d). Megakaryocyte numbers were reduced, and consistent with this, platelet counts were lower in A-463-treated Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4Δ mice compared with floxed control animals 24 h post injection (Figures 3a and b). These results prompted us to explore functional redundancy between BCL-2 and BCL-XL in the control of cell survival in megakaryocytes and platelets.

Figure 3
figure 3

BCL-XL inhibition leads to thrombocytopaenia and apoptosis of MCL-1/BCL-2 deficient megakaryocytes in vivo. Platelet (a) and bone marrow megakaryocyte counts (b) 2 and 24 h post i.p. administration of the BCL-XL-selective BH3 mimetic drug A-1155463.7 (A-463) 5 mg/kg or vehicle in Bcl2fl/fl Mcl1fl/fl and Bcl2Pf4Δ/Pf4Δ Mcl1Pf4Δ/Pf4 mice. Each symbol represents an individual mouse. **P<0.005; ***P<0.001. Data are presented as mean±S.D. (c) Apoptotic bone marrow megakaryocytes with pyknotic nuclei in H&E sections. (d) Representative images of H&E-stained spleen (lower right panel) and bone marrow (all other panels) megakaryocytes 2 h post A-463 or vehicle treatment. Scale bar: 20 μm. Pyknotic nuclei are indicated by white arrows

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Inhibition of BCL-2 does not exacerbate thrombocytopaenia in BCL-XL-deficient mice

We administered the BCL-2-selective antagonist ABT-199 (100 mg/kg) to BclxPf4Δ/Pf4Δ and floxed control littermates by oral gavage and measured platelet and megakaryocyte counts 6 and 24 h post treatment. Conditional deletion of BCL-XL in the megakaryocytic lineage causes a profound shortening in platelet lifespan, consequent thrombocytopaenia and reactive megakaryocytosis.5 Consistent with this, vehicle-treated BclxPf4Δ/Pf4Δ mice exhibited thrombocytopaenia and increased megakaryocyte counts (Figures 4a and b). No significant exacerbation of these deficient platelet and megakaryocyte numbers were observed when BclxPf4Δ/Pf4Δ mice were treated with ABT-199 (Figures 4a and b). To complement the pharmacological approach, we generated Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ double knockout mice. Similar to their BclxPf4Δ/Pf4Δ littermates, Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ animals were present at weaning at ~75% of the number expected (25 observed/33 expected). Platelet counts in adult Bcl2+/+ BclxPf4Δ/Pf4Δ and Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ mice were identical (Figure 4c). In Bclx heterozygotes (BclxPf4Δ/fl), deletion of one or both alleles of Bcl2 had no additional impact on platelet numbers and lifespan (Figure 4d). Megakaryocyte counts in bone marrow and spleen were increased in adult Bcl2+/+ BclxPf4Δ/Pf4Δ and Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ mice relative to control animals (Figure 4e). We did observe a modest, but statistically significant, decrease in bone marrow megakaryocytes, and an increase in splenic megakaryocytes in Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δanimals relative to Bcl2+/+ BclxPf4Δ/Pf4Δ counterparts. However, these mice were bred in different colonies, which likely explains the minor differences. As expected, and similar to BclxPf4Δ/Pf4Δ mice, megakaryocyte ploidy was increased in Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ animals (Figure 4f).

Figure 4
figure 4

Thrombocytopaenia in Bclx-deficient mice is not exacerbated by BCL-2 inhibition. Platelet (a) and bone marrow megakaryocyte counts (b) 6 and 24 h post oral administration of the BCL-2-selective BH3 mimetic ABT-199 (A-199) 100 mg/kg or vehicle control. n=3–4 mice per group, except BclxPf4Δ/Pf4Δ 24 h vehicle where n=2. Platelet counts are shown on a log scale. (c) Platelet counts (log scale) in Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/Pf4Δ, Bcl2+/+ BclxPf4Δ/Pf4Δ and floxed control mice at 7–10 weeks of age. (d) Platelet survival curves in Bcl2Pf4Δ/Pf4Δ BclxPf4Δ/fl and Bcl2Pf4Δ/fl BclxPf4Δ/fl mice. Platelets were labelled via i.v. injection of a DyLight 488-conjugated anti-CD42c mAb. n=4 mice per genotype. Time 0 (100%) was set at 1 h post injection. (e) Numbers of morphologically recognisable megakaryocytes in H&E-stained bone marrow and spleen sections. n=8–17 mice per genotype. (f) Ploidy distribution profile of CD41+ bone marrow cells, as determined by flow cytometry. n=4 mice per genotype. *P<0.05; **P<0.005; ***P<0.001. Data represent mean±S.D.

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Pharmacological inhibition of BCL-XL, but not BCL-2, induces megakaryocyte apoptosis

The results from our in vivo studies suggested that genetic or pharmacological antagonism of BCL-2 has no adverse effect on megakaryopoiesis. To test this directly in isolated primary cells, we treated foetal liver or bone marrow-derived mouse megakaryocytes with ABT-737, ABT-199 or A-463. It has been previously reported that in cultured primary mouse megakaryocytes, deletion of Bcl-x or treatment with ABT-737 triggers loss of cell viability, Caspase-3/7 activity and a failure of proplatelet formation.5 In line with these data, we found that the BCL-XL-selective inhibitor A-463 induced dose-dependent Caspase-3/7 activation in wild-type foetal liver-derived megakaryocytes (Figure 5a). Consistent with its increased potency against BCL-XL,27 A-463 treatment at a concentration of 2.5 μM induced more caspase activity in megakaryocytes than ABT-737 at 5 μM. In contrast, treatment with the BCL-2 antagonist ABT-199 (2.5 μM) had no effect. Combination treatment with A-463 and ABT-199 did not amplify Caspase-3/7 activity beyond that seen with A-463 treatment alone. Similarly, loss of Bcl2 did not render bone marrow-derived megakaryocytes more sensitive to A-463 (Figure 5b).

Figure 5
figure 5

BCL-XL inhibition induces cell death in megakaryocytes in vitro. (a) Caspase-3/7 activity expressed as fold-increase in wild-type foetal liver-derived BSA gradient-purified megakaryocytes 5 h post treatment with A-1155463.7 (A-463) 0.01–2.5 μM, ABT-199 (A-199) 2.5 μM, ABT-737 (A-737) 5 μM or vehicle (DMSO) was assessed by using the Caspase-3/7 Glo (Promega) kit. n=4–5 biological replicates, three independent experiments. (b) Caspase-3/7 activity expressed as fold-increase in bone marrow-derived BSA gradient-purified Bcl2Pf4Δ/Pf4Δ and Bcl2fl/fl megakaryocytes 5 h post treatment with A-463 (5 μM) or vehicle (DMSO) was assessed by using the Caspase-3/7 Glo kit. Data shown are representative of two independent experiments. *P<0.05; **P<0.005; ***P<0.001. Data represent mean±S.D.

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Discussion

In this study, we analysed platelet production in mice with a megakaryocytic lineage-specific deletion of Bcl2 alone or in combination with deletion of Mcl1 or Bclx. In addition, selective BH3 mimetics inhibiting BCL-2 or BCL-XL were assessed. Our genetic and pharmacological studies demonstrate that BCL-2 is dispensable for platelet production both at steady state and under conditions of stress. Moreover, platelet survival in vivo was not affected by genetic loss of BCL-2 or its pharmacological inhibition. This aligns with initial reports on the effects of ABT-199 in patients,15, 16 and bodes well for the clinical development of BCL-2 antagonists. Our experiments confirm the importance of BCL-XL and the ancillary role of MCL-1 in maintaining megakaryocyte viability and BCL-XL in sustaining platelet survival.5

Our data indicate that BCL-2 is dispensable for platelet survival in adults, and in neonates as well. The latter findings are consistent with a recent study from Liu and colleagues,19 which demonstrated that although neonatal platelets display elevated BCL-2 protein levels and extended survival compared with adult platelets, BCL-2 was not the primary molecule facilitating this effect. We have further demonstrated that conditional deletion of Bcl2, on its own or in concert with Mcl1, had no impact on neonatal platelet counts.

BCL-2 has recently been linked to myeloid progenitor cell survival.28 As Cre-dependent recombination utilising the Pf4-Cre transgenic mouse is restricted to megakaryocytes, platelets23 and a small fraction of late megakaryocyte progenitors,29 we could not use this model to address the role of BCL-2 in the earliest stages of megakaryopoiesis. However, mice carrying the constitutive deletion of Bcl2 did afford this opportunity. Germline loss of BCL-2 leads to polycystic kidney disease, lymphopaenia, grey fur (due to premature death of melanocytes), growth retardation and early mortality.24, 30, 31 Even with the complex co-morbidities of this model, our results show that young Bcl2−/− and control mice exhibited similar megakaryocyte and platelet counts, indicating that loss of BCL-2 in progenitor cells does not significantly affect the megakaryocytic lineage at steady state in young animals. Although it is increasingly clear that ABT-199 used as a single agent within the therapeutic window will not kill platelets and megakaryocytes, potential effects on progenitor cells with subsequent thrombocytopaenia may become apparent when used in combination with certain chemotherapeutics. This is pertinent as clinical trials are currently evaluating the safety and efficacy of ABT-199 in combination with proteasome inhibitors in multiple myeloma,32 hypomethylating agents in acute myeloid leukaemia,33 and alkylating or antimitotic drugs in non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia and follicular lymphoma34, 35 known to cause thrombocytopaenia through bone marrow suppression or inhibition of proplatelet formation.36, 37

Despite dose-limiting thrombocytopaenia, BCL-XL antagonism has shown encouraging results in certain solid tumours.12 38 Combination trials with Navitoclax and kinase inhibitors (MEK, RAF and BRAF) are underway for advanced or metastatic solid tumours, including small-cell lung, colon, pancreatic, rectal and liver cancer. Limited information is available on the effects of this group of kinase inhibitors on megakaryocytes and platelets, although MEK inhibition has been associated with thrombocytopaenia.39 Hence, for combination treatments using BCL-XL antagonists and kinase inhibitors, it will be imperative to closely monitor platelet counts, as both platelet production and platelet survival may be affected. With the recent development of BCL-XL–specific BH3 mimetics,25, 26, 27 there is a need to find means of sustaining platelet counts in order to allow safe dose escalation without increased risk of bleeding.40 Although BCL-2 is not required for platelet survival, we recently showed that overexpression of BCL-2 in blood cells extends platelet lifespan in adult mice,20 similarly to that observed in animals lacking the essential mediators of intrinsic apoptosis, BAK and BAX.5, 9 One could imagine that one approach to facilitating the safe administration of BCL-XL antagonists might be transfusion of platelets either overexpressing BCL-2, or lacking BAK/BAX, thus rendering them resistant to BCL-XL inhibition. Recent advances in this field of research, including development of human-induced pluripotent stem cell-derived megakaryocytes generating platelets,41 HLA-universal platelets,42 synthetic micro-vessels43 and a novel microfluidic bioreactor design enabling ex vivo platelet production,44 might allow such manipulation in the not-too-distant future.

Materials and Methods

Mice

Bcl2-/-,24 Bcl2 floxed,22 Mcl1 floxed,9 Bclx floxed45 and Pf4-Cre23 mice have been previously described. All mouse lines had been backcrossed onto the C57BL/6 background for at least 10 generations prior to this study. Mice were 7–12-weeks old or as otherwise stated. All animal experiments complied with the regulatory standards of, and were approved by, the Walter and Eliza Hall Institute (WEHI) Animal Ethics Committee.

Materials

Dimethyl sulfoxide (DMSO) and propidium iodide were from Sigma-Aldrich, St. Louis, MO, USA. ABT-7377 was provided by Abbott Laboratories, Abbott Park, IL, USA. ABT-19915 and A-1155463.727 were provided by AbbVie, North Chicago, IL, USA. The enhanced chemiluminescence system was from Merck Millipore (Kilsyth, VIC, Australia), the protease inhibitor cocktail, Complete, was from Roche (Basel, Switzerland), 4–12% Bis-Tris gels (NuPAGE) were from Invitrogen Life technologies (Carlsbad, CA, USA), and calibration beads 3.5–4.0 μm were from Spherotech Inc (Lake Forest, IL, USA). Antibodies used for immunoblotting included: mouse anti-BCL-2 (Clone 7; recognises mouse, rat, chicken, dog and human) from BD Transduction Laboratories (Franklin Lakes, NY, USA), rat anti-MCL-1 (clone 19C4-15; recognises mouse and human, WEHI mAb Facility, Parkville, VIC, Australia) and anti-actin-HRP from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for flow cytometry included: X488 from Emfret Analytics GmbH and Co. KG (Eibelstadt, Germany); fluorescently conjugated anti–mouse CD41 (MWReg30) from BD. Anti-platelet serum was purchased from Cedarlane (Burlington, Ontario, Canada).

Haematology

Automated cell counts were performed on blood collected by cardiac puncture or from the retro-orbital plexus into Microtainer tubes containing EDTA (Sarstedt, Ingle Farm, SA, Australia), using an Advia 2120 haematological analyser (Siemens, Munich, Germany). Megakaryocytes were counted manually in sections of sternum and spleen stained with haematoxylin and eosin (H&E) with a minimum of 10 high-power fields ( × 200) analysed. Images were acquired on a Nikon Eclipse E600 microscope equipped with AxioCam MRc5 (Zeiss, Oberkochen, Germany) and AxioVision 4.8. Scale bars were inserted with Image J. Acute thrombocytopaenia was induced by injection of anti-platelet serum and assessed as described.46

Primary megakaryocyte culture

Foetal livers were removed at embryonic day (E) 13.5 and transferred into Dulbecco’s modified Eagle’s medium (high glucose version) with 10% foetal calf serum (Gibco, Paisley, UK). Bone marrow was flushed into Dulbecco’s modified Eagle’s medium with 2% foetal calf serum. The cells were lineage-depleted by incubation with a mix of biotinylated antibodies (CD4, CD2, CD3, CD5, CD8, Ter119, B220, CD19, Gr-1, Ly6G, F4/F8, CD127; WEHI mAb Facility) in KDS-BSS 2% foetal calf serum, followed by anti-biotin magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and MAC LS columns (Miltenyi Biotec) in EDTA-KDS-BSS 0.5% foetal calf serum. Single cell suspensions were cultured for 3–5 days at 5 × 105 cells per ml in serum-free medium47 supplemented with 100 ng/ml murine thrombopoietin (WEHI) at 37 °C, 5% CO2, and mature megakaryocytes purified using a BSA gradient as described.5

Megakaryocyte ploidy

Bone marrow was harvested from femora of 8–10-week-old mice into 1 ml of CATCH medium and megakaryocyte ploidy was studied by staining with CD41-FITC mAb and propidium iodide, as described.5

Caspase activity assay

BSA gradient-purified megakaryocytes (3 × 104 cells per ml in serum-free medium with TPO) were seeded into 96-well plates and then incubated at 37 °C, 5% CO2 with or without the addition of ABT-737 (5 μM), ABT-199 (2.5 μM) or A-1155463.7 (0.01–5 μM). Caspase-Glo 3/7 reagents (Promega, Madison, WI, USA) were added to megakaryocytes after 5 h of treatment. The luminescence of each sample was determined in a plate-reading LumiSTAR Galaxy luminometer (BMG Labtech, Ortenberg, Germany).

Platelet preparation

Murine blood was obtained by cardiac puncture into 0.1 volume of Aster-Jandl anticoagulant (85 mM sodium citrate, 69 mM citric acid, and 20 mg/ml glucose, pH 4.6).48 Platelet-rich plasma was obtained by centrifugation at 125 × g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 × g for 8 min. Platelets were washed by two sequential centrifugations at 860 × g for 5 min in 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose and 12.5 mM sucrose, pH 6.0 (buffer A). The platelet pellet was resuspended in 10 mM Hepes, 140 Mm NaCl, 3 mM KCl, 0.5 mM MgCl2, 10 mM glucose and 0.5 mM NaHCO3, pH 7.4 (buffer B).

Platelet turnover studies

Mice were injected i.v. with 0.15 μg/g body weight of X488 (Emfret), a rat-derived IgG against the platelet CD42c (GPIbβ) receptor, and platelet lifespan was measured as previously described.48 Platelets were identified in Platelet-rich plasma as being CD41+ by flow cytometry and the proportion of X488+ platelets remaining at each time point was assessed.

SDS-PAGE and western blot analysis

Platelets were lysed in NP40 lysis buffer and megakaryocytes were lysed in RIPA buffer. Proteins were separated on 4–12% Bis-Tris gels (NuPAGE; Invitrogen) under reducing conditions, transferred onto Immobilon-P membranes (Micron Separation), and immunoblotted with various Abs (see above), followed by incubation with secondary HRP-conjugated Abs and ECL.

Platelet counts in newborn mice

Newborn (day P2) mice were given paw tattoos to assist identification. Newborn mice were weighed on P5, 7 and 10 prior to blood collection. Genotyping was performed by PCR using DNA obtained on P2. For blood collection, the anterior facial vein was punctured using a 30-gauge needle. Five microlitres of blood were collected using a micropipette and dispensed into an EDTA blood tube (Sarstedt) with PBS to a total volume of 200 μl. Automated cell counts were performed on blood collected from newborn mice using an Advia 2120 haematological analyser (Siemens). Reticulated platelets in newborn mice were enumerated using staining with thiazole orange.48

In vivo administration of BH3 mimetics

Mice were treated via oral gavage with 100 mg/kg ABT-199 administered as a single dose. A stock solution of ABT-199 (10 mg/ml) was diluted in 60% phosal 50 PG (standardised phosphatidylcholine concentrate with at least 50% PC and propylene glycol; Phospholipid GmbH, Cologne, Germany), 30% polyethylene glycol 400 and 10% ethanol. Blood and sterna were collected at 6 and 24 h after treatment. Alternatively, mice were injected i.p. with 5 mg/kg A-1155463.7 administered as a single dose. A stock solution of A-1155463.7 (12.5 mg/ml) in DMSO was diluted by addition of 30% final volume of dosing solution Cremophor ELP:ethanol (ratio 2 : 1) (Cremophor ELP, Sigma-Aldrich) and 5% dextrose in H2O (to reach final volume). Blood, sterna and spleens were collected at 2 and 24 h after treatment. Sternum and spleen sections were stained with H&E.

Statistical analyses

Statistical significance between two treatment groups was analysed using an unpaired Student’s t test with two-tailed P values. One-way ANOVA with the Bonferroni multiple comparison test was applied where appropriate (GraphPad Prism Software Version 6.0b, La Jolla, CA, USA). *P<0.05; **P<0.005; ***P<0.001 or as otherwise stated. Data are presented as mean±S.D.