BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation by Yulia Kaluzhny, Guangyao Yu, Shishinn Sun, Paul A. Toselli, Bernhard Nieswandt, Carl W. Jackson, and Katya Ravid Blood Volume 100(5):1670-1678 September 1, 2002 ©2002 by American Society of Hematology

BclxL transgene expression in megakaryocytes BclxL transgene expression in megakaryocytes.(A) PCR-based cDNA amplification, showing transgene expression in BM of transgenic mice, as described in “Materials and methods.” Reverse-transcribed, amplified DNA was electrophoresed, transferred onto a nylon m... BclxL transgene expression in megakaryocytes.(A) PCR-based cDNA amplification, showing transgene expression in BM of transgenic mice, as described in “Materials and methods.” Reverse-transcribed, amplified DNA was electrophoresed, transferred onto a nylon membrane, and analyzed with 32P-labeled probes as indicated. The reaction was carried out in the absence or presence of reverse transcriptase (RT) for transgenic lines (Tg) and wild-type (WT) mice. SV40 probe detects the transgene only, BclxL probe detects the endogenous and transgenic BclxL at similar positions, and both PF4 and GAPDH are used to evaluate and normalize for the efficiency of the RT reaction. The lower panel with SV40 probe shows twice the time of the exposure of the film as the top panel to permit visualization of the band in Tg12. Data are representative of 3 experiments. (B) BM cells derived from transgenic (Tg) or control (WT) mice were cytospun (as a dense layer of cells to allow focus on the rare megakaryocytes) and Cy3-labeled with rabbit antibodies to human BclxL (which also reacts with mouse BclxL). Transgenic megakaryocytes, identified based on size and morphology, displayed higher levels of BclxL, as compared with control (examinations were done blindly). The staining appeared cytosolic, as also described by others.42 The data are representative of 2 transgenic lines (2 mice per line in duplicates; original magnification, × 400). (C) Freshly derived BM cells were cytospun onto a slide and subjected to immunohistochemistry with anti-BclxL (middle panel). Megakaryocytes were identified based on staining with an antibody to CD41 (top panel) and DNA was stained with Hoechst (lower panel). No staining was observed with secondary antibody alone and no BclxL-specific fluorescence was observed on staining with CD41 alone and vice versa (not shown). The micrographs are of a representative field, further demonstrating enhanced, lineage-specific expression of BclxL in a transgenic line (original magnification, × 400). Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

A decrease in the number of megakaryocytes undergoing apoptosis is detected in BclxL transgenic mice.BM cultures of transgenic line 19 (Tg19) or control (WT) mice were enriched for megakaryocytes by indirect immunomagnetic negative separation. A decrease in the number of megakaryocytes undergoing apoptosis is detected in BclxL transgenic mice.BM cultures of transgenic line 19 (Tg19) or control (WT) mice were enriched for megakaryocytes by indirect immunomagnetic negative separation. Following this procedure, there are still numerous, contaminating BM cells. (A) Apoptotic cells were detected by dark staining with the ApopTag Plus Peroxidase in situ Apoptosis Detection kit. Arrows point to some of the nonapoptotic megakaryocytes (original magnification, × 200). The experiment shown is representative of 3 performed. Terminal dioxyl transferase (TdT) enzyme control was always included in the study, because contaminant magnetic beads often give a positive signal. (B) Isolated megakaryocytes were cytospun and stained with Cy3-labeled rabbit antibodies to human BclxL (which also reacts with mouse BclxL; original magnification, × 400). In the center of the WT panel are proplatelet-forming cells with poor BclxL expression. The arrows point to cellular extensions (as also demonstrated in detail in Figure 7), which do not appear in megakaryocytes with high abundance of BclxL (eg, Tg19; indicated by arrowheads). Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

Megakaryocytes are more numerous in BM of BclxL transgenic mice, but no increase in platelet level is detected.(A) BM was harvested from femurs of WT or transgenic mice and cytospun; megakaryocytes were identified by in situ staining for acetylcholinesteras... Megakaryocytes are more numerous in BM of BclxL transgenic mice, but no increase in platelet level is detected.(A) BM was harvested from femurs of WT or transgenic mice and cytospun; megakaryocytes were identified by in situ staining for acetylcholinesterase as described in “Materials and methods.” The data are presented as percentage of WT megakaryocytes, presented as averages ± SD for 4 different experiments (4 mice from each group), each performed in duplicates. The actual average number for WT is 52.3 ± 15.4 megakaryocytes/5 × 105 BM cells. Values were also obtained in MGDF-injected mice. (B) Platelet counts from the peripheral blood of WT and transgenic mice. The data are shown as percentage of WT values with averages ± SD for 8 mice for Tg12; for 16 mice each for Tg13, Tg16, and Tg19; and for 24 mice for WT. The actual average number for WT is 1.2 × 106 platelets in 1 μL blood. Values were also obtained in MGDF-injected mice. Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

Platelet recovery is slower in transgenic mice subjected to experimentally induced ITP.The data are averages ± SD for 6 mice for Tg19, 3 mice for Tg16, and 8 wild-type (WT) mice. Platelet recovery is slower in transgenic mice subjected to experimentally induced ITP.The data are averages ± SD for 6 mice for Tg19, 3 mice for Tg16, and 8 wild-type (WT) mice. The differences between the transgenic lines and control were statistically significant (P < .01), as derived by t tests. Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

Transmission electron micrographs of megakaryocytes derived from control or BclxL transgenic mice.Micrographs show megakaryocytes from WT control (A,B), Tg19 (C,D), and Tg16 (E,F) mice. Transmission electron micrographs of megakaryocytes derived from control or BclxL transgenic mice.Micrographs show megakaryocytes from WT control (A,B), Tg19 (C,D), and Tg16 (E,F) mice. At least 50 megakaryocytes from 3 different mice (control and transgenic) were analyzed. The nucleus (Nu) and demarcation membrane system (dms) are labeled. Note the poor dilation of demarcation membranes in panels C through F. Arrows point to peripheral sheet of cytoplasm which appearance is consistent with platelet shedding in control cells (A,B). Note the extensive outer ring of cytoplasm in panels C and D, and especially in panels E and F. Granule numbers are comparable in control and transgenic megakaryocytes. Original magnifications, × 4500 in panels A, C, and E; × 12 500 in panels B, D, and F. Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

Effects of Mpl ligand on platelet and megakaryocyte levels and structure in the BclxL transgenic and control mice.(A) Level of circulating platelets on day 5 after a single injection of PEG-rmMGDF (50 μg/kg) in the peripheral blood of WT and Tg19 mice. Effects of Mpl ligand on platelet and megakaryocyte levels and structure in the BclxL transgenic and control mice.(A) Level of circulating platelets on day 5 after a single injection of PEG-rmMGDF (50 μg/kg) in the peripheral blood of WT and Tg19 mice. Numbers are averages ± SD for 3 different mice. (B) Number of acetylcholinesterase-positive cells in mouse BM after injection of PEG-rmMGDF (50 μg/kg). The numbers are averages ± SD for 2 mice, each with 3 determinations. Details are as in the legend for Figure 3. (C) Transmission electron micrograph of megakaryocytes from WT and Tg19 mice after PEG-rmMGDF injection. Original magnification, × 16 000 in the top panel and × 6800 in the lower panel. Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology

The frequency of megakaryocytes capable of forming proplatelets is decreased in BM cultures derived from BclxL transgenic mice.BM cultures of Tg19 or WT mice were enriched for megakaryocytes by indirect immunomagnetic negative separation. The frequency of megakaryocytes capable of forming proplatelets is decreased in BM cultures derived from BclxL transgenic mice.BM cultures of Tg19 or WT mice were enriched for megakaryocytes by indirect immunomagnetic negative separation. Isolated megakaryocytes were cultured for 3 days as described in “Materials and methods” and subjected to examination by phase contrast microscopy (A-D). Note 3 different types of cytoplasmic extensions for WT megakaryocytes: beaded proplatelets (A), pseudopodialike (B), and long, thin extensions (C), whereas mainly one type was observed for Tg19—long, thin extensions (D). Original magnification, × 600 for panel A and × 400 for panels B, C, and D. (E).The frequency of megakaryocytes making proplatelets was monitored. The data are averages ± SD for 3 experiments, with 2 wells analyzed in each. Yulia Kaluzhny et al. Blood 2002;100:1670-1678 ©2002 by American Society of Hematology