Rapid In Vivo Consumption and Ex Vivo Phagocytosis of WASP(−) Platelets.

Abstract Patients with the Wiskott-Aldrich Syndrome (WAS) have thrombocytopenia and increased platelet consumption rates, and may have reduced platelet production rates. WASP(−) mice have been reported to have only a mild thrombocytopenia. We find that when fully crossed onto the C57Bl/6J background, WASP(−) mice have a >50% reduction in platelet counts. Ex vivo labeled WASP(−)platelets are consumed 2 to 4-fold faster than are WT platelets in WT mice, and with exponential rather than linear kinetics. Clearance rates of WASP(−) platelets in WT mice, and vice versa, indicate that rapid consumption is due to factors both intrinsic and extrinsic to platelets. In vivo biotinylation demonstrates consumption rates comparable to those seen with ex vivo labeling, and shows a normal rate of consumption of WASP(−) reticulated (immature) platelets. Reticulated platelet counts are reduced, indicating that their production rate is reduced. Megakaryocytes are increased in spleen and bone marrow, and in the latter their ploidy distribution is normal, suggesting that impaired platelet production occurs at the level of thrombopoiesis. The absolute turnover rates of mature and reticulated platelets, however, indicate that maturation of the latter can account for only a fraction of the former’s turnover in either WT or WASP(−) mice. A subset of WASP(−) mice show an increased fraction of reticulated platelets and more severe thrombocytopenia, and some members of this subset also express serum anti-platelet antibodies. CMFDA-labeled WASP(−) platelets opsonized with anti-CD61(IgG1) antibody are more susceptible to ex vivo phagocytosis by bone marrow derived macrophages (BMDM) than WT platelets, and as susceptible as CD47(−/−) platelets. After opsonization with 6A6(IgG2b) antibody, WASP(−) platelets are also taken up more rapidly than WT platelets by BMDM, but less rapidly than are CD47(−/−) platelets. The in vivo consumption rate of WASP(−) platelets in WT recipients is more accelerated by opsonization with anti-CD61 antibody than is that of WT platelets. Increased phagocytosis is not due to (A) altered levels of the targeted antigens or of CD47 (B) increased exposure of phosphatidyl serine, or (C) antibody-induced activation as assayed by CD62P expression. Increased phagocytosis is not due to selective sensitivity to IgG1 vs IgG2b antibodies, as opsonization with an engineered 6A6(IgG1) antibody leads to reduced phagocytosis for both CD47(−/−) and WASP(−) platelets. Anti-CD61 opsonized platelets deficient in both WASP and CD47 show markedly increased ex vivo phagocytosis compared to platelets deficient in either protein, suggesting that platelet WASP does not function to amplify signals from platelet CD47 through macrophage SIRP-alpha. These results raise the possibility that the binding of low affinity or low titer antibodies that might have no effect on WT platelets could cause thrombocytopenia when the platelets lack WASP. Alternatively, rapid phagocytosis of opsonized WASP(−) platelets could promote a self-reinforcing cycle of increased host antigen presentation and increased immune responses to host antigens.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Blood ◽

10.1182/blood.v126.23.420.420 ◽

2015 ◽

Vol 126 (23) ◽

pp. 420-420

Author(s):

Melissa M. Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley Elizabeth Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Severe Thrombocytopenia ◽

Extrinsic Factors ◽

Platelet Production ◽

Platelet Counts

Abstract Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.

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High-Vorticity with Periodic Flow Enhances in Vitro Biogenesis of Healthy Platelets from iPSC-Derived-Megakaryocytes

Blood ◽

10.1182/blood.v128.22.2181.2181 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2181-2181

Author(s):

Yukitaka Ito ◽

Sou Nakamura ◽

Tomohiro Shigemori ◽

Naoshi Sugimoto ◽

Yoshikazu Kato ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Ex Vivo ◽

Annexin V ◽

Poor Quality ◽

Flow Chamber ◽

Physical Parameters ◽

Platelet Production

Abstract Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (<10-15%) and shortened bleeding time post transfusion into NOG mice and rabbits with thrombocytopenia, comparable to human blood product platelets. Regarding the platelet production using WAVE bag system (GE healthcare, UK), the system is already clinically available for cord blood cell expansion in most countries, but lacks adequate levels of vorticity and shear strain/stress. Accordingly, the produced platelets had high Annexin V levels (i.e., 50-65%) as well as diminished yield efficiency (P<0.001). In conclusion, our study has uncovered the novel biophysical aspect of platelet biogenesis. The application of the new set of physical parameters in constructing large sized bioreactors shall facilitate the industrialization of platelet production. Disclosures Eto: Megakaryon Co. Ltd.: Research Funding.

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Bortezomib Induced Thrombocytopenia Might Be Due to the Inhibition of Proplatelet Formation of Megakaryocyte.

Blood ◽

10.1182/blood.v116.21.3696.3696 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3696-3696

Author(s):

Kazunori Murai ◽

Shugo Kowata ◽

Akiko Abo ◽

Tatsuo Oyake ◽

Kenichi Nomura ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Studies ◽

Bone Marrow Cells ◽

Platelet Production ◽

Platelet Counts ◽

Proplatelet Formation ◽

Marrow Cells

Abstract Abstract 3696 Background: Bortezomib is potent and reversible proteasome inhibitor that has been extensively used for multiple myeloma. Several clinical studies demonstrated that overall response rates using bortezomib alone to relapsed or refractory patients with multiple myeloma were 33 to 50%. The most common grade 3 adverse event was a cyclic thrombocytopenia, which was reported in 20–30% of patients in several clinical studies. The mechanism by which bortezomib causes thrombocytopenia remains unknown. In this study, we evaluated the effect of bortezomib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method: All animal procedures were approved by the Institutional Animal Care and Use Committee in Iwate Medical University. Male ddY at 8 weeks of age mice were used in all experiments. In vivo experiments: (a) The mice received 2.5 mg/kg bortezomib via tail-vein injection. Blood was obtained and the following experiments were carried out at day 2, 4, 6, 8, 10 after intravenous injection (n=9, each group). Complete blood counts were measured. Reticulated platelet (RP) was analyzed by flow cytometry using thiazole orange (TO) to evaluate platelet kinetics. Plasma TPO level were measured by ELISA. Bone marrow megakaryocyte's number and morphology from femur in bortezomib- and control-treated mice were observed by microscopy. Femur was fixed in 10% buffered formalin, decalcificated, embedded in paraffin and stained for Hematoxylin-Eosin (H-E). (b) Bortezomib (2.5 mg/kg) was administrated via tail-vein to mice. After 24hr, bone marrow cells were cultured in MegaCult®-C at 5% CO2 and 20% O2for 7 days. The megakaryocytic colonies (CFU-Megs-in vivo) were counted. In vitro experiments: (c) Bone marrow cells, obtained from non-treated mice, were cultured at 37°C in 5% CO2and 20% O2 for 7 days with bortezomib (0.01, 0.1, 1, 10, 100 ng/ml). CFU-Megs were counted (CFU-Megs-in vitro). (d) Proplatelet formation: Murine megakarocytes were partially purified from bone marrow using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte)well) and cultured in IMDM in duplicates, supplemented with 1 × ITS-G (Life technologies) and each concentration of bortezomib (0.01, 0.1, 1, 10, 100 ng/ml), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Results: (a) Control mice did not have any significant change in platelet counts, % reticulated platelets and plasma TPO levels at days 0, 2, 4, 6, 8, 10. While, bortezomib treated mice (2.5mg/kg) had a significant reduction in platelet counts at day 2 (470 ± 210 × 109/L. P<0.001), at day 4 (667 ± 118 × 109/L, P<0.001). The platelet counts returned to normal value at day 6 (903 ± 548 × 109/L) and day 10 (1122 ± 187 × 109/L). RP (%) began to increase at day 6 (8.8 ± 4.0 %). Plasma TPO levels tend to increase at day 4. Means megakaryocytes's number in one field of femur was similar in between bortezomib non-treated and –treated mice. The megakaryocytes were similar in morphology at each day, too. (b) CFU-Megs-in vivo were similar in number between bortezomib non-treated and –treated mice (38.0 ± 6.1 vs 34.5 ± 5.6 per 1 × 105 bone marrow cells respectively). (c) CFU-Megs-in vitro were not decreased significantly at 0.001 to 1 ng/ml and decreased significantly (p<0.01) at 10 and 100 ng/ml of bortezomib. (d) Proplatelet formation (PPF) were decreased significantly at 0.01, 0.1, 1, 10, 100 ng/ml bortezomib (0 mg)ml: 25.2 ± 4.8%, 0.01ng/ml: 23.8 ± 4.9%, 0.1 ng/ml: 18.4 ± 3.1% p<0.01, 1 ng/ml: 13.2 ± 3.8% p<0.001, 10 ng/ml: 13.3 ± 2.1% p<0.001, 100ng/ml: 5.9 ± 1.4 % p<0.001). Discussion & Conclusion: Bortezomib did not adversely affect on megakaryocytic prognitors nor megakaryocytes. It did inhibit PPF, that is, the step of platelet production, even when bortezomib plasma concentration levels have gone down. Plasma TPO level showed an inverse relationship against circulating platelet counts. Based on the evidence in which Cmax of plasma bortezomib concentration was under 100 ng/ml in bortezomib-injected mice (2.5mg/kg), bortezomib induced thrombocytopenia might be due to the inhibition of proplatelet formation of megakaryocyte. Disclosures: No relevant conflicts of interest to declare.

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Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish

Blood Advances ◽

10.1182/bloodadvances.2018021865 ◽

2018 ◽

Vol 2 (23) ◽

pp. 3418-3427 ◽

Cited By ~ 5

Author(s):

Megan S. Rost ◽

Ilya Shestopalov ◽

Yang Liu ◽

Andy H. Vo ◽

Catherine E. Richter ◽

...

Keyword(s):

Ex Vivo ◽

Severe Thrombocytopenia ◽

Wild Type ◽

Transcription Activator ◽

Platelet Production ◽

Adult Kidney ◽

Depth Study ◽

And Function

AbstractThe NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in nfe2, we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and nfe2 null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in nfe2 mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.

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T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes

Blood ◽

10.1182/blood.v124.21.94.94 ◽

2014 ◽

Vol 124 (21) ◽

pp. 94-94

Author(s):

Renata Grozovsky ◽

Silvia Giannini ◽

Haley Ramsey ◽

Martha Sola-Visner ◽

Karin M Hoffmeister

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Platelet Production ◽

Platelet Counts ◽

Cell Counts

Abstract Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis. Disclosures No relevant conflicts of interest to declare.

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Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Blood ◽

10.1182/blood-2019-128133 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2378-2378 ◽

Cited By ~ 1

Author(s):

Gulab Fatima Rani ◽

Olivier Preham ◽

Ian Hitchcock ◽

Paul Kaye

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Visceral Leishmaniasis ◽

Conflicts Of Interest ◽

Experimental Models ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Production ◽

Platelet Counts ◽

Splenic Macrophages

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Translational approaches to functional platelet production ex vivo

Thrombosis and Haemostasis ◽

10.1160/th15-07-0570 ◽

2016 ◽

Vol 115 (02) ◽

pp. 250-256 ◽

Cited By ~ 12

Author(s):

Christian A. Di Buduo ◽

Alessandra Balduini ◽

David L. Kaplan

Keyword(s):

Bone Marrow ◽

Developmental Process ◽

Ex Vivo ◽

Platelet Production ◽

Life Threatening ◽

Human Use ◽

And Function ◽

Tissue Models ◽

Insight Into

SummaryPlatelets, which are released by megakaryocytes, play key roles in haemostasis, angiogenesis, immunity, tissue regeneration and wound healing. The scarcity of clinical cures for life threatening platelet diseases is in a large part due to limited insight into the mechanisms that control the developmental process of megakaryocytes and the mechanisms that govern the production of platelets within the bone marrow. To overcome these limitations, functional human tissue models have been developed and studied to extrapolate ex vivo outcomes for new insight on bone marrow functions in vivo. There are many challenges that these models must overcome, from faithfully mimicking the physiological composition and functions of bone marrow, to the collection of the platelets generated and validation of their viability and function for human use. The overall goal is to identify innovative instruments to study mechanisms of platelet release, diseases related to platelet production and new therapeutic targets starting from human progenitor cells.

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Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production

Blood ◽

10.1182/blood.v97.6.1635 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1635-1643 ◽

Cited By ~ 31

Author(s):

Lia E. Perez ◽

Henry M. Rinder ◽

Chao Wang ◽

Jayne B. Tracey ◽

Noel Maun ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Ex Vivo ◽

Human Platelets ◽

Scid Mice ◽

In Vivo Model ◽

Specific Flow ◽

Platelet Production

The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units–megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 × 109/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.

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Immature platelet values indicate impaired megakaryopoietic activity in neonatal early-onset thrombocytopenia

Thrombosis and Haemostasis ◽

10.1160/th09-03-0148 ◽

2010 ◽

Vol 103 (05) ◽

pp. 1016-1021 ◽

Cited By ~ 27

Author(s):

Hannes Hammer ◽

Christoph Bührer ◽

Christof Dame ◽

Malte Cremer ◽

Andreas Weimann

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Intensive Care ◽

Early Onset ◽

Absolute Number ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Immature Platelet Fraction ◽

Neonatal Thrombocytopenia ◽

Immature Platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

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