Number Of Megakaryocytes In Bone Marrow Research Articles

Qing-Fei-Pai-Du Decoction ameliorated coagulopathy and thrombocytopenia in endotoxaemia rats and enhanced platelet production from megakaryocytes

BackgroundsThe Qing-Fei-Pai-Du Decoction (QFPDD) was reported to ameliorate the inflammatory coagulopathy and thrombocytopenia in COVID-19 patients. This work investigated the effects of QFPDD in endotoxaemia rats and then further studied its influence on platelet production using cultured megakaryocytes. MethodsThe coagulation indexes and platelet aggregation reactivity of LPS-induced endotoxemia rats with or without QFPDD treatment were analyzed. The platelet parameters, the expression of thrombopoietin (TPO) in the liver and the kidney, number of the megakaryocytes in bone marrow smear of rats were checked. The maturation and platelet production of human megakaryoblastic MEG-01 cells treated with QFPDD or isochlorogenic acid A (ISA), an active component of QFPDD, were observed. ResultsQFPDD improved the coagulation indexes of LPS-induced endotoxaemia rats and decreased the hyper-reactivity of platelets. QFPDD ameliorated LPS-induced decrease in platelet number, partially alleviated increase in liver TPO expression and exhibited no influence on increase in the number of bone marrow megakaryocytes. QFPDD promoted maturation and production of platelet-like particles of MEG-01 megakaryocytes. The effects of QFPDD on MEG-01 megakaryocytes might be related to activation of AKT pathway and up-regulation of related transcription factors. ISA also could induce the maturation of MEG-01 cells. ConclusionsQFPDD effectively ameliorated coagulopathy and thrombocytopenia in rats under inflammatory state. The mechanisms of QFPDD in ameliorating thrombocytopenia might be based on both decreasing effects on platelet depletion in coagulation and inducing effects on maturation and platelet production of megakaryocytes. ISA might be one of the components which contribute to the effects of QFPDD on megakaryocytes.

A Single Institutional Experience Using Avatrombopag to Manage Severe and Refractory Treatment Related Thrombocytopenia in Solid Tumor and Hematologic Malignancy Patients

Background:Cancer therapy induced thrombocytopenia (CTIT) is a common complication of systemic treatment for cancer, both in solid tumors and hematologic malignancies. Some patients had persistent severe thrombocytopenia that failed to respond to thrombopoietin receptor (TPO-R) agonists such as eltrombopag, hetrombopag or recombinant human thrombopoietin (rhTPO). Avatrombopag, a second generation TPO-R agonist, has been approved for the treatment of thrombocytopenia in patients with chronic liver disease scheduled to undergo invasive procedures and primary chronic immune thrombocytopenia (ITP). Recent studies testified the effect of avatrombopag in ITP patients not responding to romiplostim and/or eltrombopag. In this study, we share the experience of the off-label use of avatrombopag in the management of CTIT with grade 4 thrombocytopenia, and assessed the efficacy as well as safety of the drug. Methods: This study retrospectively evaluated patients with severe and refractoryCTIT treated at Qilu Hospital of Shandong University between 2021-2023 who received avatrombopag. Severe and refractoryCTIT met all of the following conditions: platelet count <25 × 10 9/L as grade 4 thrombocytopenia; previously unresponsive to rhTPO or TPO-R agonists other than avatrombopag. Patients' baseline characteristics were summarized in Table 1, including tumor types, dosing and duration of avatrombopag administration, and response at 4 weeks. The primary outcome was achievement of a response measured through (1) the proportion of patients with platelet count ≥50×10 9/L and increased by ≥ 100% above baseline at 4 weeks after medication and throughout the medication, (2) the time from avatrombopag initiation to first achievement of platelet count ≥50 × 10 9/L and increased by ≥ 100%, (3) the median maximum platelet count throughout avatrombopag administration. Safety was assessed by monitoring treatment emergent adverse events such as venous thromboembolism (VTE) development during treatment. Results: A total of 28 patients were included with mean age 59 years (range 27 to 78). The median duration of severe CTIT pre-avatrombopag was 4.5 weeks (range 2 to 96). The overall median time on avatrombopag support was 13 weeks (range 0.7 to 148). The median maximum dose was 50 mg QD (range 20 mg QD to 60 mg QD). Treatment with avatrombopag led to significantly higher median platelet count compared with baseline (137.5 × 10 9/L vs. 12 × 10 9/L; p < 0.0001) (Figure 1), and the response rate was 86%. The median time to response was 3.5 weeks (range 1 to 19). Of the total 24 solid tumor patients, 22 (92%) achieved a platelet response. Fifteen of the 28 patients (54%) met the response criteria at 4 weeks. With respect to disease subgroup, patients with breast cancer on avatrombopag had higher platelet count compared to ovarian cancer subgroup (p = 0.048), while the duration of severe CTIT pre-avatrombopag in the two groups was similar. At 4 weeks, the response group showed an increased tendency in the median number of bone marrow megakaryocytes in smear at baseline than the non-response group (9.5 vs. 2; p = 0.069). In assessing incidence of adverse events, 1 patient were noted to be on anticoagulation for a documented VTE at six days post-avatrombopag discontinuation. Conclusions: Avatrombopag was effective and safe in patients with severe and refractory CTIT. Future, prospective study is needed to investigate the efficacy of TPO-R agonists in patients with severe and refractory CTIT.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

Dual specificity tyrosine phosphorylation regulated kinase 1B inhibition promotes megakaryocyte polyploidization and platelet production.

Platelets are produced from mature megakaryocytes which undergo polyploidization and proplatelet formation. Cell cycle regulation plays a crucial role in megakaryocyte terminal differentiation especially in polyploidization. Dual specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) controls cell cycle progression in cancer cells. The objective of this study was to determine DYRK1B function in megakaryocyte maturation and platelet production. A DYRK1B knock out mouse (DYKO) was generated with increased peripheral platelet count compared to the wild type (WT) mouse without affecting megakaryocyte numbers in bone marrow. Polyploidy and proplatelet formation were significantly enhanced when DYRK1B was depleted in vitro. DYRK1B inhibition promoted megakaryocyte maturation by simultaneously upregulating cyclin D1 and downregulating P27. Furthermore, there was platelet restoration in two mice disease models of transient thrombocytopenia. In summary, DYRK1B plays an important role in megakaryocyte maturation and platelet production by interacting with cyclin D1 and P27. DYRK1B inhibition has potential therapeutic value in transient thrombocytopenia treatment.

Tanshinone IIA Has a Potential Therapeutic Effect on Kawasaki Disease and Suppresses Megakaryocytes in Rabbits With Immune Vasculitis.

Background and ObjectiveIt is urgent to find out an alternative therapy for Kawasaki disease (KD) since around 20% patients are resistant to intravenous immunoglobulin (IVIG) or aspirin. Tanshinone IIA is the active component of the traditional Chinese medicine Danshen (Salvia miltiorrhiza), which has anti-inflammatory and anti-platelet properties; however, whether or not tanshinone IIA has a therapeutic effect on KD remains unclear. Therefore, the present study aimed to examine the effect of tanshinone IIA on KD patients and rabbits with immune vasculitis, and to identify the potential mechanisms with special emphasis on megakaryopoiesis and megakaryocytic apoptosis.MethodsKawasaki disease patients were recruited and prescribed with tanshinone IIA in the absence or presence of aspirin and IVIG, and the inflammatory responses and platelet functions were determined. Megakaryocytes (MKs) isolated from rabbits with immune vasculitis and human megakaryocytic CHRF-288-11 cells were treated with tanshinone IIA to examine the colony forming unit (CFU) and apoptosis, respectively. Microarray assay was conducted to identify potential targets of tanshinone IIA-induced apoptosis.ResultsTanshinone IIA reduced the serum levels of C-reactive protein (CRP), interleukin (IL)-1β, IL-6, and P-selectin in KD patients; such inhibitory effect was more significant compared to aspirin and IVIG. It also dose-dependently lowered the levels of tumor necrosis factor (TNF)-α and IL-8 in peripheral blood mononuclear cells isolated from KD patients. In rabbits with immune vasculitis, tanshinone IIA significantly reduced the serum levels of proinflammatory cytokines and platelet functions. In addition, tanshinone IIA significantly decreased the number of bone marrow MKs and inhibited the Colony Forming Unit-Megakaryocyte (CFU-MK) formation. In human megakaryocytic CHRF-288-11 cells, tanshinone IIA induced caspase-dependent apoptosis, probably through up-regulating TNF receptor superfamily member 9 (TNFRSF9) and the receptor (TNFRSF)-interacting serine/threonine-protein kinase 1 (RIPK1), which may contribute to its anti-platelet and anti-inflammatory properties.ConclusionTanshinone IIA exerts better anti-inflammatory and anti-platelet effects in treating KD patients than aspirin and IVIG. It attenuates immune vasculitis likely by inhibiting IL-mediated megakaryopoiesis and inducing TNFRSF9/RIPK1/caspase-dependent megakaryocytic apoptosis. The findings therefore suggest that tanshinone IIA may be a promising alternative therapy for the treatment of KD.

Eltrombopag for Treatment of Thrombocytopenia Following Hematopoietic Stem Cell Transplantation

Objective:This study aimed to evaluate the efficacy and safety of eltrombopag (ELT) in the treatment of thrombocytopenia following hematopoietic stem cell transplantation (HSCT).Materials and Methods:Forty-eight patients treated with ELT for thrombocytopenia after allogeneic or autologous transplantation at the Erciyes University Bone Marrow Transplantation Center between July 2017 and July 2021 were evaluated retrospectively.Results:Forty-eight HSCT recipients were included in this study. Thirty (62.5%) patients were evaluated as having experienced delayed platelet recovery (DPR) and 18 (37.5%) patients as having experienced secondary failure of platelet recovery (SFPR). The median platelet count before ELT treatment was 13x109/L (range: 3-20x109/L). Twenty-three patients responded to treatment and the cumulative incidence of successful platelet recovery was 48%. Patients with both DPR and SFPR responded, but patients with DPR had a higher response rate (50% vs. 44%). The median platelet count of the 23 responding patients was 12x109/L (5-19x109/L) before treatment and 68x109/L (52-266x109/L) after treatment (p<0.0001). While the number of bone marrow megakaryocytes before treatment was adequate in 22 (46%) cases, it was decreased in 26 (54%) cases. Patients with adequate bone marrow megakaryocytes had a better response rate than those without (77% vs. 23%, p<0.0001). The group with adequate megakaryocytes responded to treatment at a median of 33 days (range: 9-174 days). Patients with decreased megakaryocytes responded at a median of 55 days (30-164 days) (p=0.002). No drug-related side effects were observed in any patients.Conclusion:This real-life experience demonstrates that ELT is an effective and safe treatment option for thrombocytopenia after HSCT. The adequacy of bone marrow megakaryocytes before ELT treatment was an important factor affecting response to treatment.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/μm), or high-LET 56Fe-ions (148 keV/μm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.

The α2-adrenergic receptor pathway modulating depression influences the risk of arterial thrombosis associated with BDNFVal66Met polymorphism

Depression is associated with thrombotic risk and arterial events, its proper management is strongly recommended in coronary artery disease (CAD) patients. We have previously shown that the Brain-Derived Neurotrophic Factor (BDNF)Val66Met polymorphism, related to depression, is associated with arterial thrombosis in mice, and with an increased risk of acute myocardial infarction in humans. Herein, expanding the previous findings on BDNFVal66Met polymorphism, we show that desipramine, a norepinephrine reuptake-inhibitor, rescues behavioral impairments, reduces the arterial thrombosis risk, abolishes pathological coagulation and platelet hyper-reactivity, normalizes leukocyte, platelet, and bone marrow megakaryocyte number and restores physiological norepinephrine levels in homozygous knock-in BDNF Val66Met (BDNFMet/Met) mice. The in vitro data confirm the enhanced procoagulant activity and the alpha2A-adrenergic receptor (α2A-ADR) overexpression found in BDNFMet/Met mice and we provide evidence that, in presence of Met variant, norepinephrine is crucial to up-regulate procoagulant activity and to enhance platelet generation. The α2-ADR antagonist rauwolscine rescues the prothrombotic phenotype in BDNFMet/Met mice and reduces procoagulant activity and platelet generation in cells transfected with BDNFMet plasmid or exposed to pro-BDNFMet peptide. Finally, we show that homozygous BDNFMet/Met CAD patients have hyper-reactive platelets overexpressing abundant α2A-ADR. The great proplatelet release from their megakaryocytes well reflects their higher circulating platelet number compared to BDNFVal/Val patients. These data reveal an unprecedented described role of Met allele in the dysregulation of norepinephrine/α2A-ADR pathway that may explain the predisposition to arterial thrombosis. Overall, the development of α2A-ADR inhibitors might represent a pharmacological treatment for depression-associated thrombotic conditions in this specific subgroup of CAD patients.

Immature platelet levels correlate with disease activity and predict treatment response of thrombocytopenia in lupus patients.

The immature platelet fraction (IPF) represents recently produced platelets in bone marrow and this parameter is increased in patient with primary immune thrombocytopenia (ITP). We investigated the associations between IPF and absolute immature platelet count (AIPC), and clinical parameters in systemic lupus erythematosus (SLE), which has more complex pathological mechanisms than in primary ITP. Patients with SLE were retrospectively reviewed at the University of Tokyo Hospital from May, 2012 to January, 2021. The correlations between clinical parameters and the number of immature platelets were assessed with Spearman's rank correlation coefficients. A multiple logistic regression model was used to identify the independent clinical parameters for IPF and AIPC. The difference in the distribution of time for a complete response (CR) after prednisolone (PSL) administration was also evaluated by log-rank test. A total of 282 SLE patients were enrolled, and 12.41% of those patients showed thrombocytopenia. IPF correlated with clinical parameters such as platelet count (r = -0.58), AIPC (r = 0.64) and systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) (r = 0.24). SLEDAI-2K [odds ratio (OR) (per unit increase), 1.07; 95% CI, 1.013 - 1.13] and thrombocytopenia (OR, 32.23; 95% CI, 11.072 - 93.80) were independent clinical parameters to account for IPF increase. IPF correlated with the number of bone marrow megakaryocytes (n = 19, r = 0.57). Notably, the probability of CR in response to PSL in AIPC-high patients was higher than in AIPC-low patients (hazard ratio, 4.62; 95% CI, 1.07 - 20.02). IPF correlated with disease activity of SLE and represented platelet production in the bone marrow, whereas AIPC predicted a rapid response to steroids in thrombocytopenic patients with SLE.

Usefulness of Thrombopoietin Receptor Agonists for Thrombocytopenia after Allogeneic Stem Cell Transplantation. An Eight-Year Single Center Experience

IntroductionProlonged isolated thrombocytopenia (PT) and secondary failure of platelet recovery (SFPR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are well known complications that could lead to a potentially lethal bleeding. To date, there are not specific guidelines for the treatment of post allo-HSCT thrombocytopenia, being platelet transfusion the mainstay of the management. Thrombopoietin receptor agonists (TRA), e.g. Eltrombopag (ETP) and Romiplostim have been approved for the treatment of primary immune thrombocytopenia and thrombocytopenia in other conditions (e.g. aplastic anemia) but information on their role in the allo-HSCT is scarce. The aims of our study were a) to analyze the effectiveness, in terms of platelet recovery and length of the response, and b) the safety profile of TRA for the treatment of thrombocytopenia after allo-HSCT in our Institution.MethodsWe performed a retrospective study on 467 patients who underwent to allo-HSCT from 2010 to 2017 in our center, of which a total of 17 patients were treated with TRA as compassionate use for thrombocytopenia after peripheral blood allo-HSCT. In order to be subsidiary to receive a TRA, patients had to be in complete remission with complete chimerism. Patients with thrombocytopenia related to cytomegalovirus and/or thrombotic microangiopathy were excluded. PT was defined as the engraftment of all peripheral blood cell lines but with a platelet (PLT) count <20,000/μL or dependence on PLT transfusions for more than 60 days after allo-HSCT. SFPR was defined as a decline in PLT count <20,000/μL for 7 consecutive days, or requirement of transfusion support after achievement of sustained PLT count ≥50,000/μL without transfusions for 7 consecutive days post-HSCT. The effectiveness or response consisted on achieving platelet recovery (≥20,000/μL) and transfusion independence of at least 7 days.ResultsPatient characteristics are detailed in table 1. One patient was excluded due to short course of treatment that was not evaluable. Platelet recovery and transfusion independence was achieved in 9 out of 16 patients (56%) with a median of 71 days (range: 8-176 days). Three of these responder-patients (R-patients) were switched to an alternative TRA in order to achieve response. Most of the R-patients had SFPR (n=7; 78%). Four patients (44%) were previously treated with mesenchymal stromal cells (MSC; n=2), immunoglobulins (n=3) and/or rituximab (n=1). The median of treatment duration was 181 days (21-350) and all R-patients (n=9) discontinued TRA. At the last follow-up, with a median of 282 days (67-1617), 7 out 9 patients (78%) maintained the response [P=152,000/μL (11,000-358,000)] and 6 (67%) are alive.Among NR-patients (n=7; 43.7%), the median duration of TRA treatment was 14 days (8-82) and 3 patients (43%) were previously treated (MSC and/or immunoglobulins). The main characteristics of NR-patients related to poor response were: active acute graft-versus host disease (GVHD) (n=2; p=0,038), active chronic GVHD (n=1; p=0,025), decreased number of bone marrow megakaryocytes (<++) (n=6; p>0,05), 5 (71%) required platelet transfusion frequency >2 times per week (p>0,05) and all NR-patients received packed red blood cell transfusions (p=0,09). In this group, at the last follow-up, with a median of 86 days (9-144), all except one patient died due to infection (n=5; 83%) or relapse disease (n=1). Finally, no adverse events were related to TRA.ConclusionsTreatment of thrombocytopenia after allo-HSCT with TRA is feasible. Further prospective studies are necessary to determine if these treatments may be considered as a potentially therapeutic alternative. [Display omitted] DisclosuresSanchez-Guijo:Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Effect of Indirubin and Sheng-Xue-Xiao-Ban Capsule (SXXBC) on Promoting Peripheral Platelet in ITP Model Mice

To compare the effect of Sheng-Xue-Xiao-Ban Capsule (SXXBC) and indirubin to the peripheral platelets of the Idiopathic thrombocytopenic purpura (ITP) model mouse. The ITP mouse model was established by the method of passive immunization. SXXBC and indirubin were used for intervention treatment. Then the hemorrhagic phenomena of ITP mice were observed and the numbers of peripheral platelets, hemoglobin and white blood cells, bone marrow megakaryocytes and their classification and coagulation function were detected and compared. The improvement rate of hemorrhage in SXXBC group was 40% for small dose, 60% for medium dose and 80% for high dose, while the improvement rate of hemorrhage in indirubin group was 30% for small dose, 50% for medium dose and 60% for high dose. There was no statistically significant difference in the improvement rate of hemorrhage between the two groups (P＞0.05). Compared with the model control group, PLT and Hb increased in different doses of SXXBC and indirubin group 4th-8th day after drug intervention (P＜0.05, 0.01). However, there was no significant difference between the different doses of SXXBC group and indirubin group (P＞0.05). Compared with the model control group, the WBC in each group was significantly lower (P＜0.05, 0.01) on the 4th-8th day after drug intervention; However, there was no statistical significance between the two groups of SXXBC and indirubin (P＞0.05). Compared with the model control group, the total number of megakaryocytes in each treatment group were decreased (P＜0.05, P＜0.01), in which the number of primary megakaryocytes in the large and medium dose groups of SXXBC and indirubin were decreased (P＜0.05, 0.01), and the number of juvenile megakaryocytes in the large dose group of SXXBC and indirubin were also decreased (P＜0.05). The number of granular megakaryocytes were decreased in each intervention groups (P＜0.05, 0.01), and the number of thromocytogenic megakaryocyte was increased in the high and medium dose groups of SXXBC and indirubin (P＜0.01). The time of prothrombin was shortened in the high and medium dose groups of SXXBC and indirubin (P＜0.05), and the fibrinogen (FIB) content in the high and medium dose groups of SXXBC was close to that of the normal control group. Both of the SXXBC and the indirubin standard all show good hemostatic effects. Indirubin shows a positive effect on increasing the peripheral platelet and hemoglobin in ITP model mice, regulating the immune response, reducing the total number of bone marrow megakaryocytes, increasing the thromocytogenic megakaryocyte, and increasing coagulation function.

Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked.

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss-of-function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b-B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b-/- ) mice. Although male and female Mpig6b-/- mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b-/- mice developed progressive splenomegaly starting at 8 weeks of age. Micro-computed tomography (μCT) of femurs showed that female Mpig6b-/- mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b-/- mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10-week-old female Mpig6b-/- mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b-/- mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b-/- mice also demonstrated improvement in spleen weight compared to sham-operated Mpig6b-/- mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte-driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Slc35a1 deficiency causes thrombocytopenia due to impaired megakaryocytopoiesis and excessive platelet clearance in the liver.

Sialic acid is a common terminal residue of glycans on proteins and acidic sphingolipids such as gangliosides and has important biological functions. The sialylation process is controlled by more than 20 different sialyltransferases, many of which exhibit overlapping functions. Thus, it is difficult to determine the overall biological function of sialylation by targeted deletion of individual sialyltransferases. To address this issue, we established a mouse line with the Slc35a1 gene flanked by loxP sites. Slc35a1 encodes the cytidine-5’-monophosphate (CMP)-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation. Here we report our study regarding the role of sialylation on megakaryocytes and platelets using a mouse line with significantly reduced sialylation in megakaryocytes and platelets (Plt Slc35a1– /–). The major phenotype of Plt Slc35a1–/– mice was thrombocytopenia. The number of bone marrow megakaryocytes in Plt Slc35a1–/– mice was reduced, and megakaryocyte maturation was also impaired. In addition, an increased number of desialylated platelets was cleared by Küpffer cells in the liver of Plt Slc35a1–/– mice. This study provides new insights into the role of sialylation in platelet homeostasis and the mechanisms of thrombocytopenia in diseases associated with platelet desialylation, such as immune thrombocytopenia and a rare congenital disorder of glycosylation (CDG), SLC35A1-CDG, which is caused by SLC35A1 mutations.

Development of Liver-Specific Thrombopoietin Targeted Sirnas: Impact on Platelet Count, Megakaryocyte Mass, and Hematopoietic Progenitors in Normal and MPN Murine Models

The Thrombopoietin (THPO)/ thrombopoietin receptor (MPL) signaling axis is not only critical for the generation of platelets and megakaryocytes, but also for the maintenance of hematopoietic stem cells (HSC) and the bone marrow niche. MPL is expressed on primitive HSC, HSC progenitors, megakaryocytes, platelets, osteoblasts and osteoclasts, clonal hematopoietic stem cells and many leukemias. THPO production is constitutive but is also increased by inflammatory cytokines. Sustained exposure to high levels of THPO not only enhances platelet production, but also has a profound effect on HSC and the bone marrow microenvironment. Excess THPO/MPL signaling, whether driven by inflammatory cytokines, or due to mutations in THPO, JAK2, MPL or CALR, is associated with HSC expansion, megakaryocyte hypertrophy and increased platelet count, excess release of megakaryocyte and platelet-based cytokines such as TGF-beta and PDGF-alpha, and the development of stromal myofibroblasts that drive tissue fibrosis and anemia.We developed a robust and clinically validated RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent N-acetylgalactosamine (GalNAc) conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous injection. Since liver is the major source of THPO expression, we utilized GalNAc-siRNA technology to develop siRNAs targeting THPO for evaluation in wild type mice and murine models of myeloproliferative neoplasms (MPN). Active siRNAs were identified by in vitro screening in primary mouse hepatocytes and the 12 best siRNAs were evaluated in vivo in normal mice to select the most potent siRNA. THPO liver mRNA levels were reduced by up to 80% after a single subcutaneous THPO siRNA dose, with no effect on THPO mRNA expression in other organs (kidney, and bone marrow, both of which had marginal THPO expression compared to liver). Circulating TPO levels were reduced by 80% by day 7 and were suppressed for up to 28 days post a single dose treatment. Platelet counts were reduced to 60% of baseline by day 14, and a further reduction to more than 70% of baseline was observed with every other week dosing. No changes in red blood cell or white blood cell subsets were observed. Platelet reduction was accompanied by a reduction in megakaryocyte mass, as evidenced by a 50% decrease in the number of bone marrow megakaryocytes in THPO siRNA treated mice compared to controls. Mice treated every other week with TPO siRNA for three months demonstrated sustained circulating THPO protein and platelet count reductions, and a significant reduction in bone marrow HSC, Lin-Sca1+Kit- (LSK) and multipotent progenitor (MPP) frequency. Evaluation of impact THPO silencing on MPN phenotypes in transgenic JAK2V617F mice is ongoing. THPO silencing is a potential novel targeted therapeutic approach that may be beneficial in benign and malignant conditions in which deregulated THPO/MPL signal transduction drives disease pathology. DisclosuresNo relevant conflicts of interest to declare.

Hypercholesterolemia impairs megakaryopoiesis and platelet production in scavenger receptor BI knockout mice

Background and aimsThrombocytopenia in scavenger receptor BI (SR-BI) knockout mice is suggested to result from augmented platelet clearance induced by elevated intracellular unesterified cholesterol (UC) levels. We hypothesize that SR-BI deficiency may also influence platelet production at the level of its precursor cell in the bone marrow, the megakaryocyte. MethodsIn this study, we compared megakaryopoiesis and platelet production in SR-BI knockout and wild-type mice. ResultsIn line with our hypothesis, megakaryocytes from SR-BI knockout mice exhibited UC accumulation while no accumulation of UC was detectable in wild-type megakaryocytes. Bone marrow expression of transcription factors involved in megakaryocyte maturation was induced, but megakaryocyte counts were unchanged in bone marrow of SR-BI knockout mice. Interestingly, we did find a striking 62% decrease (p < 0.01) in proplatelet production by SR-BI knockout megakaryocytes. SR-BI knockout mice displayed an impaired increase in circulating platelet concentrations and bone marrow megakaryocyte numbers upon thrombopoietin challenge. Importantly, megakaryocytes from normolipidemic bone marrow-specific SR-BI knockout mice exhibited a normal ability to produce proplatelets. Moreover, bone marrow-specific deletion of SR-BI did not impair the thrombopoietin response or induce thrombocytopenia, confirming that absence of megakaryocyte SR-BI does not underlie the thrombocytopenic phenotype in total body SR-BI knockout mice. ConclusionsIn conclusion, the elevation of plasma unesterified cholesterol levels impairs megakaryopoiesis and platelet production in SR-BI knockout mice. Our findings suggest that, in addition to an increased platelet clearance, a decrease in platelet production may also, in part, explain the thrombocytopenic phenotype associated with SR-BI deficiency in mice.

The high bone mass phenotype of Lrp5-mutant mice is not affected by megakaryocyte depletion

Bone remodeling is a continuously ongoing process mediated by bone-resorbing osteoclasts and bone-forming osteoblasts. One key regulator of bone formation is the putative Wnt co-receptor Lrp5, where activating mutations in the extracellular domain cause increased bone formation in mice and humans. We have previously reported that megakaryocyte numbers are increased the bone marrow of mice carrying a high bone mass mutation (HBM) of Lrp5 (Lrp5G170V). Since megakaryocytes can promote bone formation, we addressed the question, if the bone remodeling phenotype of Lrp5G170V mice is affected by megakaryocyte depletion. For that purpose we took advantage of a mouse model carrying a mutation of the Mpl gene, encoding the thrombopoietin receptor. These mice (Mplhlb219) were crossed with Lrp5G170V mice to generate animals carrying both mutations in a homozygous state. Using μCT, undecalcified histology and bone-specific histomorphometry of 12 weeks old littermates we observed that megakaryocyte number was remarkably decreased in Mplhlb219/Lrp5G170V mice, yet the high bone mass phenotype of Lrp5G170V mice was not significantly affected by the homozygous Mpl mutation. Finally, when we analyzed 24 weeks old wildtype and Mplhlb219 mice we did not observe a statistically significant alteration of bone remodeling in the latter ones. Taken together, our results demonstrate that an increased number of bone marrow megakaryocytes does not contribute to the increased bone formation caused by Lrp5 activation.

Abstract 29: Chronic Stress Predisposes to Thrombosis by Abnormal Megakaryopiesis: Protective Effect of Apocynin

Introduction: Psychological stress (e.g. anxiety and depression) has been identified as an important trigger of acute coronary syndromes (ACS), as a consequence of enhanced coagulation and of hyper-reactive platelets. Changes in redox balance, alteration of genes regulating antioxidant systems, including NADPH oxidase, and increased production of reactive oxygen species (ROS) have been measured in both chronic stress and ACS. However, the mechanisms by which chronic stress affects platelet activation and predisposes to thrombosis are not well known. Hypothesis: We hypothesized that Apocynin, an inhibitor of NADPH oxidase influences the alteration of megakaryopoiesis and activation of platelets induced by chronic stress in mice. Methods and Results: We show the NADPH/NADP + ratio in bone marrow (BM) of mice exposed to forced swimming for 4 days (5 min twice/day) is markedly reduced compared to control mice, and that Apocynin treatment (2.4 mg/ml in drinking water for 4 days) prevents this alteration. Chronic stress leads to an abnormal megakaryopoiesis increasing the number of BM megakaryocytes (MKs) and affecting circulating platelets. MKs of stressed mice show an advanced maturation state (e.g. nuclear/cytoplasmic ratios and expression of CD42d), and an enhanced ability to produce ROS. Interestingly, a higher number of large and reticulated platelets with marked functional activation (e.g. integrin α IIb β3 and P-selectin expression, and platelet/leukocyte aggregates) is detected after chronic stress. In addition, Apocynin prevents ROS MKs generation and decreases the total number of MKs without affecting the percentage of CD42d + cells. Finally, the inhibitor of NADPH oxidase activity reduces the hyper-activation of platelets and the enhanced susceptibility to FeCl3-induced arterial thrombosis in stressed mice. Conclusion: Apocynin treatment, reducing ROS generation in MKs, restores the physiological bone marrow megakaryopoiesis and platelet behaviour, and it prevents the detrimental effect of chronic stress on atherothrombosis. These data suggest a potential use of NADPH oxidase inhibitors in the occurrence of thrombosis associated with chronic stress. Studies in human will verify the clinical impact of these findings.

Neonatal mice with necrotizing enterocolitis-like injury develop thrombocytopenia despite increased megakaryopoiesis.

BackgroundThrombocytopenia is frequently encountered in infants with necrotizing enterocolitis (NEC). To develop a preclinical model of NEC-related thrombocytopenia, we measured serial platelet counts in 10-day-old (P10) mouse pups with trinitrobenzene sulfonic acid (TNBS)-induced NEC-like injury. We also measured platelet volume indices, immature platelet fraction (IPF), and megakaryocyte number/ploidy in these animals.MethodsPlatelet counts, platelet volume indices, and IPF were measured in control (N=65) and TNBS-treated pups (N=104) using an automated hematology analyzer. Bone marrow megakaryocyte number, ploidy and CD41 expression were measured by flow cytometry. These findings were confirmed in a small cohort of P3 mice with NEC-like injury.ResultsMurine pups with TNBS-mediated NEC-like injury developed thrombocytopenia at 15–24h after exposure to TNBS. Intestinal injury was associated with increased platelet volume indices (mean platelet volume, platelet-to-large cell ratio, and platelet distribution width), and IPF, indicating increased thrombopoiesis. These mice also showed increased megakaryocyte number, ploidy, and CD41 expression, indicating increased megakaryocyte differentiation.ConclusionsSimilar to human NEC, murine NEC-like injury was also associated with decreased platelet counts. There was evidence of increased megakaryocyte differentiation and thrombopoiesis, which favors peripheral consumption of platelets as the likely mechanism of thrombocytopenia in these animals, over decreased platelet production.

Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib

Mutations of calreticulin (CALR) are detected in 25–30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

Eltrombopag for Treatment of Thrombocytopenia after Allogeneic Hematopoietic Cell Transplantation

Persistent thrombocytopenia is a common complication after allogeneic hematopoietic cell transplantation (HCT). Eltrombopag is an oral thrombopoietin receptor agonist whose efficacy against persistent thrombocytopenia after allogeneic HCT has not been well characterized. This retrospective study evaluated the safety and efficacy of eltrombopag in 12 consecutive patients with persistent thrombocytopenia after allogeneic HCT. Eltrombopag was started at 12.5 mg once daily and the dose was increased by 12.5 mg daily every week until platelet counts exceeded 50,000/μL. Five patients had prolonged isolated thrombocytopenia (PIT) and 7 patients had secondary failure of platelet recovery (SFPR). The cumulative incidence rate of successful platelet recovery to ≥50,000/μL without transfusion support was 60% in PIT patients and 71% in SFPR patients. No patients discontinued the drug because of adverse events or intolerability. Notably, the rate of platelet recovery was higher (100% versus 58%; P = .0017) and recovery was faster (median, 33 days versus 137 days; P = .0078) in patients with normal numbers of bone marrow megakaryocytes before starting eltrombopag than in those with decreased numbers of megakaryocytes. Eltrombopag is a promising treatment for both PIT and SFPR after allogeneic HCT. The number of megakaryocytes in bone marrow before eltrombopag treatment may predict the response to eltrombopag.