Enhance Platelet Production Research Articles

LMAN1 serves as a cargo receptor for thrombopoietin.

Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MK) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. LMAN1 and MCFD2 form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion. In this study, we showed that LMAN1 deficient mice (with complete germline LMAN1 deficiency) exhibited mild thrombocytopenia, whereas the platelet count was entirely normal in mice with approximately 7% Lman1 expression. Surprisingly, mice deleted for Mcfd2 did not exhibit thrombocytopenia. Analysis of peripheral blood from LMAN1 deficient mice demonstrated normal platelet size and normal morphology of dense and alpha granules. LMAN1 deficient mice exhibited a trend toward reduced MK and MK progenitors in the bone marrow. We next showed that hepatocyte-specific but not hematopoietic Lman1 deletion results in thrombocytopenia, with plasma TPO level reduced in LMAN1 deficient mice, despite normal Tpo mRNA levels in LMAN1 deficient livers. TPO and LMAN1 interacted by co-immunoprecipitation in a heterologous cell line and TPO accumulated intracellularly in LMAN1 deleted cells. Altogether, these studies confirmed the hepatocyte as the cell of origin for TPO production in vivo and were consistent with LMAN1 as the endoplasmic reticulum cargo receptor that mediates the efficient secretion of TPO. To our knowledge, TPO is the first example of an LMAN1-dependent cargo that is independent of MCFD2.

Compound K Promotes Megakaryocytic Differentiation by NLRP3 Inflammasome Activation.

Platelets are essential blood components that maintain hemostasis, prevent excessive bleeding, and facilitate wound healing. Reduced platelet counts are implicated in various diseases, including leukemia, hepatitis, cancer, and Alzheimer's disease. Enhancing megakaryocytic differentiation is a promising strategy to increase platelet production. Compound K (CK), a major bioactive metabolite of ginsenosides from Panax ginseng, has demonstrated anti-cancer and neuroprotective properties. In this study, we investigated the effects of CK on megakaryocytic differentiation and apoptosis in chronic myeloid leukemia (CML) cell lines K562 and Meg-01. CK treatment significantly upregulated the mRNA expression of key megakaryocytic differentiation markers, including CD61, CD41, and CD42a, and promoted the formation of large, multinucleated cells in K562 cells. Additionally, flow cytometry analysis revealed that CK at 5 µM induced apoptosis, a critical process in thrombocytopoiesis, in both K562 and Meg-01 cells. RT2 Profiler PCR array analysis further identified a marked increase in the expression of genes associated with the activation of the NLRP3 inflammasome in CK-treated K562 and Meg-01 cells. This study is the first to demonstrate that CK promotes megakaryocytic differentiation and apoptosis through the activation of the ERK/EGR1 and NLRP3 inflammasome pathways. These findings suggest that CK may enhance platelet production, indicating its potential as a therapeutic candidate for platelet-related disorders and other associated diseases.

Thrombopoietin Receptor Agonists in Post-Hematopoietic Cell Transplantation Complicated by Prolonged Thrombocytopenia: A Comprehensive Review.

Hematopoietic cell transplantation (HCT) is a well-established procedure that has become a therapeutic mainstay for various hematological conditions. Prolonged thrombocytopenia following HCT is associated with a significant risk of morbidity and mortality, yet no universally recognized treatment protocol exists for such a complication. First-generation thrombopoietin receptor (TpoR) agonists as well as second-generation agents are known for their role in enhancing platelet production, and their use is expanding across various thrombocytopenic conditions. Therefore, we conducted this comprehensive review of the literature to provide an updated evaluation of the use of TpoR agonists and explore their efficacy and safety in the treatment of extended post-HCT thrombocytopenia. The literature search was conducted using PubMed database from 1996 through December 2023, using a predefined strategy with medical subject headings terms. We identified 64 reports on the utility of TpoR agonists, five of them were randomized controlled trials and the rest were retrospective observational studies and case series, with a total number of 1730 patients. Second-generation TpoR agonists appear more convenient than subcutaneous recombinant human thrombopoietin (rhTpo) as they can be orally administered and exhibit similar efficacy in platelet recovery, as indicated by recent trial results. Among these agents, avatrombopag, unlike eltrombopag, does not require any dietary restrictions, which could be more favorable for patients. However, eltrombopag remains the most extensively studied agent. TpoR agonists had promising effects in the treatment of post-HCT thrombocytopenia with a good safety profile so far, highlighting the potential benefit of their use.

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.

Effectiveness and Safety of Hetrombopag in the Management of Radiotherapy-Induced Thrombocytopenia in Patients with Gynecological Malignancies

Background: Radiotherapy is a cornerstone of treatment strategies for gynecological malignancies, However, while effective against neoplastic cells, pelvic radiotherapy frequently incurs collateral damage to normal tissues, especially to the bone marrow. This unintended consequence often precipitates thrombocytopenia, a condition characterized by diminished platelet levels, thereby elevating the risk of bleeding and posing hurdles for the continuity of anticancer regimens. Addressing this clinical challenge, hetrombopag, an innovative, orally administered non-peptide thrombopoietin receptor agonist, presents promise. It works by kindling megakaryocyte proliferation and differentiation, thereby enhancing platelet production via the activation of thrombopoietin receptor dependent STAT and MAPK signaling pathways. Previous studies have documented its efficacy in expediting platelet recovery in patients with chemotherapy-induced thrombocytopenia, thereby curtailing the duration of the condition and ensuring the execution of planned chemotherapy cycles. This study aims to investigate the therapeutic effectiveness and safety profile of hetrombopag in the treatment of radiotherapy-induced thrombocytopenia within a real-world context. Methods: This retrospective study encompassed gynecological cancer patients who developed thrombocytopenia (platelet counts<100×10 9/L) following radiotherapy and subsequently underwent treatment with hetrombopag from January 2022 to June 2023. The key inclusion criteria included an age of ≥18 years, the development of thrombocytopenia after radiotherapy or a combination of radiotherapy and other anticancer interventions, the administration of hetrombopag as monotherapy or as part of a combined regimen with other platelet-boosting agents, and the availability of follow-up data. The primary efficacy outcome was the proportion of patients exhibiting a platelet response (an elevation in platelet counts to ≥75×10 9/L) within 14 days. Results: Our cohort consisted of 28 gynecological cancer patients, encompassing 21 (75.0%) cases of cervical cancer, 3 (10.7%) of endometrial cancer, 1 (3.6%) of ovarian cancer, and 3 (10.7%) of other gynecological malignancies. The patients' median age was 58 years (range: 29-76 years), with 50% in clinical stage III/IV. Among them, 19 had received radiotherapy, 8 had undergone concurrent chemoradiotherapy, and 1 had received radiotherapy plus targeted therapy and immunotherapy. The baseline platelet count prior to hetrombopag treatment was 54.1±16.5×10 9/L. Hetrombopag was administered at a dose of 2.5mg/day as monotherapy in 32.1% of patients and combined with recombinant human thrombopoietin or recombinant human interleukin 11 in 67.9% of patients. The observed platelet response rate was 53.6% within 7 days and increased to 75.0% by the 14-day mark. The median time to platelet response was 7 days (95% confidence interval: 5-14). Following the introduction of hetrombopag as monotherapy or part of a combination regimen, we noted a steady uptick in patients' platelet counts. The mean platelet counts on days 3, 5, 7, 9, 11, and 14 were 62.3, 74.9, 78.7, 88.3, 87.9, and 147.9 ×10 9/L, respectively (Figure). Throughout the treatment period, no adverse events related to hetrombopag were reported. Conclusion: In conclusion, our findings provide preliminary evidence for the effectiveness and safety of hetrombopag, administered either as monotherapy or in combination, for managing radiotherapy-induced thrombocytopenia. Given the retrospective design of this study, further corroboration is needed through large-scale, prospective, randomized, and controlled clinical trials.

Association of Platelet Thromboxane Inhibition by Low-Dose Aspirin With Platelet Count and Cytoreductive Therapy in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once‐daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once‐ vs. twice‐ or three‐times daily low‐dose aspirin in inhibiting platelet thromboxane (TX) A2 production, as reflected by serum (s) TXB2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once‐daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P < 0.01, n = 84), but not in patients on cytoreduction. Patients in the lowest sTXB2 quartile were older, more often on cytoreductive drugs, had lower platelet count and Janus‐Associated Kinase2 (JAK2)‐V617F allele frequency as compared with patients in the upper sTXB2 quartiles. After 2 weeks of a twice‐ or 3‐times daily aspirin regimen, the association between the platelet count and sTXB2 became similar in cytoreduced and non‐cytoreduced patients. In conclusion, the platelet count appears the strongest determinant of TXA2 inhibition by once‐daily low‐dose aspirin in ET, with different patterns depending of cytoreductive treatment. More frequent aspirin dosing restores adequate platelet inhibition and reduces interindividual variability, independently of cytoreduction.

Recent lessons learned for ex-vivo platelet production.

Platelet transfusion can be life-saving but carries a risk of infection or alloimmunization and is limited by insufficient donor sources and restricted unit shelf life. Generating sufficient platelets in vitro to replace a unit of collected blood remains a challenge. Here, we examine the latest advances in the regulation of megakaryocyte maturation and expansion along with platelet formation and survival. We also discuss alternative therapies investigated to induce platelet production. Recent studies examined candidate niche cells in the bone marrow microenvironment for promoting platelet formation and developed an explant-based bioreactor to enhance platelet production ex vivo. Chemical inhibitors were examined for their ability to promote megakaryocyte maturation and expansion. Microparticles from megakaryocytes or platelets were found to improve megakaryocyte maturation and platelet formation. Membrane budding was identified as a novel mode of platelet formation. Lastly, a chemical inhibitor to improve cold-stored platelets was identified. Recent advances in the regulation of megakaryocyte expansion and platelet production provide exciting promise for the development of improved approaches to generate platelets in vitro. These findings bring the field one step closer to achieving the ultimate goal of creating a unit of platelets without the need for donation.

Refractory Coronary Stent Thrombosis in a Patient with Chronic Immune Thrombocytopenia on Eltrombopag.

Immune thrombocytopenia (ITP) is a rare autoimmune disorder, caused by immunologic destruction and insufficient compensatory production of platelets. The management and treatment of ITP depends on estimated risks including platelet count, symptomatic severity, and duration of disease.1 One of the more recent second-line therapies for enhancing platelet production in patients with ITP is the use of thrombopoietin receptor agonist (TPO-RA), such as eltrombopag.1 However, some randomized controlled trials have reported severe adverse effects of arterial and venous thromboembolic events from using TPO-RA, but the pathophysiology and risk factors have not been well elucidated.2 Regarding thromboembolic events, a clinical dilemma arises when a patient with acute coronary syndrome (ACS) presents with ITP from TPO-RA treatment because a balance must be maintained between bleeding events and thromboembolism. Here, we report a case of recurrent fatal coronary thrombosis in a middle-aged woman with chronic ITP on eltrombopag.

Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Prolonged Isolated Thrombocytopenia Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By N-Acetyl-L-Cysteine

BackgroundProlonged isolated thrombocytopenia (PT) is a frequent complication with low platelet count or dependence on platelet transfusions for more than 60 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PT increased risk of bleeding and had poor overall survival. However, the exact mechanisms underlying PT remain unclear. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) not only support hematopoietic stem cells (HSCs) but also play an important role in differentiation and proliferation of megakaryocytes (MKs), thereby enhancing platelet production in BM microenvironment. In addition, our previous study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PT after allo-HSCT. However, little is known about the functional role of BM EPCs and BM HSCs in patients with PT after allo-HSCT. In addition, in vivo and in vitro studies reported that the anti-oxidant -N-acetyl-L-cysteine (NAC) repaired the endothelial functions and enhanced engraftment of HSCs through prevention of exogenous ROS accumulation.Nevertheless, whether in vitro treatment of NAC is able to improve the functions of BM EPCs from patients with PT has never been investigated.AimsTo evaluate the function of BM EPCs and HSCs in patients with PT. Moreover, to investigate the effect of NAC on the number and function of the cultivated BM EPCs in patients with PT and its underlying molecular mechanisms.MethodsThree cohorts were included: patients with PT (N=25), patients with good graft function (N=25), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=25). BM mononuclear cells were cultivated in fibronectin pre-coated plates with EGM-2-MV-SingleQuots and 10% fetal bovine serum at 37°C for 7 days and characterized as EPCs by CD34(+)/CD133(+)/KDR(+) using flow cytometry. The number and functions of EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining,migration, tube formation test and apoptosis. ROS level was evaluated by flow cytometry. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. 1mM NAC was administrated to cultivated BM EPCs from patients with PT 24 hours before tested on day 7. EPCs-MKs co-culture system and Colony-forming unit (CFU)-MKs assays were used to evaluate the supporting effect that NAC exposure on MKs in vitro.ResultsHuman BM EPCs was characterized by the spindle shape and expression of CD34, CD309 and CD133 at day 7 of cultivation. Dysfunctional BM EPCs, which were characterized by impaired migration, angiogenesis, and higher levels of reactive oxygen species(ROS) and apoptosis, were revealed in patients with PT. Activation of p38 was detected in BM EPCs from patients with PT. Furthermore, there was no significantly difference in functions of CD34+ cells between patients with PT and GGF, except higher level of ROS and lower number of CFU-GEMM, suggesting an impaired process of megakaryocytopoiesis in patients with PT. Furthermore, the number and function of BM EPCs derived from patients with PT were enhanced by NAC treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The platelet number and percentage and functions of MKs characterized by apoptosis, ploidy and CFU-MKs were further improved by NAC in BM MKs and EPCs co-culture system from patients with PT. NAC demonstrated similar improvement effect on the number and function of BM EPCs as p38 inhibitor from patients with PT.ConclusionThe dysfunction of BM EPCs in patients with PT was improved by in vitro treatment of NAC through down-regulation of the p38 MAPK pathway, which further augment megakaryocytopoiesis and platelet production in patients with PT. These data suggest that NAC may serve as an effective therapeutic option to improve platelet production through repair of BM EPCs in patients with PT. Further prospective clinical trials will be conducted in the future. DisclosuresNo relevant conflicts of interest to declare.

Hematologic Response to Romiplostim Treatment Is Associated with Stimulation of Primitive Stem/Progenitor Cells and Stromal Cells in Patients with Aplastic Anemia Refractory to Immunosuppressive Therapy: A 2-Year Interim Exploratory Analysis of a Phase 2 Clinical Trial

Introduction:Romiplostim (RMP) is a thrombopoietin-mimetic protein that enhances platelet production. RMP has already been approved in numerous countries for treatment of immune thrombocytopenia. We previously reported the efficacy and safety of RMP through a 1-year treatment period in patients with aplastic anemia (AA) that was refractory to immunosuppressive therapy (IST). In this study, we report the interim exploratory analysis of the influence of RMP after a 2-year treatment period (105 weeks) on the hematopoietic stem cells (HSCs) and progenitor cells, and on mesenchymal stromal cells, a specialized HSC niche, in patients with AA who were refractory to IST.Methods:This study is a multicenter, randomized, open-label, parallel, comparative, dose-finding study in South Korea (NCT02094417). Eligible patients with AA, refractory to prior IST, were randomized into 4 dose groups that received 1, 3, 6, or 10 μg/kg of RMP for the initial 8-week treatment. After the initial treatment, the dose was adjusted from 1 to 20 μg/kg to maintain their platelet response. After week 53, if the blood cell count improved and was maintained at a predefined range, the dose was tapered and the patient was considered to be in a “tapering-dose” status; if the dose was decreased to 0 µg/kg at this point, the patient was considered to be off the drug. The range was defined as follows: platelet count, > 50x109/L; hemoglobin concentration, > 10.0 g/dL; and neutrophil count, > 1.0x109/L. The blood cell response was defined based on the absolute cell count. To examine the association between cellular changes in the bone marrow in response to RMP and the clinical course of the patients with AA, the primitive HSC and progenitor cell subsets were analyzed by flow cytometry and the colony formation assay at the baseline and every 6 months after initiation of RMP. Further, changes in the mesenchymal stromal cells within the bone marrow were also analyzed by the CFU-F assay.Results:A total of 35 subjects were randomized; of these, 18 received RMP beyond 1 year, and 10 are still receiving it over 2 years.At week 105, the platelet response was observed in all 10 subjects, erythroid response in 9 subjects, neutrophil response in 5 subjects, and tri-lineage response in 5 subjects.Of the ongoing 10 subjects, 7 have achieved “tapering-dose” status, and 2 have achieved “off-the-drug” status. In addition, The RMP was suspended in 1 subject due to excessive increase in platelet-count (>400x109/L). This subject has also been exhibiting the tri-lineage response.At week 53, the median counts of all colony assays were higher in subjects with any blood cell response than in those without the response. When we compared the serial colony assays in the subjects with any blood cell response at week 53 up to week 105, the colony counts of BFU-E, CFU-E, CFU-M, and CFU-GM gradually increased until week 77 from the baseline, and then declined at week 105. Further, the colony counts of CFU-F also exhibited a similar pattern. The colony counts of CFU-G and CFU-MK increased until week 53, and then decreased. However, the CD34+CD38- cells steadily increased throughout up to week 105 from the baseline.Conclusion:These data indicate that RMP influences the stimulation of primitive HSC (CD34+CD38- cells) and bone marrow microenvironment (HSC niche), and enhances the differentiation of primitive HSCs to late progenitor cells resulting in hematologic response in the patients with AA.Although further follow up is ongoing the up to the end of 3-year study period, but this interim analysis may strongly implies that the RMP might be beneficial for patients with AA, refractory to IST. DisclosuresNo relevant conflicts of interest to declare.

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

AbstractAlthough cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Novel physico-chemical regulation mechanism enables the production of 100 billion platelets

Since induced pluripotent stem (iPS) cell-derived blood products can be produced from any individual, they are expected to complement current transfusion products. However, a main problem is how to produce 10 U platelet preparations. Therefore, we established an immortalized megakaryocyte cell line (imMKCL) from iPS cells. We also found that turbulent flow was an essential physical factor for platelet generation in vivo. This knowledge enabled us to obtain 100 billion functional platelets from imMKCL using an 8 L bioreactor. We propose that the enhanced platelet production in the bioreactor occurs due to the turbulent flow that promoted the release of stress-induced cytokines.

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.

CCL5 Enhances Megakaryocyte Differentiation and Development

The importance of platelets in clot formation is well understood, but the role of platelets in the inflammatory response is becoming increasingly apparent. Platelet count increases during acute inflammatory stress; platelets also facilitate inflammation by releasing chemokines and cytokines from their granules. Our lab has previously shown that platelet derived chemokine ligand 5 (CCL5, RANTES), an inflammatory chemokine that is stored in platelets and released upon activation, acts to increase megakaryocyte maturation and proplatelet production in vitro. Furthermore, our lab found that CCL5 treated megakaryocytes have enhanced phosphorylation of proteins in the AKT pathway, such as BAD, which when phosphorylated induces pro-survival signaling. This indicates that CCL5-induced increase of platelet production is mediated by suppression of apoptosis, but the exact mechanism of CCL5 on megakaryocytes is not understood.To more completely asses the global impact of CCL5 on megakaryocytes, we performed proteomic analysis of CCL5-treated megakaryocytes and found that, as expected, CCL5 increases the expression of several pro-survival proteins in the AKT pathway. We have now confirmed increased phosphorylation of BAD by western blot in megakaryocytes treated with CCL5. In addition, we found that CCL5 treatment protects megakaryocytes from etoposide-induced apoptosis. Together, our data support the model of CCL5-induced inhibition of mitochondrial apoptosis via BAD phosphorylation. Interestingly, proteomic analysis also revealed an increase in proteins involved in quantity and differentiation of hematopoietic progenitor cells, as well as differentiation of megakaryocytes. To test whether CCL5 can also affect the differentiation of hematopoietic progenitor cells, we cultured mouse bone marrow with 100 ng/mL CCL5 and TPO and found a small but sustained increase in the number of megakaryocytes in vitro. To better understand the effect of CCL5 on skewing of hematopoietic cells in a physiological setting, we serially injected mice with 0.025 mg/kg or 0.05 mg/kg CCL5 for one week. Flow cytometry of bone marrow from these mice showed an increase in the number of Pre-MK E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150+) and Pre-CFU E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105+/CD150+) populations and a decrease in the number of Pre GM (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150-) cells in CCL5 treated mice. Interestingly, the CCL5 treated mice did not have a significant inflammatory response, as was measured by complete blood count, indicating that these hematopoietic changes are independent from inflammation. These data suggest that CCL5 may act to skew hematopoiesis toward a megakaryocyte-erythroid lineage and away from a granulocyte lineage in non-inflamed mice.Our proteomics data and in vitro live-dead assay suggest that CCL5 treatment protects against apoptosis through increased BAD phosphorylation, potentially leading to increased megakaryocyte maturation and proplatelet formation. Furthermore, CCL5 may have a dual action in both enhancing platelet production via pro-survival signaling and skewing hematopoiesis toward the megakaryocyte-erythroid lineage. Hematopoietic skewing was seen in CCL5 treated mice despite not having increased inflammation, suggesting that CCL5 itself does not cause a significant inflammatory response. Nonetheless, CCL5 has previously been shown to increase in an inflammatory setting, indicating that the effects seen here could be triggered by systemic inflammation in a different model. Further investigation of the mechanisms leading to these phenotypes may reveal novel therapeutic options to enhance megakaryocyte development and increase platelet counts in patients with thrombocytopenia. DisclosuresBattinelli:Sanofi: Consultancy.

Sugar makes neutrophils RAGE: linking diabetes-associated hyperglycemia to thrombocytosis and platelet reactivity.

Diabetes mellitus is associated with an increased risk for cardiovascular disease, but the link between hyperglycemia and atherothrombotic disease is not completely understood. Patients with diabetes often show hyporesponsiveness to antiplatelet therapies, and it has been suggested that hyperreactive reticulated platelets underlie this altered therapeutic response. In this issue of the JCI, Kraakman et al. uncover a previously unknown link between hyperglycemia and enhanced platelet production and reactivity. The authors demonstrate that high blood glucose levels trigger neutrophil release of S100 calcium-binding protein A8/A9 (S100A8/A9), which binds to the receptor for advanced glycation end products (RAGE) on Kupffer cells, ultimately leading to increased thrombopoietin (TPO) production in the liver. TPO causes megakaryocyte proliferation and increased platelet production. This study demonstrates the importance of glycemic control and identifies potential therapeutic targets in the normalization of platelet numbers and function in diabetes.

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.

An IGF2BP3-Cdk9 Pathway Governs the Human Fetal-Adult Megakaryocyte Transition

Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK.In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant.In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production. DisclosuresNo relevant conflicts of interest to declare.

PDGFR Inhibitor Imatinib May be a Potent Drug in Treating Essential Thrombocythemia

Approximates 80% of essential thrombocythemia (ET) patients carrying JAK2, CALR, MPL mutations. However, there is still 20% of ET patients are negative for all three mutations. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) and its receptor (PDGFR) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis. Therefore, we speculated that PDGF/PDGFR may plays an important role in megakaryocyte/platelet related disease, such as ET. In this study, we found an increase of PDGF-BB and PDGFR expression in ET patients comparing to the normal persons. We then used the adjusted dosage of PDGF-BB (1.5ug/kg/d) to establish an ET mimic mouse model. We found that PDGF-BB can enhance platelet production in vivo significantly, while PDGFR inhibitor imatinib can block this effect. Bone marrow histology data also confirmed the megakaryopoiesis effect of PDGF, as shown by an increased number of megakaryocytes in PDGF-treated mouse. The addition of imatinib can block the effect, evident by a decrease number of megakaryocytes and an increase of apoptosis. Furthermore, we demonstrated that PDGF-BB activated theP-JAK2, the P-STAT3 and the P-AKT expression in the megakaryocytic cell line CHRF-288, while addition of imatinib can reduce the phosphorylation level of all three genes. Our results suggested that the overexpression of PDGF-BB and PDGFR may be important in the pathogenesis of ET, especially in the JAK2-mutation negative ET. The elevated PDGF-BB activates PDGFR with subsequently activation of the JAK2/ STAT3 and PI3K/AKT pathway. Imatinib may have an effect on treating ET via blocking PDGFR and the following JAK2/STAT3 and PI3K/AKT pathway. This study suggests that PDGF/PDGFR may be involved in the pathogenesis of essential thrombocythemia, and the expression of PDGF-BB and PDGFR is potential to be a diagnostic index of ET. Blockage of PDGFR by imatinib may be a new therapeutic target for ET. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

AbstractHuman blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRBS111L) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRBS111L encompassed within the substrate/cofactor [α/β dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXβ tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.