The Application of Ethnomedicine in Modulating Megakaryocyte Differentiation and Platelet Counts.

Megakaryocytes (MKs), a kind of functional hematopoietic stem cell, form platelets to maintain platelet balance through cell differentiation and maturation. In recent years, the incidence of blood diseases such as thrombocytopenia has increased, but these diseases cannot be fundamentally solved. The platelets produced by MKs can treat thrombocytopenia-associated diseases in the body, and myeloid differentiation induced by MKs has the potential to improve myelosuppression and erythroleukemia. Currently, ethnomedicine is extensively used in the clinical treatment of blood diseases, and the recent literature has reported that many phytomedicines can improve the disease status through MK differentiation. This paper reviewed the effects of botanical drugs on megakaryocytic differentiation covering the period 1994-2022, and information was obtained from PubMed, Web of Science and Google Scholar. In conclusions, we summarized the role and molecular mechanism of many typical botanical drugs in promoting megakaryocyte differentiation in vivo, providing evidence as much as possible for botanical drugs treating thrombocytopenia and other related diseases in the future.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.

BACKGROUND: Non-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PURPOSE: This study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. METHODS: A potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. RESULTS: A natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. CONCLUSION: TMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation.

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA). In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

The plant hormone abscisic acid stimulates megakaryocyte differentiation from human iPSCs in vitro.

In the clinic, the supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, many scientists have established the derivation of functional platelets from CD34+ cells or human pluripotent stem cells (PSCs). However, the yield of platelets is still far below what is required. Here we found that the plant hormone abscisic acid (ABA) could increase the generation of megakaryocytes (MKs) and platelets from human induced PSCs (hiPSCs). During platelet derivation, ABA treatment promoted the generation of CD34+/CD45+ HPCs and CD41+ MKs on day 14 and then increased CD41+/CD42b+ MKs and platelets on day 19. Moreover, we found ABA-mediated activation of Akt and ERK1/2 signal pathway through receptors LANCL2 and GRP78 in a PKA-dependent manner on CD34+/CD45+ cells. In conclusion, our data suggest that ABA treatment can promote CD34+/CD45+ HPC proliferation and CD41+ MK differentiation.

Long-acting PGE2 and Lisinopril Mitigate H-ARS.

Thrombocytopenia is a major complication in hematopoietic-acute radiation syndrome (H-ARS) that increases the risk of mortality from uncontrolled hemorrhage. There is a great demand for new therapies to improve survival and mitigate bleeding in H-ARS. Thrombopoiesis requires interactions between megakaryocytes (MKs) and endothelial cells. 16, 16-dimethyl prostaglandin E2 (dmPGE2), a longer-acting analogue of PGE2, promotes hematopoietic recovery after total-body irradiation (TBI), and various angiotensin-converting enzyme (ACE) inhibitors mitigate endothelial injury after radiation exposure. Here, we tested a combination therapy of dmPGE2 and lisinopril to mitigate thrombocytopenia in murine models of H-ARS following TBI. After 7.75 Gy TBI, dmPGE2 and lisinopril each increased survival relative to vehicle controls. Importantly, combined dmPGE2 and lisinopril therapy enhanced survival greater than either individual agent. Studies performed after 4 Gy TBI revealed reduced numbers of marrow MKs and circulating platelets. In addition, sublethal TBI induced abnormalities both in MK maturation and in in vitro and in vivo platelet function. dmPGE2, alone and in combination with lisinopril, improved recovery of marrow MKs and peripheral platelets. Finally, sublethal TBI transiently reduced the number of marrow Lin-CD45-CD31+Sca-1- sinusoidal endothelial cells, while combined dmPGE2 and lisinopril treatment, but not single-agent treatment, accelerated their recovery. Taken together, these data support the concept that combined dmPGE2 and lisinopril therapy improves thrombocytopenia and survival by promoting recovery of the MK lineage, as well as the MK niche, in the setting of H-ARS.

Platelet enhancement by Carica papaya L. leaf fractions in cyclophosphamide induced thrombocytopenic rats is due to elevated expression of CD110 receptor on megakaryocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Carica papaya leaf juice/decoction has been in use in folk medicine in Srilanka, Malaysia and in few parts of India for enhancing the platelet counts in dengue. In Siddha medicine, a traditional form of medicine in India, papaya leaf juice has been used for increasing the platelet counts. Papaya leaf has been reported to enhance blood volume in ancient Ayurveda books in India. Carica papaya leaf is well known for its platelet enhancement activity. Although many preclinical and clinical studies have demonstrated the ability of papaya leaf juice for platelet enhancement, but the underlying mechanisms are still unclear. AIM OF THE STUDY: The study is aimed at identifying the key ingredients of papaya leaf extract and elucidate the mechanism (s) of action of the identified potent component in mitigating thrombocytopenia (Thp). MATERIALS AND METHODS: C. papaya leaf juice was subjected for sequential fractionation to identify the anti-thrombocytopenic phytochemicals. In vivo, stable thrombocytopenia was induced by subcutaneous injection of 70 mg/kg cyclophosphamide (Cyp). After induction, rats were treated with 200 and 400 mg/kg body weight papaya leaf juice and with identified fractions for 14 days. Serum thrombopoietin level was estimated using ELISA. CD110/cMpl, a receptor for thrombopoietin on platelets was measured by western blotting. RESULTS: Administration of cyclophosphamide for 6 days induced thrombocytopenia (210.4 ± 14.2 × 103 cells/µL) in rats. Treating thrombocytopenic rats with papaya leaf juice and butanol fraction for 14 days significantly increased the platelet count to 1073.50 ± 29.6 and 1189.80 ± 36.5 × 103 cells/µL, respectively. C.papaya extracts normalized the elevated bleeding and clotting time and decreased oxidative markers by increasing endogenous antioxidants. A marginal increase in the serum thrombopoietin (TPO) level was observed in Cyp treated group compared to normal and treatment groups. Low expression of CD110/cMpl receptor found in Cyp treated group was enhanced by C. papaya extracts (CPJ) and CPJ-BT. Furthermore, examination of the morphology of bone marrow megakaryocytes, histopathology of liver and kidneys revealed the ability of CPJ and fractions in mitigating Cyp-induced thrombocytopenia in rats. CONCLUSION: C. papaya leaf juice enhances the platelet count in chemotherapy-induced thrombocytopenia by increasing the expression of CD110 receptor on the megakaryocytes. Hence, activating CD110 receptor might be a viable strategy to increase the platelet production in individuals suffering from thrombocytopenia.

Photobiomodulation therapy for thrombocytopenia by upregulating thrombopoietin expression via the ROS-dependent Src/ERK/STAT3 signaling pathway.

BACKGROUND: Chemotherapy-induced thrombocytopenia (CIT) can increase the risk of bleeding, which may delay or prevent the administration of anticancer treatment schedules. Photobiomodulation therapy (PBMT), a non-invasive physical treatment, has been proposed to improve thrombocytopenia; however, its underlying regulatory mechanism is not fully understood. OBJECTIVE: To further investigate the mechanism of thrombopoietin (TPO) in megakaryocytopoiesis and thrombopoiesis. METHODS: Multiple approaches such as western blotting, cell transfection, flow cytometry, and animal studies were utilized to explore the effect and mechanism of PBMT on thrombopoiesis. RESULTS: PBMT prevented a severe drop in platelet count by increasing platelet production, and then ameliorated CIT. Mechanistically, PBMT significantly upregulated hepatic TPO expression in a thrombocytopenic mouse model, which promoted megakaryocytopoiesis and thrombopoiesis. The levels of TPO mRNA and protein increased by PBMT via the Src/ERK/STAT3 signaling pathway in hepatic cells. Furthermore, the generation of the reactive oxygen species was responsible for PBMT-induced activation of Src and its downstream target effects. CONCLUSIONS: Our research suggests that PBMT is a promising therapeutic strategy for the treatment of CIT.

Isolation, Cryopreservation, and Characterization of iPSC-Derived Megakaryocytes.

Current research in the field of transfusion medicine is focused on developing innovative approaches to generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.

Astragalus polysaccharide has a protective effect on hematopoiesis in an irradiated mouse model and decreases apoptosis in megakaryocytes.

Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiation­induced myelosuppressive mouse model. Colony­forming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and anti­apoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day 21 following treatment, as well as platelets and white blood cells at day 14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that tri­lineage hematopoiesis was preserved in the ASPS­ and thrombopoietin (TPO)­treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPS­treated group was similar to that of the TPO­treated group. Additionally, in vitro experiments indicated that treatment with 100 µg/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase­3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.

Cissus quadrangularis-Induced Thrombocytopenia in a Renal Allograft Recipient.

An elderly male renal allograft recipient presented with thrombocytopenia. He had a kidney transplant for diabetic kidney disease and was on azathioprine and prednisolone. He had taken Cissus quadrangularis capsules for backache. A bone marrow aspiration to evaluate the cause of thrombocytopenia showed megakaryocyte hyperplasia, suggesting peripheral destruction. Repeat platelet counts after stopping Cissus quadrangularis showed normal levels.

A natural small molecule induces megakaryocytic differentiation and suppresses leukemogenesis through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells.

Kaempferol-3-O-rhamnoside (KOR) has multiple potency involved in anti-cancer, anti-inflammatory and antibacterial actions. However, the potential roles of KOR and the analogues isolated from the leaves of Cyclocarya paliurus in anti-erythroleukemia remain unclear. In the present study, KOR and the two analogues (Kaempferol-3-O-(4″-O-acetyl-a-L-rhamnopyranoside) (KLR) and (kaempferol-3-O-α-L-(4″-E-p-coumaroyl) rhamnoside) (KCR) were isolated from leaves of Cyclocarya paliurus. Cell viability assay showed that KCR exerted an excellent anti-erythroleukemia activity. We observed that KCR not only significantly increased the percentage of G2 phase and apoptotic cells compared with control group, but also induced megakaryocytic differentiation in HEL and K562 cells by flow cytometry, indicating that KCR might inhibit cell proliferation through inducing differentiation-mediated apoptosis and cell cycle arrest. Mechanism investigation revealed that KCR treatment obviously increased phosphorylation levels of PKCδ and ERK1/2 as well as GATA1 expression. Taken together, these findings demonstrate that KCR induces megakaryocytic differentiation and suppresses leukemogenesis at least partly through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells. KCR may also serve as a promising natural compound for human erythroleukemia treatment.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

A ß1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production.

During maturation, megakaryocytes (MKs) express ß1-tubulin (TUBB1) and rearrange their microtubule components to enlarge, form proplatelets, and eventually release platelets. The development of a platform to identify in vitro conditions that would efficiently promote MK development could potentially enable large-scale platelet production. Here, we show that an immortalized MK cell line (imMKCL) genetically modified to express the ß1-tubulin-Venus reporter provides a practical system to efficiently monitor the in vitro production of platelet-like particles (PLPs). The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9, and the expression was visualized by Venus fluorescence intensity. This imMKCL reporter line was then used for high-throughput drug screening. We identified several compounds that significantly improved the efficiency of PLP production in vitro under feeder-free conditions and showed a significant tendency to recover platelets in vivo in a mouse thrombocytopenia model induced by anti-GPIbα antibody administration. Interestingly, most of these compounds, including a WNT signaling pathway inhibitor, Wnt-C59, antagonized the aryl hydrocarbon receptor (AhR) to increase PLP production, confirming the crucial role of AhR inhibition in MK maturation. Consistently, small interfering RNA treatment against AhR increased the Venus intensity and PLP production. TCS 359, an FLT3 inhibitor, significantly increased PLP production independently of FLT3 or AhR. This study highlights the usefulness of the ß1-tubulin reporter MK line as a useful tool to study the mechanisms underlying thrombopoiesis and to identify novel inducers of ex vivo platelet production.

Justicia adhatoda induces megakaryocyte differentiation through mitochondrial ROS generation.

BACKGROUND: Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts. PURPOSE: To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation METHODS: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR. RESULTS: At a concentration of 40 µg/ml, the leaf extracts of Justicia adhatoda for 72 h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels. CONCLUSION: Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.

Investigation of Iron Metabolism for Regulating Megakaryopoiesis and Platelet Count According to the Mechanisms of Anemia.

BACKGROUND: Iron deficiency anemia (IDA) is characterized by depletion of total body iron stores or a poor supply of plasma iron. By contrast, chronic inflammation makes iron unavailable for hematopoiesis through a cytokinemediated cascade and leads to a condition known as anemia of chronic disease (AOC). However, the laboratory data regarding the regulatory role of iron metabolism on platelet count has not been fully discussed yet. In this study, we investigated the relationship between iron status and platelet production according to different anemic mechanisms representing different iron metabolisms. METHODS: The study included a total of 759 specimens. Blood samples were obtained through venipuncture. The complete blood count was measured using an Advia 2120 (Siemens Healthcare Diagnostics Inc., USA). Biochemical indices including iron level were estimated using a Toshiba chemical analyzer (Toshiba, Japan). RESULTS: In the AOC group, we found a significant relationship between platelet count and serum iron level (p < 0.27), whereas there was no correlation in the IDA group. Moreover, when the AOC patient group was subdivided by serum iron level, a remarkable difference was observed as follows. The platelet count was significantly correlated with serum iron level only in the AOC group with decreased serum iron levels (serum iron < 50 µg/dL) (p < 0.0001), while there was no correlation in the AOC group with normal serum iron levels (serum iron 50 - 100 µg/dL). CONCLUSIONS: Iron deficiency in AOC involves upregulated hepcidin production induced by elevated inflammatory cytokines. This can cause increased iron sequestration in macrophages and decreased iron absorption for bone marrow. The condition of decreased megakaryocytic iron supply makes megakaryocytes with higher ploidy which can release more platelets than lower ploidy. Moreover, reactive thrombocytosis in inflammatory states occurs by cytokine cascades involving interleukin 6 and thrombopoietin in AOC. These two features may enhance thrombocytosis in patients of AOC with decreased iron level. In the future, further study should be performed to elucidate regulating mechanism of iron metabolism for megakaryopoiesis in AOC patients, and guide proper supplemental therapy of iron to decrease thrombotic risk due to reactive thrombocytosis in various kinds of anemia.

R-lipoic acid overdosing affects platelet life span via ROS mediated autophagy.

R-lipoic acid (ALA), a powerful antioxidant valuable for the treatment of diabetes and its complications, has been reported to exhibit an antiplatelet activity in vitro. The aim of this study was to investigate the effect and mechanism of ALA on platelets in vivo. Sprague-Dawley (SD) male rats were intravenously administered with low-dose ALA (20 mg/kg/d), high-dose ALA (80 mg/kg/d) and saline, respectively. Platelets count and bone marrow smear were evaluated and the expressions of markers related to apoptosis and autophagy were measured. Platelet clearance analysis was conducted out on mice. The results showed that high-dose ALA administration could significantly decrease platelet count by 43% compared with control group, whereas, megakaryocytes showed no difference in the number. Moreover, high-dose ALA administration led to significant reduction in half-life of circulating platelets, indicative of enhanced rate of platelet clearance. Interesting, high-dose ALA administration could increase the level of reactive oxygen species (ROS) in platelets and induce autophagy without affecting apoptosis. Our finding also showed that high ALA-induced autophagy in platelets was mediated by class III PtdIns3K activity, which could be reversed by 3-methyladenine (3-MA). Moreover, AKT and MAPK/ERK pathways were also observed to be involved in the regulation of autophagy in platelets. Thus, high-dose ALA could induce autophagy in platelets through modulating the activity of class III PtdIns3K, which was associated with decreased count of circulating platelets and shortened lifespan of platelets.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl.

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract.

We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of CD41+/DAPI- cells (platelets), but had no effect on the formation of CD41+/DAPI+ cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MARE-luciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not influence the development of hematopoietic stem cells into megakaryocytes.

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

PURIFICATION AND FRACTIONAL ANALYSIS OF METHANOLIC EXTRACT OF WEDELIA TRILOBATA POSSESSING APOPTOTIC AND ANTI-LEUKEMIC ACTIVITY.

BACKGROUND: Wedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity. MATERIALS AND METHODS: The methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components. RESULTS: Out of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC. CONCLUSION: The single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis.