The role of blood platelets in nucleoside metabolism: regulation of megakaryocyte development and platelet production

In higher vertebrates, different types of blood cells develop from common precursors. Mammals are unique in possessing two types of blood cells — erythrocytes and platelets — which lack nuclei. Although platelets display consistent and easily-recognisble morphological and ultrastructural characteristics and show exreme metabolic and functional versatility, they are not true cells, being produced by fragmentation of giant polyploid precursors called megakaryocytes. At present, the physiological mechanisms which regulate megakaryocyte development and platelet production are not well understood.

Platelets are actively involved in metabolism of purine derivatives and a significant platelet role in pyrimidine metabolism has also been demonstrated (see previous papers). Here an attempt is made to integrate information about platelet involvement in nucleic precursor metabolism with current concepts of haematopoiesis, particularly megakaryocyte development and platelet production.

It is concluded (i) that megakaryocytic cels are immediate descendents of haematopoietic stem cells which have become polyploid as a result of genetic damage or metabolic imbalances, (ii) megakaryocytes and platelets are the ultimate regulators of stem cell development because they control the availability of thymidine and (iii) that the production of megakaryocytes and platelets is a physiological safety mechanism which prevents fixation of genetic damage and protects other cells from potentially cytotoxic and genotoxic stimuli.     

Large generation of megakaryocytes from serum-free expanded human CD34 cells

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34⁺ cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.     

Transfer of adenine nucleotides between the releasable and nonreleasable compartments of rabbit blood platelets

The metabolic pool of adenine nucleotides in platelets can be labeled by incubating platelets for 1 h in vitro with [14C]adenosine or [32P]orthophosphate. When these platelets are treated with thrombin, the adenine nucleotides released are not labeled. Because of this, Holmsen's suggestion of a metabolically inert pool of granule nucleotides has been generally accepted. We have found that upon incubation of labeled rabbit platelets for longer times (up to 6 h) in vitro, or upon reinjection and reharvesting at times up to 66 h, the releasable pool of adenine nucleotides becomes labeled. Because the rates of 32p and 14C incorporation into this releasable pool are similar, it seems likely that these labels enter the granules as ATP. Equilibrium between the metabolic and granule pools is complete by 18 h. When rabbit platelets are labeled in vivo by intravenous injection of [32P]orthophosphate, peak labeling occurs between 60 and 70 h; this corresponds to their maturation time. The platelets probably incorporate 32P during their production in the megakaryocytes. The specific radioactivity of the adenine nucleotides in the releasable (granule) pool of these platelets is the same as the specific radioactivity in the nonreleasable (metabolic) pool. Since inorganic phosphate in platelets (and undoubtedly in the megakaryocytes) exchanges with inorganic phosphate in plasma, and since the radioactivity of the latter decreases rapidly, the adenine nucleotides in the two pools must exchange to maintain the same specific radioactivity. Transfer of adenine nucleotides into storage granules may represent a general phenomenon because it has been observed in the chromaffin cells of the adrenal medulla also.     

Megakaryocyte precursors (pro- and megakaryoblasts) in bone marrow tissue from patients with reactive thrombocytosis, polycythemia vera and primary (essential) thrombocythemia. An immunomorphometric study

To determine the number of megakaryocyte precursors (pro- and megakaryoblasts), an immunomorphometric study was performed on paraffin-embedded trephine biopsies of the bone marrow using a monoclonal antibody against platelet glycoprotein IIIa. Eighteen control specimens from patients with no evidence of any hematological disorder and a normal platelet count were selected and assessed together with the same number of specimens from patients with reactive thrombocytosis, polycythemia vera rubra (P. vera) or primary (essential) thrombocythemia (PTH). A strikingly proportionate increase in early megakaryocytes occurred in all patients enrolled in this study, compared with the controls. Moreover, there were no significant correlations between counts for precursors or total megakaryocytes per square millimeter of bone marrow with the corresponding values for platelets. This indicates that despite an orderly increase in immature forms in the bone marrow, the number of platelets circulating in the blood is influenced by other additional factors, such as the expanded platelet pool in the enlarged spleen. The non-disproportionate expansion of megakaryocyte precursors extends previous findings on progenitor cells of this lineage in vitro, particularly in PTH. Histological evaluation of the bone marrow of patients with P. vera and PTH indicated that megakaryopoiesis proceeded to the production of appropriate mature forms with no obvious excess of very small or blastic elements.     

Suppressive Effects of Anthrax Lethal Toxin on Megakaryopoiesis

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34⁺ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.     

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.     

Megakaryocyte development is normal in mice with targeted disruption of Tescalcin

BackgroundTescalcin is an EF-hand calcium-binding protein that interacts with the Na+/H + exchanger 1 (NHE1). Levay and Slepak recently proposed a role for tescalcin in megakaryopoiesis that was independent of NHE1 activity. Their studies using K562 and HEL cell lines, and human CD34 + hematopoietic stem cells suggested an essential role for tescalcin in megakaryocyte differentiation.ObjectiveTo study the role of tescalcin in megakaryocyte development using a murine model of megakaryopoiesis.MethodsWe generated a mouse with targeted disruption of tescalcin and investigated megakaryocyte development.ResultsTescalcin-deficient mice had a normal number of megakaryocytes and platelets. The morphology, polyploidization profile, and expression of Fli-1 in bone marrow-derived megakaryocytes were also normal.ConclusionTescalcin does not appear to be necessary for normal megakaryocyte development.     

Phytosphingosine promotes megakaryocytic differentiation of myeloid leukemia cells

We report that phytosphingosine, a sphingolipid found in many organisms and implicated in cellular signaling, promotes megakaryocytic differentiation of myeloid leukemia cells. Specifically, phytosphingosine induced several hallmark changes associated with megakaryopoiesis from K562 and HEL cells including cell cycle arrest, cell size increase and polyploidization. We also confirmed that cell type specific markers of megakaryocytes, CD41a and CD42b are induced by phytosphingosine. Phospholipids with highly similar structures were unable to induce similar changes, indicating that the activity of phytosphingosine is highly specific. Although phytosphingosine is known to activate p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis, the signaling mechanisms involved in megakaryopoiesis appear to be distinct. In sum, we present another model for dissecting molecular details of megakaryocytic differentiation which in large part remains obscure. [BMB Reports 2015; 48(12): 691-695]     

Signaling by the Mpl receptor involves IKK and NF-kappaB

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.     

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.     

Weibel-Palade bodies in pig megakaryocytes

Originally described in vascular endothelial cells, Weibel-Palade bodies were considered as specific of this cellular type, as they have never been reported elsewhere. Weibel-Palade bodies serve as storage granules for von Willebrand factor which is stored in microtubular form. Besides endothelial cells von Willebrand factor is also synthetized by bone marrow megakaryocytes. Von Willebrand factor has been located in alpha-granules of megakaryocytes and blood platelets. We describe true Weibel-Palade bodies in pig megakaryocytes, and also alpha-granules which look like an evolutionary form of Weibel-Palade bodies. Von Willebrand Factor is most likely stored in microtubular form in these two types of structure. This is supported by the absence of microtubules in these granules in cells obtained from pigs homozygous for the von Willebrand disease (lacking totally this protein).     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

Thrombopoietin induces histidine decarboxylase gene expression in c-mpl transfected UT7 cells

The leukemic cell line UT7 is endowed with both megakaryocyte and basophil differentiation potential, as judged by its capacity to respond to PMA by displaying megakaryocytic and basophilic markers and to produce histamine by neosynthesis. Herein, we addressed the question whether the biological activities characteristic of basophil differentiation were still induced when c-mpl-transfected UT7 cells received a specific megakaryocytic differentiation signal delivered by thrombopoietin (TPO). Surprisingly, we found that histamine synthesis did effectively occur in response to the growth factor. This activity was not associated with megakaryopoiesis since it was not detected in megakaryocytes generated from CD34(+) cells cultured in the presence of TPO. Comparing different c-mpl-transfected cell lines, we found that the amount of histamine generated in response to TPO correlated with their responsiveness to PMA, but not with their level of c-mpl expression, thus revealing an intrinsic basophil differentiation potential. Both PMA- and TPO-induced histamine synthesis was reduced by PKC and MEKs inhibitors, indicating that the induction occurred through a common signalling pathway.     

Arachidonic acid and Docosahexanoic acid enhance platelet formation from human apheresis-derived CD34+ cells

An Aberration in megakaryopoiesis and thrombopoiesis, 2 important processes that maintain hemostasis, leads to thrombocytopenia. Though platelet transfusions are used to treat this condition, blood banks frequently face a shortage of platelets. Therefore, methods to generate platelets on a large scale are strongly desirable. However, to generate megakaryocytes (MKs) and platelets (PLTs) in numbers sufficient for clinical application, it is essential to understand the mechanism of platelet production and explore efficient strategies accordingly. We have earlier reported that the N-6 and N-3 poly-unsaturated fatty acids (PUFAs), Arachidonic acid (AA)/Docosahexanoic acid (DHA) have beneficial effect on the generation of MKs and PLTs from umbilical cord blood derived CD34⁺ cells. Here we tested if a similar effect is observed with peripheral blood derived CD34⁺ cells, which are more commonly used in transplantation settings. We found a significant enhancement in cell numbers, surface marker expression, cellular ploidy and expression of cytoskeletal components during PLT biogenesis in cultures exposed to media containing AA/DHA than control cultures that were not exposed to these PUFAs. The test cells engrafted more efficiently in NOD/SCID mice than control cells. AA/DHA appears to have enhanced MK/PLT generation through upregulation of the NOTCH and AKT pathways. Our data show that PUFAs could be valuable additives in the culture system for large scale production of platelets for clinical applications.     

Abnormalities of megakaryocytes in myelitis and chronic myeloproliferative diseases

A planimetric study of megakaryopoiesis in various chronic myeloproliferative diseases (CMPD) was performed and the results compared with those from controls and myelitis of rheumatic origin. Morphometric measurements included at least 200 megakaryocytes in each case observed in Giemsa-stained semithin sections of resin-embedded core biopsies. Twenty specimens were evaluated from the controls and inflammatory disorders and from each of the following CMPD: 1, chronic granulocytic leukaemia (CGL); 2, polycythaemia vera (P. vera); 3, chronic megakaryocytic-granulocytic myelosis without or with minimal increase in reticulin fibre content (CMGM); 4, myelofibrosis or osteomyelosclerosis (MF/OMS). Megakaryocytes were classified as follows: 1, normal megakaryocytes at all stages of maturation; 2, giant forms; 3, microforms; 4, intussusceptions; 5, a-nuclear cytoplasmic fragments; 6, naked nuclei or necrotic forms. The results of this study demonstrate obvious abnormalities of megakaryopoiesis in addition to the increase in absolute numbers of megakaryocytes per marrow area and their different sizes as reported earlier (Thiele et al. 1982). Aberrations are particularly conspicuous when pure granulocytic proliferation or neoplasia of CGL is compared with the so-called mixed cellularity of megakaryocytes and granulocytes in CMGM including MF/OMS. Abnormalities of the giant forms of megakaryocytes are especially evident and comprise irregular cellular and nuclear perimeters (as calculated by a modified shape factor) in the two latter entities (CMGM-MF/OMS). This remarkable feature is associated with a disorganization of nuclear development and/or a disproportionate nuclear-cytoplasmic ratio which has never been observed in CGL previously. In combination with this striking cellular anomaly, which is compatible with an extreme amoeboid shape of giant forms in CMGM and MF, intussuceptions and a-nuclear cytoplasmic fragments are frequently encountered. The final stage of megakaryopoiesis, i.e. naked nuclei, are increased in number in all CMPD, probably because of enhanced proliferation and platelet shedding. Naked nuclei are often small in CGL (as remnants of the frequent micromegakaryocytes) and large in P. vera and CMGM/MF (depending on the high incidence of giant megakaryocytes in these latter disorders).     

hESCs/hiPSCs体外诱导产生红细胞的研究进展及其临床应用的展望

人类胚胎干细胞和多功能诱导性干细胞的诞生,标志着干细胞研究已经跨入了全新的应用时代。干细胞研究领域的一个重要方向是特定谱系成熟细胞的定向诱导分化。在诸多的血细胞中,成熟红细胞因为无核而携带着最小量的遗传物质,可能作为最早的干细胞治疗产品而应用于输血替代治疗。最近,干细胞向造血细胞（包括红细胞）的研究正方兴未艾。但由于方法学上的偏差,诱导产生的红细胞的成熟度各有所不同。该文在结合了作者实验室的工作经验的基础上,对目前人类多潜能干细胞向红细胞特定诱导分化的方法做了综合的描述,并提出了该研究领域亟需解决的重大科学问题。