Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s.

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.     

Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation, and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis

Rapid proliferation of atypical megakaryoblasts is a characteristic of megakaryoblastic leukemia. Cells from patients with this disorder and cell lines established from this type of leukemia showed the presence of gelsolin but the absence of scinderin expression, 2 filamentous actin-severing proteins present in normal megakaryocytes and platelets. Vector-mediated expression of scinderin in the megakaryoblastic cell line MEG-01 induced a decrease in both F-actin and gelsolin. This was accompanied by increased Rac2 expression and by activation of the PAK/MEKK.SEK/JNK/c-jun, c-fos transduction pathway. The Raf/MEK/ERK pathway was also activated in these cells. Transduction pathway activation was followed by cell differentiation, polyploidization, maturation, and apoptosis with release of platelet-like particles. Particles expressed surface CD41a antigen (glycoprotein IIb/IIIa or fibrinogen receptor), had dense bodies, high-affinity serotonin transport, and circular array of microtubules. Treatment of particles with thrombin induced serotonin release and aggregation that was blocked by CD41a antibodies. PAC-1 antibodies also blocked aggregation. Exposure of cells to PD98059, a blocker of MEK, inhibited antigen CD41a expression, increases in cell volume, and number of protoplasmic extensions. Cell proliferation and cell ability to form tumors in nude mice were also inhibited by the expression of scinderin. MEG-01 cells expressing scinderin had the same fate in vivo as in culture. Thus, when injected into nude mice, they entered apoptosis and released platelet-like particles. The lack of scinderin expression in megakaryoblastic leukemia cells seems to be responsible for their inability to enter into differentiation and maturation pathways characteristic of their normal counterparts.     

Platelet-like particle formation in the human megakaryoblastic leukaemia cell lines, MEG-01 and MEG-01s

The platelet-sized particle formation in the human megakaryoblastic leukaemia cell line MEG-01 and its subline MEG-01s was examined. MEG-01 and MEG-01s cells spontaneously released platelet-sized particles into the culture medium, in which the cells occasionally extended cytoplasmic processes similar to those of megakaryocyte proplatelets. Scanning electron microscopic images showed cytoplasmic processes elongated from blebs on the MEG-01 and MEG-01s cell surface and were constricted between segments of platelet size. Immunofluorescence staining with anti-tubulin antibody showed that the cytoplasmic processes contained microtubules that were organized into a ring, which is a characteristic of circulating platelets. Some platelet-sized particles, probably released by ruptures at the sites of the process constriction, were metabolically active in an MTT assay (about 50%). Some particles also expressed the platelet-specific glycoproteins GPIIb, IIIa and GMP-140. Rarely, in response to thrombin, particles underwent a shape change from spherical to a shape with irregular membrane protrusions and fine filopodia, and aggregating with one another. The particles also had increased GMP-140 (P-selectin) expression with the addition of thrombin. These results show the usefulness of the MEG-01 and MEG-01s cell lines for the study of thrombopoiesis.     

Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01

OBJECTIVE: Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dependent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to study how PGHS-1 expression is regulated in platelets. MATERIALS AND METHODS: Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures. RESULTS: We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population. CONCLUSION: We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.     

Expression of H-related antigen on human megakaryocytes and megakaryocytic leukemia cells.

A mouse monoclonal antibody (MoAb), MG-2, was produced by immunizing a characterized human megakaryoblastic cell line, MEG-01. Since MG-2 reacted with erythrocytes of all ABH blood groups except Oh (O Bombay), and since anti-H MoAb inhibited MG-2 binding to MEG-01 cells, MG-2 is considered to recognize a molecule closely related to blood group antigen H. MG-2 reacted more strongly with normal smaller sized megakaryocytes than with larger sized ones, and not with platelets. The expression of the intrinsic H-related antigen on MEG-01 cells decreased concomitant to megakaryocytic differentiation induced by phorbol esters. This H-related antigen was expressed on leukemia cells with the megakaryocytic features from blast crisis of chronic myelogenous leukemia and acute megakaryoblastic leukemia.     

TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+ entry channel to intracellular alkalosis

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG-01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.     

Cholinergic drugs inhibit in vitro megakaryopoiesis via the alpha7-nicotinic acetylcholine receptor

Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca²⁺ channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.     

On the origin of matrix metalloproteinase-2 and -9 in blood platelets

To date, several matrix metalloproteinases (MMPs) have been identified in human platelets. In most research studies, the platelets are obtained using the isolation method from plasma by centrifugation and washing. The metalloproteinase content in the platelets can be affected by the isolation technique and the leukocyte contamination. In this work, we studied the influence of the isolation method on the detection of platelet MMPs and explore the expression of these enzymes in megakaryoblastic MEG-01 cells. We investigated the expression of mRNAs encoding for MMP-2 and -9 in platelets and MEG-01 cells. Using gelatin zymography and western blotting, we examined the expression and release of MMP-2 and 9 by platelets and MEG-01 cells and checked whether the amount of the released MMPs depends on the volume of tested platelet and leukocyte contamination. To investigate the MMP-2 expression profile, we used zymography and flow cytometry. Platelets, in contrast to the MEG-01 cells, neither contain mRNA for MMP-2 nor -9. The platelets contain pro-MMP-2 and release it during the activation. The population of uncontaminated (leukocytes<0.02%) platelets contained no MMP-9 or the active form of MMP-2. We have observed that the activity of MMP-2 in platelet lysate is proportional to their mean volume and that the MMP-2 activity may not be detected if very small platelets are examined. We conclude that the detection of gelatinases in platelets depends on platelet isolation techniques and the degree of leukocyte contamination.     

Identification of Gz alpha as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes

G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein alpha subunit referred to as Gz alpha. Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different Gz alpha-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the Gi alpha family. Northern blotting with a Gz alpha-specific probe prepared from retinal Gz alpha showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding Gz alpha was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of Gi alpha 2' the most abundant of the Gi alpha species present in platelets. The complete sequence of the platelet homolog to Gz alpha was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal Gz alpha. The paucity of Gz alpha protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize Gz alpha from these cells or that Gz alpha appears at a later stage in megakaryocyte development than does Gi alpha.     

Biosynthesis and secretion of beta-thromboglobulin by a human megakaryoblastic cell line (MEG-01).

The de-novo synthesis and secretion of beta-thromboglobulin (BTG) by a human megakaryoblastic cell line (MEG-01) were studied by measuring and immunoblotting of BTG in culture supernatant and immunoprecipitation of radiolabeled BTG synthesized after incubation with [35S]methionine. It was demonstrated that BTG synthesized by MEG-01 was secreted into culture media in a monomer form having a molecular weight of 8,800. Furthermore, we purified BTG from culture medium of MEG-01 with a heparin affinity column and compared BTG from MEG-01 with that from normal platelets. The molecular weights of BTG purified from both sources were 8,800 using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results provide direct evidence for the synthesis and secretion of BTG by megakaryocytes.     

Establishment of a novel human megakaryoblastic leukemia cell line, MEG- 01, with positive Philadelphia chromosome

A megakaryoblastic cell line, designated MEG-01, was established from the bone marrow of a patient with blast crisis of Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia. MEG-01 cells grew in single-cell suspension with a doubling time of 36 to 48 hours. Under the usual culture conditions, approximately half of the cells adhered to the culture flask with extention of pseudopods. MEG-01 cells were positive for the periodic acid-Schiff reaction, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for myeloperoxidase, alpha- naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and alkaline phosphatase. Ultrastructural platelet peroxidase was positive in MEG-01 cells. Cytoplasmic factor VIII (FVIII)-related antigen was weakly positive in larger MEG-01 cells by both an indirect immunofluorescent technique with monoclonal antibodies and a direct immunoperoxidase technique using horseradish peroxidase-conjugated conventional rabbit anti-human FVIII antibody. Platelet glycoprotein (GP) IIb/IIIa antigen was uniformly demonstrated on the surface of MEG- 01 cells by both indirect immunofluorescent and immunoperoxidase techniques using antiplatelet GP IIb/IIIa monoclonal antibodies; platelet GP lb antigen was demonstrated only in the cytoplasm of larger MEG-01 cells. MEG-01 cells possessed no markers for B or T lymphocytes or for myeloid cells. Chromosome analysis of this cell line revealed a human male hyperdiploid karyotype with a modal chromosome number of 56 to 58. The Ph1 chromosome was observed in all karyotypes analyzed. This novel human megakaryoblastic cell line may provide a useful model for the study of human megakaryopoiesis and of the biosynthetic mechanisms of proteins unique to megakaryocytic lineage.     

Varied expressions of receptors on the human megakaryoblastic cell line MEG-01 by monoclonal antibodies

We herein describe the regulation of specific receptors on the megakaryoblastic cell line MEG-01 by means of fluorescence-activated cell sorting (FACS IV) with a panel of monoclonal antibodies (MAbs). MEG-01 cells expressed GpIIb/IIIa (CD41a) and OKM5 (CD36) antigens on their cell surface, whereas they showed only a little expression of GPIb (CD42b), suggesting that these are megakaryoblastic cells. The up regulation of von Willebrand factor receptor (GPIb) and thrombospondin receptor (GPIV) and the down regulation of fibrinogen receptor (GPIIb/IIIa) and C3bi receptor (CD11b) were found by incubation with MAbs AN51 (CD42b), OKM5 (CD36), J15 (CD41a), and OKM1 (CD11b), respectively. This phenomenon was enhanced in the Ca2+ containing medium, except for the experiment with OKM5 (CD36) MAb. We thus suggest that several kinds of receptors on the surface of MEG-01 cells are dexterously regulated through stimulation from outside the cell.     

Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.     

Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts

Background P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression.Methods Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed.Results Nicotine, at concentrations of 0.1–1.0 μM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 μM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers.Conclusion Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. Condensed Abstract The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1–1.0 μM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 μM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.     

Human platelets exhibit chemotaxis using functional N-formyl peptide receptors

OBJECTIVE: Activated platelets participate in inflammatory and microbicidal processes by upregulation of surface selectins, shedding of CD40 ligand, and release of platelet microbicidal proteins and microparticles. Given their myeloid lineage, we hypothesized that platelets express functional N-formyl peptide receptors and respond to the bacterially derived chemotactic peptide N-formyl peptide with gradient-driven chemotaxis. METHODS AND RESULTS: Here we show specific binding of N-formyl peptides to the surface of activated platelets. Platelet expression and function of the formyl peptide receptor, FPR, was verified by RT-PCR of the differentiated megakaryocyte MEG-01 cell line, immunoblotting of platelet proteins, and calcium mobilization in platelets with formyl peptide binding. Furthermore, we demonstrate gradient-driven chemotaxis of platelets by video microscopy and transwell migration toward formyl peptides. We also show that endogenous formyl peptides, released by eukaryotic mitochondria from necrotic cells, induce chemotaxis using formyl peptide receptors expressed by thrombin-activated platelets. Conversely, supernatants from cells undergoing apoptotic cell death do not induce platelet chemotaxis. Platelet chemotaxis to formyl peptides was blocked with FPR-specific antibody as well as by pertussis toxin inhibition of the formyl peptide G-coupled receptor. CONCLUSION: These data establish a new role for platelets in host defense and suggest reexamination of their active function in microbicidal and other host defense activities.     

Cholinergic drugs inhibit in vitro megakaryopoiesis via the alpha7-nicotinic acetylcholine receptor

Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca(2+) channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In?vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in?vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.