Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS-PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.     

Characterization of the megakaryocyte secretory response: studies of continuously monitored release of endogenous ATP

Megakaryocytes share a number of structural and chemical properties with their progeny, blood platelets. With the availability of highly purified preparations of megakaryocytes isolated from guinea pig bone marrow, it is now also possible to study functional aspects of these cells. The present work reports the first study of the release of endogenously stored materials in megakaryocytes. Guinea pig megakaryocytes isolated to 75%-90% purity were exposed to thrombin or calcium ionophore (A23187) and the release of ATP was continuously monitored with the luciferin-luciferase reaction. Both maximal extent and initial rate of release were studied. Thrombin-induced release was half-maximal at thrombin concentrations of 0.2-0.5 NIH U/ml. At 4 U/ml thrombin, maximal release was 538 +/- 147 nmole ATP/10(9) megakaryocytes. A23187 induced half-maximal responses at concentrations of 7-8 microM. ATP release by ionophore showed a nearly absolute requirement for extracellular calcium, with release by thrombin showing only a partial calcium dependence. Following overnight culture, the response to thrombin was unchanged, whereas ATP release in response to ionophore was consistently increased (p less than 0.01). By comparison of maximally releasable ATP with total cellular ATP content, the storage pool of ATP in megakaryocytes was determined to comprise only 2%-6% of total megakaryocyte ATP, in contrast to an ATP storage pool of 20%-30% in guinea pig platelets. This difference may reflect further entry of ATP into the storage pool compartment or an enhanced ability of the cell to recognize and respond fully to platelet stimuli as the megakaryocyte reaches full maturity.     

Expression of integrins in human bone marrow

Expression of integrins, a superfamily of glycoprotein alpha/beta heterodimers which integrate the cytoskeleton with the extracellular matrix and/or mediate cell-cell adhesive interactions, was examined on normal and leukaemic bone marrow cells by immunohistochemistry and immunotransmission electron microscopy (immuno-TEM). Among the beta 1/VLA molecules studied, VLA-2 and 6 were expressed on megakaryocytes and platelets, while VLA-4 was present on 40% of haemopoietic cells, including monocytes, erythroblasts and immature cells; this molecule was typically localized at sites of intercellular contact, as seen by immuno-TEM, suggesting it may be involved in interactions among haemopoietic cells during differentiation. In human long-term bone marrow cultures (LTBMC), VLA-1 and 3 were present respectively on 35% and 40% of the adherent cells which included fibroblasts and endothelial cells, as shown by double-labelling experiments; VLA-2 was expressed only on a subpopulation of fibroblasts. beta 2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA-DR+/myeloperoxidase- early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of beta 3/cytoadhesin molecules was restricted to megakaryocytes and platelets and, in the adherent layer of LTBMC, to endothelial cells. The regulated expression and specific localization of integrins in the bone marrow suggest that these molecules may have a role in normal haemopoiesis.     

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.     

Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.     

Changes in Megakaryocyte Development Following Thrombocytopenia

S ummary . Rats were made thrombocytopenic by total-body X-irradiation and changes in megakaryocyte development studied in shielded tibial bone marrow. The platelet count fell from the fourth day after irradiation to reach a minimum value of 19.5% of the initial count 7 days after irradiation. No changes in megakaryocyte development were observed 5 days after irradiation. Megakaryocyte size was increased at day 7, and the increment maintained until day 12. Megakaryocyte numbers were only significantly increased 12 days after irradiation, but differential counts and electron-microscope autoradiographs suggested increased influx at day 9. Changes in the labelling index of megakaryocytes suggested that some cells already present were undergoing one or more extra endoreduplications. No evidence was obtained for an accelerated megakaryocyte maturation rate or for a sudden release of platelets from mature megakaryocytes. Morphological changes, signifying increased synthesis of protein and demarcation membranes were apparent only in young cells 7 days after irradiation but were present in mature cells by day 12, demonstrating that the response to thrombocytopenia is confined to immature cells.     

Botrocetin agglutination of rat megakaryocytes: a rapid method for megakaryocyte isolation.

Botrocetin isolated from the venom of Bothrops jaracara has been shown by others to induce binding of von Willebrand Factor to glycoprotein Ib and thereby produce platelet agglutination in a wide range of animal species. We have found that botrocetin also facilitates the agglutination of megakaryocytes and have used this property to develop a method to isolate megakaryocytes from rat bone marrow. When botrocetin is added to a mixture of rat bone marrow, rat platelets, and rat plasma, coagglutination of megakaryocytes and platelets occurs. The agglutinated complexes, containing > 95% of the megakaryocytes, may then be separated from the remaining marrow cells by filtration. Megakaryocytes account for 39% (range 30%-48%) of the isolated cells and 83% (range 77%-88%) of the isolated cell mass. This method allows the virtually complete removal of megakaryocytes from bone marrow as well as their isolation to a high degree of purity. It should provide a useful, inexpensive, general method for the rapid isolation of megakaryocytes from multiple, small marrow samples from a wide range of species.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

The End Is Just the Beginning: Megakaryocyte Apoptosis and Platelet Release

Under influence of hematopoietic growth factors, particularly thrombopoietin (TPO), hematopoietic stem cells in the bone marrow go through a process of commitment, proliferation, differentiation, and maturation and become mature megakaryocytes. At this critical point, terminally differentiated megakaryocytes face a new fate: ending the old life as mature megakaryocytes by induction of apoptosis and beginning a new life as platelets by fragmentation of the large megakaryocyte cytoplasm. These events are as important as megakaryocyte commitment, proliferation, differentiation, and maturation, but the molecular mechanisms regulating these events are not well established. Although TPO drives megakaryocyte proliferation and differentiation and protects hematopoietic progenitor cells from death, it does not appear to promote platelet release from terminally differentiated megakaryocytes. Although mature megakaryocyte apoptosis is temporally associated with platelet formation, premature megakaryocyte death directly causes thrombocytopenia in cancer therapy and in diseases such as mvelodysplastic syndromes and human immunodeficiency virus infection. Also, genetic studies have shown that accumulation of megakaryocytes in bone marrow is not necessarily sufficient to produce platelets. All of these findings suggest that platelet release from megakaryocytes is an important and regulated aspect of platelet production, in which megakaryocyte apoptosis may also play a role. This review summarizes recent research progress on megakaryocyte apoptosis and platelet release.     

Bone marrow stromal cells produce thrombopoietin and stimulate megakaryocyte growth and maturation but suppress proplatelet formation

Production of blood cells is regulated by the interplay of various cytokines and bone marrow stromal cells. Recently, a ligand for the orphan receptor Mpl was identified as thrombopoietin (TPO), which specifically regulates megakaryocyte differentiation, and it was reported to be expressed mainly in liver and kidney. As it was found that thrombopoietin is also produced in bone marrow stromal cells, we studied further the roles of bone marrow stromal cells on megakaryocytopoiesis and platelet formation. The stromal cells stimulated growth and maturation of bone-marrow-derived megakaryocytes in the presence of thrombopoietin, and also supported growth of BaF3 cells expressing exogenous Mpl without thrombopoietin. Thrombopoietin induces drastic morphological change of megakaryocytes in bone marrow cells in vitro, ie, the formation of lengthy beaded cytoplasmic processes (proplatelet formation). However, when the purified megakaryocytes were cocultured with the stromal cells with or without thrombopoietin, most of the megakaryocytes adhered to the stromal cells and remained unchanged, while free megakaryocytes induced proplatelet formation. These observations indicated that the stromal cells in a hematopoietic microenvironment in bone marrow secrete thrombopoietin and stimulate proliferation and maturation of megakaryocytes, but the interaction of megakaryocytes with the stromal cells may suppress proplatelet formation.     

Histidine-rich glycoprotein is present in human platelets and is released following thrombin stimulation

Histidine-rich glycoprotein, and alpha 2-glycoprotein in human plasma, has been shown to interact with heparin, with the high-affinity lysine- binding site of plasminogen, with divalent cations, and is associated with the rosette formation between erythrocytes and lymphocytes. A specific enzyme-linked immunosorbent assay for histidine-rich glycoprotein has been developed and used to demonstrate that histidine- rich glycoprotein is present in human platelets. Histidine-rich glycoprotein was detected and quantified in detergent extracts of washed human platelets, with a mean level of 371 ng/10(9) platelets. Plasma histidine-rich glycoprotein, either in the platelet suspending medium or on the surface of the platelets, accounted for less than 3.4% of the detectable platelet histidine-rich glycoprotein. Histidine-rich glycoprotein was also demonstrated in human bone marrow megakaryocytes by immunofluorescence. The extent of histidine-rich glycoprotein release from platelets was dependent on the thrombin dose and correlated directly with the extent of serotonin release. The platelet and plasma histidine-rich glycoprotein were similar by immunochemical analysis. Anti-histidine-rich glycoprotein IgG did not inhibit platelet aggregation. Histidine-rich glycoprotein released by platelets following thrombin stimulation may play a significant role in modulating inflammatory events in the microenvironment of the platelet plug.     

Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice.

Granulocyte-colony stimulating factor (G-CSF) has been found to act on the neutrophilic lineage. We recently showed that human G-CSF (hG-CSF) has effects similar to early-acting cytokines such as interleukin-3 (IL-3) in the development of multipotential hematopoietic progenitors in transgenic (Tg) mice expressing receptors (R) for hG-CSF. In the present study, we examined the effects of hG-CSF on more mature hematopoietic cells committed to megakaryocytic lineage in these Tg mice. The administration of hG-CSF to the Tg mice increased the numbers of both platelets in peripheral blood and megakaryocytes in the spleen, indicating that hG-CSF stimulates megakaryopoiesis in the Tg mice in vivo. The stimulatory effect of hG-CSF was also supported by the results of studies in vitro. hG-CSF supported megakaryocyte colony formation in a dose-dependent fashion in clonal cultures of bone marrow cells derived from the Tg mice. Direct effects of hG-CSF on megakaryocytic progenitors in the Tg mice were confirmed by culture of single-cell sorted from bone marrow cells. hG-CSF showed a stronger effect on maturation of megakaryocytes in the Tg mice than that of IL-3 alone, but weaker than that of TPO alone. In addition, hG-CSF induced phosphorylation of STAT3 but not Jak2 or STAT5, while TPO induced phosphorylation of both. In contrast to TPO, hG-CSF did not enhance ADP-induced aggregation. Thus, hG-CSF has a wide variety of functions in megakaryopoiesis of hG-CSFR-Tg mice, as compared with other megakaryopoietic cytokines, but the activity of hG-CSF in megakaryocytes and platelets does not stand up to a comparison with that of TPO. Specific signals may be required for the full maturation and activation of platelets.     

Biology of human megakaryocyte factor V

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.     

Investigation of megakaryopoiesis in myelosuppressed bone marrow using immunogold-silver staining (IGSS)

Abstract: To determine the frequencies and differential counts of megakaryocytes after cytoreductive treatment in nucleated low-density (1.060 g/ml) bone marrow cells (BMNC) of dogs an immunogold-silver staining (IGSS) technique with the lineage specific monoclonal antibody 2F9 was established. This antibody recognizes the glycoprotein IIb/IIIa complex expressed on the surface of canine megakaryocytes and platelets. The IGSS technique enables not only the detection of megakaryocytes occurring at a low frequency (0.1–0.2%), but also the discrimination between the different maturation stages of megakaryocytes due to cell size, nuclear morphology and cytoplasmic staining. By the use of this technique, small lymphoid megakaryocytic cells were identified. Comparable numbers of megakaryocyte colony-forming cells in 2F9-depleted and nondepleted BMNC suspensions (25.7 + 5.0 vs. 25.3 ± 5.1 Meg-CFC/10⁵ BMNC) indicate that these small 2F9 positive cells are nonclonogenic precursors of megakaryoblasts. To prove the applicability of IGSS, serial examinations of bone marrow samples from dogs treated with recombinant human interleukin-6 (IL-6) after exposure to 2.4 Gy total body irradiation (TBI) were performed. The results of the microscopic evaluation indicate that, in the recovery phase after TBI, IL-6 induced an earlier and stronger increase in megakaryocyte frequency in comparison to the control. Interestingly, all maturation stages of the megakaryocytic lineage took part in this IL-6 induced improvement of megakaryocyte recovery.     

Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL

The principal growth factor involved in the regulation of erythropoiesis, erythropoietin (Epo), is currently under clinical trial for the treatment of anemia. Despite the advanced state of these trials, little is known about the nature and distribution of the receptor for Epo on human hemopoietic cells or about the cellular mechanisms of signal transduction. In the present study 125I-labeled recombinant human Epo has been used to demonstrate expression of saturable, high affinity binding sites for Epo on density-fractionated human bone marrow cells and on the human erythroleukemic cell line, HEL. Binding was reversible and proportional to cell number, and HEL cells were shown to express on average 34 receptors per cell (range 30-35) with an affinity of 293 pM (range 275-300 pM) at 37 degrees C in the presence of sodium azide to block receptor internalization. Autoradiographic analysis of Epo binding to human bone marrow cells showed that specific binding, measured as the difference in grain counts between total binding and binding in the presence of excess unlabeled Epo, was greatest to pronormoblasts and declined during erythroid cell maturation to undetectable levels on nucleated red cells. Autoradiography also revealed significant Epo binding to marrow megakaryocytes, which comprise less than 1% of nonerythroid cells. In contrast to erythroid cells, Epo binding to megakaryocytes increased with cell maturation, with stage IV megakaryocytes exhibiting the highest specific binding. Grain density per surface area however, remained constant during megakaryocyte maturation and was approximately 25% that on pronormoblasts.     

Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes

Considerable experimental and clinical evidence suggests a relationship between erythropoiesis and thrombopoiesis. This is supported by observations that erythropoietin (Epo), the primary regulator of erythropoiesis, can affect platelet production when injected into animals. In this study we provide experimental evidence for a direct effect of Epo on thrombopoiesis by demonstrating that 125I-labeled recombinant human Epo binds to rat and mouse bone marrow megakaryocytes. Thus, autoradiographic analysis using cold competition to measure specific binding has been used to demonstrate that Epo binding to megakaryocytes increases with megakaryocyte maturation. When corrected for cell size, Epo binding sites per unit surface area increase from Stage I megakaryoblasts to Stage II megakaryocytes, and then remain approximately constant throughout further megakaryocyte maturation. Receptor density on megakaryocytes is similar to that on pronormoblasts in the rat, and in mice is 60% that on pronormoblasts. No binding of Epo to platelets or to naked megakaryocyte nuclei was detected. Equilibrium binding studies with partially purified rat megakaryocytes (20%-40% pure), where megakaryocytes are the only significant Epo binding cell population, showed a single class of saturable, high-affinity binding sites present on average at 6500 binding sites per megakaryocyte with a KD of 287 pM. Binding of [125I]Epo to rat megakaryocytes was inhibited with an antiserum against murine erythroblasts. These results suggest that the effects of Epo on thrombopoiesis may be directly mediated through specific, high-affinity binding sites for Epo on the surface of maturing megakaryocytes.     

Immuno-electron microscopical demonstration of lysosomes in human blood platelets and megakaryocytes using anti-cathepsin D

Immunocytochemistry with affinity-purified anti-human cathepsin D was applied to ultrathin frozen sections of human bone marrow megakaryocytes and of blood platelets from peripheral blood. The fixative used was paraformaldehyde (concentration gradient 2----8%). Protein A/colloidal gold (5 and 8) particles were used as second label. Cathepsin D was localized in primary and secondary lysosomes in blood platelets and in primary and secondary lysosomes in megakaryocytes. Primary lysosomes in megakaryocytes were identified by their localization on the trans-side of the Golgi complex and secondary lysosomes by the presence of inclusions. The lysosomes in platelets differed from alpha-granules by being smaller, lacking an electron dense core, and by the presence of a transparent submembrane halo. Platelets undergoing a release reaction after stimulation with thrombin showed cathepsin-D staining in the surface-connecting tubules.     

The significance of megakaryocyte size

Normal guinea pig and human megakaryocytes in suspension were measured with an optical micrometer. The range of megakaryocyte diameters in both species was from 10 to about 65 micrometer. Approximately 20%-25% of megakaryocytes were smaLler than 20 micrometer in diameter and were mostly missed in past studies. However, virtually the entire population of megakaryocytes was larger than all but a very small percent of the other marrow cells. This size range and the existence of a visual threshold size between the megakaryocytes and nonmegakaryocytes were confirmed by flow cytometric analysis of fresh unfixed cells. On human bone marrow smears there was some flattening of all cell types, but the megakaryocytes were consistently at least minimally greater in size than almost all the nonmegakaryocytes. Normal marrow cells greater than 20 micrometer in diameter were always megakaryocytes. Cells 14-20 micrometer were still noticeably larger than the general marrow population; thus easily found, they could be examined for specific morphological criteria. Size, therefore, is a useful first criterion for the identification of megakaryocytes. The larger sizes of megakaryocytes were related to their greater DNA content per cell (polyploidy) compared to nonmegakaryocytes. The relationship between megakaryocyte size, ploidy, and maturation was examined by the simultaneous measurement for the first time of each of these parameters in the same cell. Maturation was quantitated by the new scheme based on the progressive changes in megakaryocytes nuclear configuration. Within each maturation stage the mean cell volume of guinea pig megakaryocytes doubled with each ploidy doubling. Within each ploidy group, the sizes of megakaryocytes increased with maturation stage. However, maturation and polyploidization appear to be linked; the data showed that 80% of the low ploidy (4N-8N) megakaryocytes were immature and that 95% of the platelet-shedding megakaryocytes were 16N-32N.     

Platelet receptor expression on three human megakaryoblast-like cell lines.

Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.