The control of megakaryocyte ploidy and platelet production: biology and pathology

Following experimental platelet destruction in animals, large platelets, which are more hemostatically active, are produced before any change in bone marrow megakaryocyte DNA content. When platelet production is stimulated by administration of i.v. vincristine in rats, megakaryocyte ploidy is increased, but mean platelet volume is unchanged. When platelet production and destruction are both stimulated by chronic hypoxia or administration of anti-platelet serum, mean platelet volume and megakaryocyte DNA content are both increased. Since platelet volume is determined primarily at thrombopoiesis, these results imply that mean platelet volume and megakaryocyte DNA content are under separate hormonal control. Therefore, it has been postulated that changes in mean platelet volume occur following changes in platelet production rate, whereas changes in megakaryocyte ploidy are associated with an increased rate of platelet production. In myocardial infarction, platelets have increased mean volume and reduced bleeding time more than in controls. In addition, men with myocardial infarction have increased megakaryocyte size and increased DNA content when compared to controls. These changes are similar to those observed in rabbits following cholesterol feeding. If megakaryocyte polyploidy and mean platelet volume are under separate hormonal control, this suggests that in myocardial infarction, both hormones are active--one stimulating an increased platelet size, the other stimulating the increased megakaryocyte DNA content. In contrast, patients with lymphoma exhibiting a secondary thrombocytosis have no change in mean platelet volume. However, these subjects also have larger bone marrow megakaryocytes when compared to controls. The relation between megakaryocyte size and ploidy implies that the DNA content of these cells is increased in lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human megakaryocyte ploidy.

We reviewed the literature concerning the history of determination of the ploidy of human megakaryocytes and its relationship with diseases. The ploidy of rabbit megakaryocytes was analyzed by microspectrophotometry in 1964, and the analysis of the ploidy in human megakaryocytes was first performed in 1968. Presently, microphotometry and flow cytometry are the primary methods for the evaluation of the ploidy, but they have their merits and demerits. In the ploidy of human megakaryocytes, a peak has often been reported at 16N in healthy individuals, and the next peaks have been observed at 32N and 8N. The results of ploidy analyses have been reported by many investigators to be comparable between patients with idiopathic thrombocytopenic purpura and normal subjects, but various shifts of the peaks have also been documented. The ploidy is often reported to shift to a larger ploidy class in polycythemia vera and essential thrombocythemia, but it has invariably been reported to shift to a smaller class in chronic myelogenous leukemia. In reactive thrombocytosis, the ploidy pattern was reported to be the same as that in normal individuals by some investigators but to shift to a larger ploidy by others. These differences are considered to be due to heterogeneity of the subjects. In myelodysplastic syndrome, the ploidy shifts mostly to a smaller class, but it may show various patterns. We also reviewed the ploidy in other rare hematological disorders, the relationships of the ploidy with diabetes mellitus and atherosclerotic disorders, and its changes in the ontogeny. Details of the mechanism of polyploidization and its biological significance remain unknown, and further advances in the studies of these topics are anticipated.     

The relationship between cell volume,ploidy, and functional activity in differentiating hepatocytes

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radio-immunoassay. The capacity of this cell type to store³⁵S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up^99mTC-colloid sulfur and to incorporate⁵⁹Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.     

The relationship between cell volume, ploidy. and functional activity in differentiating hepatocytes

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1 g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radioimmunoassay. The capacity of this cell type to store 35S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up 99mTC-colloid sulfur and to incorporate 59Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.     

The relationship between megakaryocyte nuclear DNA content and gene expression

Megakaryocytes are a distinct population of bone marrow cells that have the unique feature of increasing their DNA content without undergoing division. The biological effect of ploidy distribution on gene expression, receptor expression and protein synthesis is still unknown. Using molecular hybridization techniques, we have started a systematic analysis of mRNA expression in megakaryocytes for a number of proteins involved in clot formation. These data will be related to ploidy. Platelets are the unnucleated product of megakaryocytes, having their protein content derived from the precursor cell. Therefore, the understanding of the molecular mechanisms regulating megakaryocyte biology and the consequent type and reactivity of platelets produced is of fundamental importance in both physiological and pathological conditions.     

Analysis of megakaryocyte ploidy in patients with thrombocytosis

The DNA content of bone marrow megakaryocytes was analyzed in 24 patients with myeloproliferative disorders, 23 patients with secondary thrombocytosis and 15 normal volunteers using 2-color flow cytometry. Compared with normal controls, the majority of patients with secondary thrombocytosis, polycythemia vera and essential thrombocytosis exhibited a relative increase in higher ploidy (greater than 16N) cells. In contrast, patients with chronic myelogenous leukemia exhibited an increase in lower ploidy cells (less than 16N), with a modal DNA content of 8N. Patients with myeloproliferative disorders tended to show a decrease in the 16N megakaryocyte population compared with patients with secondary thrombocytosis. No correlation between ploidy distribution and platelet count was observed.     

Stimulators of megakaryocyte development and platelet production

The range of known purified and cloned growth factors and their target cells within the megakaryocytic lineage is described. Data are reviewed outlining that megakaryo-cytopoiesis appears to be controlled at two levels: (i) by feedback control via circulating factors, and (ii) by factors within the marrow itself. Hypotheses are presented about the nature of thrombopoietin, its relationship to known growth factors, especially Interleukin-6 (IL-6), and the specificity of a thrombopoietic response following change in the circulating platelet mass.     

The relationship between settled volume and displacement volume in samples of freshwater zooplankton

The results of some volume estimates of zooplankton biomassare presented. The results suggest that, with freshwater zooplankton,the relationship between settled volume and displacement volumeremains relatively constant despite seasonal variations in speciescomposition.     

Regulation of murine megakaryocyte size and ploidy by non-platelet-dependent mechanisms in radiation-induced megakaryocytopenia

Megakaryocytic macrocytosis was evaluated in mice after irradiation with 6.5 Gy 60Co gamma rays. During the second and third months after sublethal irradiation, one or more of the following abnormalities of thrombocytopoiesis was present: thrombocytopenia, megakaryocytopenia, macromegakaryocytosis, a shift to higher ploidies, and enlargement of cells within ploidy groups. After transfusion-induced thrombocytosis, reductions in megakaryocyte size were delayed or absent relative to non-irradiated mice, and there was more of a tendency to shift to lower values for megakaryocyte ploidy. Mice with radiation-induced megakaryocytopenia failed to show rebound thrombocytosis during recovery from immunothrombocytopenia, in spite of further increases in megakaryocyte size and ploidy. The findings support the hypotheses that numbers of megakaryocytes may influence the regulation of megakaryocytopoiesis even when there is an excess of platelets and that ploidy distribution is not the sole determinant of the average size of a population of megakaryocytes. After irradiation, persistent megakaryocytopenia may not severely affect platelet production under steady-state conditions, but the ability of the marrow to respond to homeostatic regulation is compromised.     

Mouse models of diseases of megakaryocyte and platelet homeostasis

Platelets are the small anuclear blood cells that are the product of megakaryocytopoiesis, the process of hematopoietic stem cell commitment to megakaryocyte production and the differentiation and maturation of these cells for platelet release. Deregulation or disruption of megakaryocytopoiesis can result in platelet deficiencies, the thrombocytopenias, with attendant risk of hemorrhage or thrombocytosis, a pathological excess of platelet numbers. Mouse models, particularly those engineered to carry genetic alterations modeling mutations associated with human disease, have provided important insights into megakaryocytopoiesis and deregulation of this process in disease. This review focuses on mouse models of diseases of altered megakaryocyte and platelet number, illustrating the profound contribution of these models in validating suspected roles of disease-associated genetic alterations, promoting discovery of new links between genetic mutations and specific diseases, and providing unique tools for better understanding of disease pathophysiology and progression, as well as resources to define drug action or develop new therapeutic strategies.     

In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice.

The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development.     

The transient relationship between pressure and volume in the pediatric pulmonary system

An accurate understanding of the relationship between pulmonary pressure and volume is required for modeling pulmonary mechanics in a variety of clinical applications. In this study the experimental techniques and mathematical formulations used to characterize viscoelastic materials are applied to characterize transient pulmonary compliance in juvenile swine. Fixed volumes of air were insufflated into 5 swine and held constant for 45 s while the transient decay in tracheal pressure was measured. An analytical model was developed using an optimization scheme that maximized the model fit to the experimental data over the entire time convolution. The initial injected volume was varied to assess the spatial and temporal linearity of the behavior. Model performance was assessed by comparing measured and predicted pressure during insufflations of erratic volume waveforms. It is concluded that the pulmonary impedance of healthy juveniles can be adequately described over a wide volume and frequency range using a relatively simple 5-parameter model that is linear both spatially and temporally.     

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression,grade and DNA ploidy in breast cancer

Quantification of c-erb B-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erb B-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erb B-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erb B-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erb B-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P=0.06). Immunohistochemistry revealed a significant relationship between c-erb B-2 expression and aneuploidy (P=0.04). Cytokeratinpositive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erb B-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erb B-2 expression at a level of more than 3200 molecules (P0.03). At the level of 3600 molecules significance was increased (P=0.004). These levels equated with between 15% and 19% of the samples being classified as c-erb B-2 positive. Application of these cut-off points showed no correlation between c-erb B-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P=0.0015), increased grade correlating with aneuploidy.     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.