Platelet Density and Size: the Interpretation of Heterogeneity

S ummary . Platelets have been separated according to buoyant density using a colloidal silica-polyvinylpyrrolidone system and subjected to electronic sizing. All density populations were found to be heterogeneous in size, the most dense platelets ranging from less than 3 fl to greater than 21 fl in both man and rat. Light platelet fractions contained no platelets greater than 13 fl in either species. Cohort labelling with [⁷⁵Se]selenomethionine showed no indication of significant change in platelet buoyant density with ageing; greater specific activity found in young, dense platelets appears to be related to increased protein synthetic activity shown in vitro and likely to occur also in their precursor megakaryocytes.
It is postulated that dense, intermediate and light platelets are released synchronously by the three different ploidy classes of megakaryocyte, that varying density indicates differing structural characteristics and presumably differences in function. The present findings do not deny the possibility that platelets decrease in size with ageing but if such occurs, it is not associated with a significant change in platelet buoyant density.     

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.     

Immature dense granules in platelets from mice with platelet storage pool disease

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.     

Transitory hypomegakaryocytic thrombocytopenia: aetiological association with ethanol abuse and implications regarding regulation of human megakaryocytopoiesis

We studied a patient with a long history of ethanol abuse who presented to the hospital with profound weakness, anaemia and thrombocytopenia. Evaluation of these problems revealed the patient's bone marrow to be hypercellular but severely iron depleted and almost totally devoid of morphologically recognizable megakaryocytes. However, we were able to detect the presence of non-morphologically recognizable, immature megakaryocytes in the same sample using an immunochemical detection technique. This circumstance allowed us to study the relative importance of both megakaryocyte maturation and peripheral blood platelet count on the production of megakaryocyte colony stimulating activity (Meg-CSA), a putative regulator of the megakaryocyte colony forming unit (CFU-M). The results of our investigations disclosed a rapid decline in serum Meg-CSA levels which preceded recovery of the platelet count and appeared to coincide with the maturation of megakaryocytes into the morphologically recognizable pool. The effect of ETOH on the patient's CFU-M cloning efficiency was also studied. ETOH in amounts up to 454 mg/dl did not inhibit cloning of the patient's peripheral blood CFU-M in plasma clot cultures. Our results suggest that regulation of Meg-CSA production is a complex function which appears to be dependent on a number of factors including the level of megakaryocyte maturation in the marrow. We also speculate that ethanol associated thrombocytopenia may occasionally be brought about by a disruption in the process of megakaryocyte maturation at the level of a progenitor more mature than the CFU-M.     

Acquired cyclic thrombocytopenia-thrombocytosis with periodic defect of platelet function

Summary A periodic fall of platelet number characterizes an acquired pathological condition named cyclic thrombocytopenia. We observed a patient in whom the episodes of thrombocytopenia (platelet number less than 50×10⁹/l) were followed regularly by thrombocytosis (700–2 300 × 10⁹ platelets/l). The period of platelet count fluctuation was about 30 d. Morphological examination of bone marrow showed the cyclic disappearance of mature and immature megakaryocytes: bone marrow cultures revealed a periodic severe defect of both multilineage and single-lineage progenitor cell growth. When platelet count was falling, a mild defect of platelet aggregation and ATP release was observed. while platelet function was normal when platelet count was rising. Prednisone. thymopentine. high-dose intravenous γ-globulin and splenectomy were without effect. After 4 years of cyclic platelet and megakaryocyte fluctuations, stable amegakaryo-cytic thrombocypenia developed and the patient died of haemorrhagic stroke.     

The relation of thrombokinetics to bone marrow megakaryocytes in idiopathic thrombocytopenic purpura (ITP).

In 23 patients with untreated idiopathic thrombocytopenic purpura (TP), the relation of thrombokinetics to quantitative determinations of megakaryocytes in bone marrow sections was studied. The megakaryocytes were classified into maturation stages, and platelet sizes were determined. Megarkaryocyte number and volume per microliter of bone marrow were significantly higher in ITP as compared to controls. The megakaryocyte number and volume were inversely related to the peripheral platelet count. Platelet production rate was significantly increased in ITP and related to the megakaryocyte number and volume. The megakaryocytes were shifted towards more immature forms in ITP, suggesting an increased turnover rate of the expanded recognizable metakaryocyte compartment. Platelet size was significantly increased in ITP, and the mean platelet diameter was 1.6 times normal. There was a significant relationship between platelet size and platelet production rate, as well as an inverse relationship between platelet size and platelet mean life-span (MLS). There was also a significant correlation between platelet size and the proportion of young megakaryocytes.     

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.     

The relationship between megakaryocyte ploidy and platelet volume in normal and thrombocytopenic C3H mice

Previous studies have suggested that platelet volume may be primarily regulated by the ploidy distribution of mature bone marrow megakaryocytes. However, earlier investigations from this laboratory using C57/BL mice have shown that in response to acute, severe, or moderate thrombocytopenia, platelet volume is regulated independently of megakaryocyte ploidy. Murine strains, including C57/BL, usually have a modal bone marrow megakaryocyte ploidy class of 16N. In contrast, the C3H mouse has a 32N modal bone marrow megakaryocyte ploidy class. We have examined the platelet count, platelet volume distribution, and bone marrow megakaryocyte ploidy distribution of C3H mice during steady-state thrombopoiesis and after depletion of platelets by antiplatelet serum. The platelet count and volume of normal C3H mice were not substantially different from those of C57/BL mice, but megakaryocyte frequency was marginally greater (p less than 0.05), and the ploidy distribution exhibited a a marked reduction in the proportion of 16N cells (p less than 0.001) and increased relative frequencies of 32N (p less than 0.001) and 64N (p less than 0.01) megakaryocytes. In response to acute severe thrombocytopenia, C3H mice demonstrated an increase in platelet volume equivalent to that previously reported for C57/BL mice, without a subsequent shift in the modal megakaryocyte ploidy class. The relative frequencies of 64N and 128N megakaryocytes increased significantly (p less than 0.005) compared to normal C3H mice, without a change in the frequency of 32N megakaryocytes. These studies indicate that during steady-state thrombopoiesis, a greater proportion of higher ploidy megakaryocytes (32N plus 64N) does not necessarily alter peripheral platelet count or platelet volume. Therefore, it appears that neither platelet volume nor count are primarily regulated by bone marrow megakaryocyte ploidy and that the magnitude of upward regulation of megakaryocyte ploidy is limited.     

Mature micromegakaryocytes: an unusual developmental pattern in term infants

We sought to gain perspective on platelet production in the fetus and the newborn by counting and characterizing megakaryocytes from available cord blood. Elutriation was used to isolate circulating megakaryocytes from umbilical arteries and veins obtained at scheduled caesarean sections of nine normal term fetuses. Megakaryocytes were identified by established criteria, their diameters measured, and maturation stages recorded. Large numbers of megakaryocytes, mostly mature, were found in both the umbilical arteries and veins, many times more than previously observed circulating in adult blood. In term infants more than a third of the mature megakaryocytes had unusually decreased nuclear lobation and were dwarf cells with diameters as small as 13 μm, which we considered to be micromegakaryocytes. The atypicality of these small but mature cells is seen as merely a leftward skewing in the development of megakaryocyte ploidies. We believe that in normal fetuses the extent of megakaryocyte ploidization and development is distinctive and probably regulated differently to the adult pattern.     

Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow: effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation

Guinea-pig bone marrow megakaryocytes were isolated using an antibody to platelet glycoprotein Ib and a second antibody conjugated to magnetic beads. The procedure yielded an average of 644,800 megakaryocytes from two guinea-pigs with an average viability of 83%. All of the platelet glycoprotein Ib positive cells also expressed the platelet glycoprotein IIb-IIIa complex. The size and ploidy of megakaryocytes isolated by this technique were analysed in the presence of 10 ng/ml of interleukin-6 (IL-6). Without IL-6 megakaryocyte size increased significantly after 24 h, but an even larger increase in size occurred in the presence of IL-6. The modal ploidy class was 16N with an average of 19% 2N, 2.6% 4N, 16.4% 8N, 50.8% 16N and 11.1% 32N cells as determined by flow cytometry. Measurements made by microspectrophotometry were in close agreement. After 24 h incubation there was a significant rise in the percentage of 2N and 32N cells. The ploidy distribution after 24 h with IL-6 was the same as the control. Megakaryocytes cultured in the absence of serum on collagen gels did not form pseudopods and fragment, as occurs with serum (Leven et al, 1987). Addition of IL-6 to the serum-free cultures caused megakaryocytes to form extensive proplatelet extensions. We conclude that large numbers of pure guinea-pig bone marrow megakaryocytes can be isolated by immunomagnetic bead selection, including low ploidy immature megakaryocytes. Spontaneous maturation occurred as evidenced by the increase in megakaryocyte size and ploidy. IL-6 altered megakaryocyte size and morphology but not ploidy, indicating that these different characteristics of megakaryocytes may be regulated separately.     

The effects of low-dose vincristine on megakaryocyte colony-forming cells and megakaryocyte ploidy

S ummary. The administration of low-dose vincristine (VCR) (0.1 mg/kg) to mice resulted in thrombocytosis without prior thrombocytopenia. No significant changes in marrow megakaryocyte numbers were found. However, after a minor early decrease, mean megakaryocyte ploidy increased, with a peak at 3 d. The number of megakaryocyte colony-forming cells (MEG-CFC) in bone marrow did not change significantly. In contrast with the effects on marrow, the concentration of megakaryocytes and the content of MEG-CFC in the spleen were significantly reduced for 1–2 d after VCR. This reduction was followed by a compensatory rise in the splenic content of MEG-CFC (peak 3-fold increase at 3 d), and 1–2 d later, an increase in splenic megakaryocytes which was concurrent with the increased platelet count. Culture of marrow and spleen cells in the presence of VCR resulted in inhibition of megakaryocyte colony formation at concentrations > 5 ng/ml and parallel reduction of the number of megakaryocytes per colony and the mean ploidy of colony megakaryocytes.
The results suggest that the thrombocytosis induced by low-dose VCR does not result simply from an effect on platelets, but reflects compensatory changes in megakaryopoiesis secondary to toxic suppression of megakaryocytes and their progenitors.     

Leukemia inhibitory factor can potentiate murine megakaryocyte production in vitro

Receptors for murine leukemia inhibitory factor (LIF) were demonstrated on immature and mature murine megakaryocytes. LIF alone had no effects in culture on the survival or proliferation of normal murine megakaryocytes or their precursors. However, combination of LIF with multipotential-colony-stimulating factor (Multi-CSF) (interleukin-3) enhanced the megakaryocyte colony formation able to be stimulated by Multi-CSF; the enhancement involved all types of megakaryocyte colony and resulted in the formation of increased numbers of megakaryocytes. These observations provide a possible basis for the observation that, when LIF is injected in vivo, elevations are observed in megakaryocyte numbers and platelet levels.     

Mature micromegakaryocytes: an unusual developmental pattern in term infants

We sought to gain perspective on platelet production in the fetus and the newborn by counting and characterizing megakaryocytes from available cord blood. Elutriation was used to isolate circulating megakaryocytes from umbilical arteries and veins obtained at scheduled caesarean sections of nine normal term fetuses. Megakaryocytes were identified by established criteria, their diameters measured, and maturation stages recorded. Large numbers of megakaryocytes, mostly mature, were found in both the umbilical arteries and veins, many times more than previously observed circulating in adult blood. In term infants more than a third of the mature megakaryocytes had unusually decreased nuclear lobation and were dwarf cells with diameters as small as 13 μm, which we considered to be micromegakaryocytes. The atypicality of these small but mature cells is seen as merely a leftward skewing in the development of megakaryocyte ploidies. We believe that in normal fetuses the extent of megakaryocyte ploidization and development is distinctive and probably regulated differently to the adult pattern.     

Megakaryocytopoiesis: characterization and regulation in normal and pathologic states

Data concerning megakaryocytopoiesis and its regulation were summarized in this report. Critical analysis of these data indicates that: (i) megakaryocytopoiesis is a complex, multiple-stage cellular and biologic process; (ii) the survival, proliferation and differentiation of progenitor cells into immature megakaryocytes are regulated mainly by interleukin-3, granulocyte-macrophage colony-stimulating factor and an as yet uncharacterized megakaryocyte colony-stimulating factor, and the maturation of immature megakaryocytes to produce platelets is regulated primarily by interleukin-6 and thrombopoietin; (iii) optimal megakaryocyte development needs adequate interactions of several growth factors with target cell population and hematopoietic microenvironment; (iv) megakaryocytopoietic inhibition is controlled essentially by megakaryocyte-platelet products such as transforming growth factor-beta, and platelet factor 4 and its related proteins; interferon-alpha and -gamma also are able to play an inhibitory role; and (v) expansion or decrease of either normal or neoplastic megakaryocyte progenitor cells, change of platelet mass and abnormalities of growth factor levels in hematopoietic tissue might result in an abnormal megakaryocytopoiesis.     

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 μg/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 μg/kg/d resulted in platelet counts 5-fold above normal. However, doses >60 μg/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.     

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.     

In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

Stimulation of megakaryocytopoiesis by acute thrombocytopenia in rats

Rats were made acutely thrombocytopenic by injection of antiplatelet serum. Marrow sections and squash preparations were made at intervals during 120 hr. Determinations were made of mitotic index, stage of maturation, ploidy level, and cell size of megakaryocytes; number and size of platelets were measured. Increased endomitosis among megakaryocytes was followed by an increase in the proportion of immature megakaryocytes, a greater average ploidy level of recognized megakaryocytes, and larger megakaryocytes. Maximum changes in these several parameters occurred between 32 and 72 hr after induction of thrombocytopenia. By 120 hr all megakarocyte parameters were near normal. For about 3 days, beginning at about 36 hr, platelet numbers increased rapidly. Average platelet size rose and returned to normal within about 60 hr. Changes in ploidy and size of megakaryocytes were measured in the immature and mature maturation stages. The results suggest that the initial stimulus in response to acute thrombocytopenia acts primarily on diploid precursors, programming them to mature into a population of megakaryocytes with an average ploidy approximately one level greater than in normal rats and a proportionate increase in cell size. The larger megakaryocytes presumably produce more platelets, accounting for a major part of the increased rate of platelet production. Since the changes in megakaryocytes begin to reverse before circulating platelet numbers have reached the normal level, reversal of the stimulus appears to be initiated by some change other than platelet mass.