Megakaryocyte Concentration Research Articles

Platelet Biogenesis in the Lung Circulation

Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals (c-mpl-/-) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl-/-animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. DisclosuresNo relevant conflicts of interest to declare.

1,25(OH) 2 vitamin D 3 ‐dependent inhibition of platelet Ca 2+ signaling and thrombus formation in klotho‐deficient mice

Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.

Rapid and Simple Procedure For Isolation and Concentration Of Human Megakaryocytes From Marrow Aspirates

BackgroundNumerical and morphologic abnormalities of megakaryocytes (MKs) are present in a variety of primary or secondary bone marrow (BM) disorders such as myeloproliferative neoplasms, myelodysplastic syndromes or ITP. Currently, these changes are assessed exclusively on microscopic preparations of marrow aspirates and biopsies and are used as criteria for disease diagnosis, classification and therapy monitoring despite the inherent subjectivity of microscopic evaluations. In contrast to other cells, adequate studies of freshly isolated MKs have proven difficult because of the relative rarity of these cells in the BM (∼0.05% of total nucleated cells) and separation techniques such as density gradients, magnetic beads, centrifugal elutriation or fluorescence activated cell sorting are labor-intensive, time-consuming or costly. The primary means for studying MKs is based on the isolation of progenitors primed with cytokines to differentiate in culture. While extremely valuable, these techniques are not directly translatable to the routine clinical arena for assessing MK pathology in human BM. Mature marrow MKs are large, polyploid cells whose size distribution overlaps minimally with that of all other marrow cells. This distinct size threshold is a discriminatory parameter for MK isolation. Thus, we developed a simple and inexpensive manual mesh filtration method for separation of MKs that allows a rapid and easy, size-based concentration and purification of these cells. MethodsWe examined 15 discarded anonymized BM aspirate samples suitable for our analysis. These samples were from patients with a variety of hematologic disorders originally submitted to our laboratory for evaluation. Sample age ranged from 1-4 days (mean 1.5 days). Samples were diluted with heparin/albumin-containing buffered saline solution. The cell suspensions were first filtered through a 70µm filter to remove large BM stromal particles and then twice (by gravity) through 9mm-diameter wetted 8 µm nylon filters placed in a simple plastic holder. This filtration procedure allowed the retention of MKs and removal of smaller cells. The MK-rich suspension was collected by rinsing and flushing the filters with the buffered solution. On average, the procedure only lasted 15 minutes. The pre-filtered, 8 µm filter-retained, and effluent cell suspensions collected were stained with an FITC-anti CD61 or PE-anti CD41a antibody [Becton Dickinson (BD)] to label MKs, concurrently with the cell permeable DNA-binding DRAQ5 (Cell Signaling Technology) to identify all nucleated cells. Final cell viability was assessed by C12Resazurin (Molecular probes). Cell analysis was performed by flow cytometry (FCM) using a CANTO II flow cytometer (BD). Absolute MK enumeration was performed using the Flow Cytometry Absolute Count Standard beads (Bangs). MK enrichment efficiency was expressed as the percentage of MKs of all nucleated cells determined by FCM. No cell lysing, fixation or centrifugation was used at any step of the MK separation procedure. ResultsThe median (range) volume of the BM aspirate samples used in this study was 0.54 (0.24-1.99) mL. MKs were identified by FCM on the basis of their large size, expression of platelet-associated antigens and DNA ploidy levels. The median (range) MK recovery was 31 (14-100) % of the original number of MKs and the yield was 9,882 (1,519-49,921) MKs per mL of BM aspirate. The median (range) fraction of MKs among all nucleated cells after filtration was 39 (14-68) %, representing a 904 (439-3029)-fold MK enrichment. The MK viability after filtration was near 100%. ConclusionsThis simple, gentle, rapid and inexpensive isolation and concentration method results in a MK recovery and purification that is comparable or better than other more elaborate techniques. Despite the inherent heterogeneity of the samples used, we obtained a reasonably good recovery of MKs per mL of marrow aspirate and more than 900-fold median MK concentration. The yield and level of purification of freshly isolated MKs obtained by this simple procedure may be useful in studies of a variety of primary or secondary marrow disorders. In particular, it should facilitate the application of analytical methods such as flow cytometry or in situ hybridization, and even be useful for biochemical or molecular testing that require adequate cell representation and purity. Disclosures:No relevant conflicts of interest to declare.

Neonatal Megakaryocytes Do Not Mature Normally In the Absence of Thrombopoietin In Vivo: Potential Role of Developmental Differences In CXCR4 Expression

AbstractAbstract 88Thrombopoietin (Tpo) and its receptor (c-mpl) constitute the main regulatory axis of megakaryocyte (MK) proliferation and maturation. Accordingly, adult Tpo and c-mpl knockout (KO) mice exhibit an 85% reduction in MK concentration, although the residual 10–15% MKs are ultrastructurally normal. The phenotype of newborn Tpo or c-mpl KO mice has not been well characterized, but we and others have described substantial molecular differences between neonatal and adult megakaryocytopoiesis, including several pathways that mediate the high proliferative rate of neonatal MK progenitors. Recently, two groups reported neonates with c-mpl mutations associated with congenital amegakaryocytic thrombocytopenia, who in the neonatal period exhibited normal numbers of immature appearing marrow MKs. The fact that these infants were severely thrombocytopenic at that time suggests that their MKs did not produce platelets normally. These reports, coupled with our recent observation that Tpo mediates the cytoplasmic maturation of human neonatal MKs, led us to hypothesize that, during fetal and neonatal life, Tpo-independent pathways predominantly stimulate MK proliferation, while MK maturation is Tpo-dependent. To test this hypothesis, we studied the characteristics of MKs generated in vivo in neonates in the absence of c-mpl. Since we have previously demonstrated that the liver is the main site of megakaryocytopoiesis in newborn mice, we evaluated MKs in the livers of c-mpl KO and WT mice (both C57BL/6) on day of life 1 and 3. As a first step, we quantified MKs immunohistochemically stained with an anti-vWF antibody, and found that MKs in the liver of newborn c-mpl KO mice were reduced by approx. 70%. Next, we examined the ultrastructure of these liver MKs by transmission electron microscopy, and categorized c-mpl KO MKs (n=28) and WT MKs (n=32) as stage I (immature), stage II (abundant alpha-granules and a developing demarcation membrane system, DMS), or stage III (platelet producing MKs, with an open DMS). According to these criteria, 50% of WT MKs were stage II, and 50% were stage III. In contrast, 22% of c-mpl KO MKs were stage I, 57% were stage II, and only 21% were stage III. Furthermore, significant ultrastructural abnormalities were found in 70% of c-mpl KO MKs, including decreased numbers of platelet granules, a very disorganized appearing closed demarcation membrane system, and/or an abnormally wide peripheral zone. Since MKs in adult mice mature normally in the absence of Tpo, we then hypothesized that our findings reflected a downregulation of Tpo-independent pathway(s) mediating MK maturation in neonates. In that regard, we recently found that the microRNA miR9 was expressed at 10- to 14-fold higher levels in murine fetal and neonatal compared to adult MKs. Since CXCR4 (the receptor for SDF-1) is a predicted target of miR9, and in view of recent studies characterizing the role of the SDF-1/CXCR4 axis as a Tpo-independent pathway that stimulates MK maturation, we evaluated CXCR4 protein expression in cultured MKs derived from murine fetal liver (E13.5), newborn liver, and adult bone marrow, by Western Blot. As predicted, CXCR4 protein levels were significantly lower in fetal and neonatal compared to adult MKs (p=0.003). To evaluate the significance of these findings in humans, we then quantified miR9 and CXCR4 protein levels in cord blood-derived and adult peripheral blood-derived human MKs (n=3 per group). Consistent with the murine findings, we found that miR9 levels were approximately 20-fold higher and CXCR4 protein levels were significantly lower in human neonatal compared to adult MKs (p<0.05 for both). Finally, to determine whether miR9 regulates CXCR4 protein expression, Meg-01 cells were nucleofected with miR9 or Cy3 (control). As hypothesized, up-regulation of miR9 resulted in a significant reduction in CXCR4 protein levels compared to control cells (p=0.02). In conclusion, our findings indicate that MKs in the neonatal period do not mature normally in the absence of Tpo, presumably due to a deficiency in Tpo-independent pathway(s) of MK maturation at this developmental stage. Our data also identified a developmental downregulation of CXCR4 protein expression by miR9 in fetal and neonatal MKs. Given the role of the SDF/CXCR4 axis mediating Tpo-independent MK maturation, this provides a potential mechanism to explain the c-mpl KO findings. Disclosures:No relevant conflicts of interest to declare.

Differences between Newborn and Adult Mice in Their Response to Immune Thrombocytopenia

Background: Sick neonates frequently develop severe thrombocytopenia. Objective and Methods: In order to test the ability of fetal mice to increase their megakaryocyte size and ploidy in response to thrombocytopenia, we injected an antiplatelet antibody (MWReg30) into pregnant mice daily for 7 days, and into nonpregnant adult mice to serve as controls. After that time, platelet counts were obtained and megakaryocytes in the bone marrow, liver, and spleen were stained with anti-von Willebrand factor antibody, individually measured, and quantified. Results: Our study demonstrated that megakaryocytopoiesis in newborn mice shares many features of human fetal/neonatal megakaryocytopoiesis, including the small size of megakaryocytes. In response to thrombocytopenia, adult mice increased megakaryocyte volume and concentration, primarily in the spleen. Newborn mice, in contrast, increased the megakaryocyte concentration in the spleen, but exhibited no increase in megakaryocyte volume in any of the organs studied. In fact, the megakaryocyte mass was significantly lower in the bone marrow of thrombocytopenic neonates than in age-matched controls. Conclusions: We concluded that fetuses have a limited ability to increase their megakaryocyte mass in response to consumptive thrombocytopenia, compared to adult mice. These observations provide further evidence for the existence of biological differences between fetal/neonatal and adult megakaryocytopoiesis.

Megakaryocyte Size and Concentration in the Bone Marrow of Thrombocytopenic and Nonthrombocytopenic Neonates

Megakaryocyte Size and Concentration in the Bone Marrow of Thrombocytopenic and Nonthrombocytopenic Neonates

Sites and kinetics of donor thrombopoiesis following transplantation of whole bone marrow and progenitor subsets

Little is known about the sites and kinetics of thrombopoiesis following bone marrow transplant. The spleen is a site of hematopoiesis in a healthy mouse, and hematopoietic activity increases in response to stress. We hypothesized that the spleen is a major site of early post-transplant thrombopoiesis. We transplanted whole bone marrow (WBM) or lineage depleted progenitor subsets fractionated based on expression of c-kit and Sca-1 from transgenic mice expressing green fluorescent protein into lethally irradiated C57BL/6 recipients. We also transplanted whole bone marrow cells into healthy and splenectomized mice. Post-transplant megakaryopoiesis was assessed by measuring circulating platelet number, percent donor-derived platelets, bone marrow cellularity, splenic weight, megakaryocyte size, and megakaryocyte concentration from hour 3 to day 28 post transplant. Following transplant, circulating donor-derived platelets were derived only from c-kit expressing subsets. Donor-derived platelets first appeared on post-transplant day five. Splenectomy reduced the number of these earliest circulating platelets. Splenic megakaryopoiesis increased dramatically from day 7-14 post-transplant. However, splenectomy accelerated platelet engraftment during this time frame. Overall, these results demonstrate that the first platelets are produced by c-kit expressing megakaryocyte progenitors in the bone marrow and spleen. After post-transplant day 5, the net effect of the spleen on thrombopoiesis is to slow engraftment due to immune effects or hypersplenism.

Megakaryocyte Size and Concentration in the Bone Marrow of Thrombocytopenic and Nonthrombocytopenic Neonates

Thrombocytopenia is frequent among sick neonates, but little is known about its underlying mechanisms. It is known, however, that neonatal megakaryocytes are smaller and of lower ploidy than their adult counterparts and that smaller megakaryocytes produce fewer platelets than larger, more polyploid, megakaryocytes. We hypothesized that neonatal megakaryocytes would not increase their size in response to thrombocytopenia, thus limiting the ability of neonates to mount a response. To test this, we obtained marrow specimens from thrombocytopenic and nonthrombocytopenic neonates and adults. Megakaryocytes were immunohistochemically stained, quantified using an eyepiece reticle, and measured using an image analysis system with incorporated electronic micrometer. We found that, after adjusting for differences in cellularity, neonates and adults had similar megakaryocyte concentrations. When samples from the same sources were compared (tibial clot and vertebral body sections in neonates, iliac crest biopsies in adults), there were also no differences in megakaryocyte concentration between thrombocytopenic and nonthrombocytopenic subjects. The megakaryocyte diameter, however, was greater in adults than in neonates (19.4 +/- 3.0 versus 15.3 +/- 1.7 microm, p<0.0001). Thrombocytopenic adults also had a higher proportion of large megakaryocytes than nonthrombocytopenic adults (p<0.001). This was not observed among thrombocytopenic neonates, suggesting a developmental limitation in their ability to increase megakaryocyte size.

Umbilical Cord Blood Produces Small Megakaryocytes After Transplantation

Delayed platelet engraftment is a major complication of umbilical cord blood (CB) transplantation. Megakaryocytes derived from CB in vitro are smaller than megakaryocytes derived from bone marrow (BM) or mobilized peripheral blood from adults. Small megakaryocyte size may contribute to delayed platelet engraftment. To test whether small size persists after transplantation, we measured megakaryocyte size, concentration, and maturational stage in BM biopsy specimens obtained after transplantation in archived BM samples from patients receiving CB (CB group, n = 10) versus mobilized peripheral blood or BM transplantation (BM group, n = 9). Megakaryocytes in the postengraftment BM samples were significantly smaller in the CB group than in the BM group (median diameter, 16.7 vs 22.0 μm). There were no significant differences in megakaryocyte concentration or maturational stage between the CB and BM groups. For the first time, we demonstrate that the attainment of adult size in CB-derived megakaryocytes is delayed after human CB transplantation.

Alfa-interferon in the treatment of essential thrombocythemia: clinical results and evaluation of its biological effects on the hematopoietic neoplastic clone

The efficacy of alfa-interferon (alfa-IFN) in essential thrombocythemia (ET) patients has been reported by several authors. The aim of this study is to assess the magnitude of the effect of alfa-IFN on the neoplastic clone. As of December 1993, 11 ET patients received alfa-IFN at a dose of 3-6 MU/s.c./day for 6 months. Ten of 11 obtained complete hematological remission (CHR) and one achieved partial hematological remission. Megakaryocyte concentration was reduced in six cases. The spleen,which was enlarged in four patients, decreased in size in two patients. Seven of eight patients who were symptomatic at diagnosis obtained resolution of symptoms. In order to obtain indications about the structural modifications induced by alfa-IFN in ET megakaryocytes (Mks), Fourier-transform infra-red microspectroscopy analysis performed on 10 single Mks of each patient, was done in seven of 11 patients; the analysis showed a reduction of A1/A2 ratios (A1 integrated area of the band at 1080 cm(-1) due to the nucleic acids absorption; A2 integrated area of the band at 1540 cm(-1) due to proteic components absorption) in five cases, and in three of these five patients A1/A2 ratios achieved normal values. After alfa-IFN treatment we did not observe any change in the methylation pattern of DNA from the granulocyte fraction. Our results confirm the efficacy of alfa-IFN in ET patients, and the decrease of A1/A2 ratios in several patients is a demonstration of the depth of the effect of alfa-IFN on the neoplastic clone. The results of clonality studies showed the persistence of clonal hematopoiesis. Whether higher alfa-IFN dose and/or more prolonged alfa-IFN therapy may allow a restoration of polyclonal hematopoiesis remains to be determined and should be explored in future clinical trials.

Megakaryocytes and Platelets as the Main Cause for Vascular Events in Chronic Myeloproliferative Disorders

SummaryMegakaryocytes are part of clonal hematopoiesis in chronic myeloproliferative disorders and are responsible for most of the clinical complications in this disease. About 30-40% of patients with polycythemia vera (PV) and essential thrombocythemia (ET) suffer from thrombotic complications, and microcirculatory disorders are common. Spontaneous bleeding mainly from the gastrointestinal tract is another complication that is especially prevalent in myelofibrosis and advanced stages of chronic myeloid leukemia.In vivo, the bone marrow is hypercellular and the concentration of megakaryocytes increased with characteristic morphological abnormalities. Megakaryocytes are enlarged and ploidy is increased in PV and ET but small mononuclear cells with decreased ploidy are a feature of CML. Despite spontaneous growth in cul-ture, megakaryocytes in chronic MPD are hypersensitive to added interleukin-3, interleukin-6 and GM-CSF.Platelets released from these megakaryocytes show abnormal morphology and ultrastructure, reflected in loss of storage granules and organelles, increased volume distribution and low buoyant density. Uptake, storage and secretion of platelet dense granule constituents is abnormal, and the plasma levels of platelet specific proteins which may also include growth factors for fibroblasts are elevated. At high platelet counts, spontaneous aggregation is observed, whereas agonist-induced aggregation in vitro with adrenaline, ADP and collagen is often defective. Platelet thromboxane generation may be stimulated, and production along the lipoxygenase pathway is decreased. Abnormalities of glycoprotein receptors and decreased fibrinogen binding have been reported but their clinical significance is uncertain. Several observations suggest that not only receptor defects but ineffective intracellular signalling may be responsible for platelet function abnormalities.No single underlying defect has been discovered that could explain this variety of pathological findings. Moreover, a combination of intrinsic megakaryocyte abnormalities and increased susceptibility of platelets to activation makes it difficult to differentiate secondary phenomena from effects of clonal hematopoiesis. How-ever, there are some clinical guidelines for therapy.Most elderly patients will be treated with cytoreductive therapy. Alkylating drugs and 32P have been shown to be leukemogenic, but even hydroxyurea may have a 10% incidence of leukemia induction after long-term therapy. Therapy with platelet-inhibitory drugs is often not sufficient to control thrombosis, and may aggravate a bleeding tendency, so that younger patients with PV and ET are increasingly treated with anagrelide or interferon alpha (A-IFN). Anagrelide is a quinazolin derivative that specifically inhibits megakaryocytopoiesis, while A-IFN may suppress clonal hematopoiesis by an unknown mechanism.

Megakaryocytopoiesis and platelet production are stimulated during late pregnancy and early postpartum in the rat

Platelet count during uncomplicated pregnancy shows considerable patient variation. To gain a better understanding of thrombocytopoiesis during pregnancy, megakaryocytes and platelets were examined during gestation and the early postpartum period, using as a model the rat. Platelet counts and megakaryocyte concentrations and DNA content distributions of timed-pregnant rats were examined at intervals from day 10 of gestation through parturition on day 22 and days 1 through 7 postpartum. Platelet survival was studied in late gestation and the early postpartum. Platelet volume was measured on gestation day 21. Platelet counts were moderately increased on gestation days 17 and 19 through 22, and on days 2 to 3 postpartum. However, the actual rate of platelet production was much higher than the platelet count suggests because the blood volume increased in late gestation to 1.5 times the nonpregnant level. Mean platelet volume and platelet volume distribution width of day 21 gestation rats were not significantly altered. Platelet survival in pregnant rats was not significantly different from that in nonpregnant females. In contrast, megakaryocyte concentration was significantly increased on gestation days 12, 17, and 19 through 21, and 2 to 3 days postpartum. In addition, in late gestation, megakaryocyte DNA content distributions displayed a marked increase in the proportion of high ploidy cells, which peaked 1 day before parturition. At that time, the proportions of 32N (43%) and 64N cells (3%) were, respectively, three and four times nonpregnant values. In contrast to megakaryocyte concentration, megakaryocyte DNA content distributions had returned to the nonpregnant pattern by day 1 postpartum. The changes in megakaryocyte DNA content distribution were accompanied by changes in megakaryocyte size. These data indicate that thrombopoiesis is substantially increased during late pregnancy, and that this increase is accomplished through an increase in megakaryocyte DNA content and size, as well as megakaryocyte number. The more rapid return of megakaryocyte DNA content than of megakaryocyte concentration to nonpregnant levels postpartum suggests that pregnancy-associated hormonal changes which produce an increase in megakaryocyte DNA content and size differ from those which cause an increase in megakaryocyte number.

Megakaryocytopoiesis and platelet production are stimulated during late pregnancy and early postpartum in the rat

Platelet count during uncomplicated pregnancy shows considerable patient variation. To gain a better understanding of thrombocytopoiesis during pregnancy, megakaryocytes and platelets were examined during gestation and the early postpartum period, using as a model the rat. Platelet counts and megakaryocyte concentrations and DNA content distributions of timed-pregnant rats were examined at intervals from day 10 of gestation through parturition on day 22 and days 1 through 7 postpartum. Platelet survival was studied in late gestation and the early postpartum. Platelet volume was measured on gestation day 21. Platelet counts were moderately increased on gestation days 17 and 19 through 22, and on days 2 to 3 postpartum. However, the actual rate of platelet production was much higher than the platelet count suggests because the blood volume increased in late gestation to 1.5 times the nonpregnant level. Mean platelet volume and platelet volume distribution width of day 21 gestation rats were not significantly altered. Platelet survival in pregnant rats was not significantly different from that in nonpregnant females. In contrast, megakaryocyte concentration was significantly increased on gestation days 12, 17, and 19 through 21, and 2 to 3 days postpartum. In addition, in late gestation, megakaryocyte DNA content distributions displayed a marked increase in the proportion of high ploidy cells, which peaked 1 day before parturition. At that time, the proportions of 32N (43%) and 64N cells (3%) were, respectively, three and four times nonpregnant values. In contrast to megakaryocyte concentration, megakaryocyte DNA content distributions had returned to the nonpregnant pattern by day 1 postpartum. The changes in megakaryocyte DNA content distribution were accompanied by changes in megakaryocyte size. These data indicate that thrombopoiesis is substantially increased during late pregnancy, and that this increase is accomplished through an increase in megakaryocyte DNA content and size, as well as megakaryocyte number. The more rapid return of megakaryocyte DNA content than of megakaryocyte concentration to nonpregnant levels postpartum suggests that pregnancy-associated hormonal changes which produce an increase in megakaryocyte DNA content and size differ from those which cause an increase in megakaryocyte number.

An Analysis of Megakaryocytopoiesis in the C3H Mouse: An Animal Model Whose Megakaryocytes Have 32N as the Modal DNA Class

The modal DNA content of normal marrow megakaryocytes from species so far examined usually has been reported to be 16N. In this report we describe an exception in the C3H mouse whose megakaryocytes have a modal DNA content of 32N. Female C3H/HEN mice had an average DNA content distribution of 14% 8N, 37% 16N, 43% 32N, and 6% 64N. Male C3H/HEN mice had somewhat higher proportions of 32N and 64N megakaryocytes (average DNA content distribution of 12% 8N, 29% 16N, 47% 32N, and 12% 64N) than females. All 11 other mouse strains examined had 16N as the modal megakaryocyte DNA content, although the proportions in the various polyploid DNA classes showed some strain variation. Megakaryocyte size was similar among all 12 strains evaluated, and mean platelet volume (MPV) of C3H/HEN mice differed from only 1 of the other 4 strains analyzed. Platelet counts of C3H/HEN mice were similar to those of six, and slightly but significantly lower than those of five other mouse strains examined. Compared with megakaryocyte concentrations of other mouse strains studied, that of C3H/HEN mice was similar to seven, somewhat higher than one, and slightly lower than three strains. Offspring from reciprocal matings of C57BL/6 and C3H/HEN mice had megakaryocyte DNA distributions intermediate between those of the parent strains, suggesting that a higher gene dosage of some component is responsible for the right-shifted megakaryocyte DNA content distribution phenotype of C3H mice. The proportions of 32N and 64N megakaryocytes increased in C3H/HEN mice in response to acute thrombocytopenia, as did those of CBA/CAJ mice used as a comparative strain. In summary, megakaryocytes of the C3H mouse have a higher average DNA content but similar platelet count, MPV, and megakaryocyte size and concentration as those of most other mouse strains. These results suggest that the number of platelets produced per unit of C3H megakaryocyte DNA is less than that for other mice.

An analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class

The modal DNA content of normal marrow megakaryocytes from species so far examined usually has been reported to be 16N. In this report we describe an exception in the C3H mouse whose megakaryocytes have a modal DNA content of 32N. Female C3H/HEN mice had an average DNA content distribution of 14% 8N, 37% 16N, 43% 32N, and 6% 64N. Male C3H/HEN mice had somewhat higher proportions of 32N and 64N megakaryocytes (average DNA content distribution of 12% 8N, 29% 16N, 47% 32N, and 12% 64N) than females. All 11 other mouse strains examined had 16N as the modal megakaryocyte DNA content, although the proportions in the various polyploid DNA classes showed some strain variation. Megakaryocyte size was similar among all 12 strains evaluated, and mean platelet volume (MPV) of C3H/HEN mice differed from only 1 of the other 4 strains analyzed. Platelet counts of C3H/HEN mice were similar to those of six, and slightly but significantly lower than those of five other mouse strains examined. Compared with megakaryocyte concentrations of other mouse strains studied, that of C3H/HEN mice was similar to seven, somewhat higher than one, and slightly lower than three strains. Offspring from reciprocal matings of C57BL/6 and C3H/HEN mice had megakaryocyte DNA distributions intermediate between those of the parent strains, suggesting that a higher gene dosage of some component is responsible for the right-shifted megakaryocyte DNA content distribution phenotype of C3H mice. The proportions of 32N and 64N megakaryocytes increased in C3H/HEN mice in response to acute thrombocytopenia, as did those of CBA/CAJ mice used as a comparative strain. In summary, megakaryocytes of the C3H mouse have a higher average DNA content but similar platelet count, MPV, and megakaryocyte size and concentration as those of most other mouse strains. These results suggest that the number of platelets produced per unit of C3H megakaryocyte DNA is less than that for other mice.

Prolonged Thrombocytosis in Mice After 5-Fluorouracil Results From Failure to Down-Regulate Megakaryocyte Concentration. An Experimental Model That Dissociates Regulation of Megakaryocyte Size and DNA Content From Megakaryocyte Concentration

Rodents treated with 150 mg/kg of 5-fluorouracil (5-FU) exhibit a marked and prolonged rebound thrombocytosis, suggesting that feedback control of one or more megakaryocyte characteristics (size, polyploidy, or concentration) is altered. To determine the changes in megakaryocytes that lead to such a profound thrombocytosis, C3H mice were injected with 150 mg/kg 5-FU, and platelet and megakaryocyte responses were examined at frequent intervals from days 1 through 25. After 5-FU injection, all megakaryocyte indices decreased, as did platelet number. However, the decrease in platelets to one third of control was greater than the decreases in megakaryocyte indices, suggesting that thrombocytopoiesis was ineffective from days 3 through 7 post 5-FU. Megakaryocyte size began to recover on day 4, followed by polyploid DNA content on day 5, and megakaryocyte concentration and platelets at 7.5 days. Megakaryocyte size peaked on days 6 through 8 (1.25 x normal), followed by megakaryocyte polyploid DNA content on day 8, megakaryocyte concentration on days 9 through 12 (2 1/2 to 3x normal), and platelets on days 12 through 15 (2x normal). Platelet levels are thought to be important in the feedback regulation of megakaryocytes; however, only polyploid DNA content distributions showed a close inverse relationship to platelet counts during both the recovery and rebound thrombocytosis phases after 5-FU. In contrast, megakaryocyte size peaked before platelet recovery commenced, while megakaryocyte concentration increased in parallel with platelets from 7.5 to 10 days post 5-FU and continued to be maintained at 2 to 3 times normal through day 13, despite platelet levels that were more than twice normal. Both megakaryocyte size and polyploid DNA content distributions shifted toward lower values in response to the rebound thrombocytosis (DNA content on day 10 and size on days 12 and 13). Splenectomy did not substantially alter the pattern of post 5-FU rebound thrombocytosis or megakaryocyte response from that seen in intact mice, indicating that splenic megakaryocytes are not responsible for the prolonged thrombocytosis seen after this drug. In summary, the prolonged thrombocytosis after 5-FU administration results from failure to down-regulate the number of precursors entering the differentiating megakaryocyte compartment. These data indicate that megakaryocyte size and DNA content are responsive to different feedback controls than megakaryocyte concentration in this model system.

Prolonged thrombocytosis in mice after 5-fluorouracil results from failure to down-regulate megakaryocyte concentration. An experimental model that dissociates regulation of megakaryocyte size and DNA content from megakaryocyte concentration

Rodents treated with 150 mg/kg of 5-fluorouracil (5-FU) exhibit a marked and prolonged rebound thrombocytosis, suggesting that feedback control of one or more megakaryocyte characteristics (size, polyploidy, or concentration) is altered. To determine the changes in megakaryocytes that lead to such a profound thrombocytosis, C3H mice were injected with 150 mg/kg 5-FU, and platelet and megakaryocyte responses were examined at frequent intervals from days 1 through 25. After 5-FU injection, all megakaryocyte indices decreased, as did platelet number. However, the decrease in platelets to one third of control was greater than the decreases in megakaryocyte indices, suggesting that thrombocytopoiesis was ineffective from days 3 through 7 post 5-FU. Megakaryocyte size began to recover on day 4, followed by polyploid DNA content on day 5, and megakaryocyte concentration and platelets at 7.5 days. Megakaryocyte size peaked on days 6 through 8 (1.25 x normal), followed by megakaryocyte polyploid DNA content on day 8, megakaryocyte concentration on days 9 through 12 (2 1/2 to 3x normal), and platelets on days 12 through 15 (2x normal). Platelet levels are thought to be important in the feedback regulation of megakaryocytes; however, only polyploid DNA content distributions showed a close inverse relationship to platelet counts during both the recovery and rebound thrombocytosis phases after 5-FU. In contrast, megakaryocyte size peaked before platelet recovery commenced, while megakaryocyte concentration increased in parallel with platelets from 7.5 to 10 days post 5-FU and continued to be maintained at 2 to 3 times normal through day 13, despite platelet levels that were more than twice normal. Both megakaryocyte size and polyploid DNA content distributions shifted toward lower values in response to the rebound thrombocytosis (DNA content on day 10 and size on days 12 and 13). Splenectomy did not substantially alter the pattern of post 5-FU rebound thrombocytosis or megakaryocyte response from that seen in intact mice, indicating that splenic megakaryocytes are not responsible for the prolonged thrombocytosis seen after this drug. In summary, the prolonged thrombocytosis after 5-FU administration results from failure to down-regulate the number of precursors entering the differentiating megakaryocyte compartment. These data indicate that megakaryocyte size and DNA content are responsive to different feedback controls than megakaryocyte concentration in this model system.

The differential diagnosis of polycythaemia--a bone marrow study (the bone marrow in polycythaemia).

Bone marrow sections from posterior iliac crest aspiration and/or trephine specimens have been examined in 39 patients with true polycythaemia, along with a variety of other clinical and laboratory data. The emphasis has been on objective assessment of cellularity and megakaryocyte concentration in a prospective four year study. In patients with untreated primary polycythaemia mean cellularity was 87.0% and 86.4% for aspirate and trephine specimens compared with 55.5% and 48.7% for secondary cases and 48.3% and 45.5% for controls. Eighty per cent of primary polycythemia patients had moderate to marked megakaryocytic hyperplasia. We conclude that, in the presence of an elevated red cell volume, marrow cellularity of greater than 75%, particularly when accompanied by megakaryocytic hyperplasia, of iliac crest aspirate or trephine specimens is sufficient per se to establish a diagnosis of polycythaemia rubra vera.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia

The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.

The Wistar Furth Rat: An Animal Model of Hereditary Macrothrombocytopenia

The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.