Occurrence of Megakaryocytes in Various Vessels and Their Retention in the Pulmonary Capillaries in Man

A total of 17 patients with hypertension undergoing renal vein or adrenal vein catheterization were investigated in order to ascertain the number of megakaryocytes in blood from the inferior vena cava, the femoral artery and a cubital vein. On an average 11.9, 3.8, and 4.5 megakaryocytes per ml were found, respectively. In blood from the inferior vena cava, 30 % of the megakaryocytes had copious cytoplasm, while megakaryocytes in arterial and cubital venous blood had sparse or no visible cytoplasm. It was demonstrated that ? of the megakaryocytes were retained in the pulmonary circulation and that at least 70% of the platelets could derive from megakaryocytes in central venous blood or the pulmonary circulation. It was found that megakaryocytes pass through a life cycle in which the differentiation take place in the bone marrow, platelet release occurs mainly in central venous blood and in the pulmonary circulation and the destruction of the megakaryocyte nucleus take place outside the bone marrow, especially in the pulmonary circulation.     

Megakaryocytes in cubital venous blood in patients with chronic myeloproliferative diseases

33 patients with chronic myelo-proliferative disease were investigated for megakaryocytes in cubital venous blood. 6 Patients had chronic myeloid leukaemia, 11 polycythaemia vera and 16 myelofibrosis. In all patients, a significantly higher number of megakaryocytes than in normal adult humans was found. In patients with chronic myeloid leukaemia and myelofibrosis, an increased number of intact megakaryocytes was found, indicating thrombopoiesis in the peripheral part of the upper extremity. 2 patients with myelofibrosis showed a % of intact megakaryocytes greater than 25, as found in blood from the inferior vena cava in normal adults. In patients with chronic myeloid leukaemia, some of the megakaryocytes were smaller than normal, whereas in myelofibrosis, some of the megakaryocytes were larger than normal, with increased lobulation of nuclei. Active thrombopoiesis in the peripheral part of the extremities can be of great importance, when splenectomy is considered in treatment of myelofibrosis.     

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.     

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

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

Circulating megakaryocytes in blood from the antecubital vein in healthy, adult humans

A total of 21 healthy, adult men and 30 healthy, adult women aged 21-73 years were investigated for circulating megakaryocytes in the antecubital vein using the saponinhaemolysis leucoconcentration technique. In the males, the number of megakaryocytes in 1.5 ml blood varied from 1 to 20, the arithmetic mean being 10 megakaryocytes. In the females the number varied from 0 to 29 megakaryocytes and the arithmetic mean was 7.6 megakaryocytes. No significant difference was found between the values for the two sexes. There was no correlation between the number of megakaryocytes and platelets or leucocytes. 99% of the observed megakaryocytes were naked nuclei or had sparse cytoplasm and 1% had copious cytoplasm. Megakaryocytes without copious cytoplasm may be regarded as normally occurring cells in the peripheral venous blood.     

Circulating megakaryocytes in patients with pulmonary inflammation and in patients subjected to cholecystectomy

30 patients with pulmonary inflammation and 17 patients subjected to cholecystectomia à froid were investigated for circulating megakaryocytes in a cubital vein using the saponin-haemolysis leucoconcentration technique. The number of circulating megakaryocytes was significantly higher in patients with pulmonary inflammation than in healthy humans. In 15 patients with bronchitis, bronchopneumonia, and leucocytosis the arithmetic mean was 15.6 megakaryocytes per 1.5 ml blood (range 3 to 47). In 15 patients with bronchitis and a normal leucocyte count the arithmetic mean was similarly 14.1 megakaryocytes per 1.5 ml blood. After cholecystectomy a significant maximum increase in the number of circulating megakaryocytes to 3 times the preoperative value was found on the third postoperative day but not on the other postoperative days. Of the observed megakaryocytes 99% had only a narrow rim of cytoplasm or were naked nuclei.     

A role of GAGs in ECM on morphogenesis of megakaryocytes

The morphogenesis of megakaryocytes that results in the formation of cytoplasmic processes is thought to be the final maturation step before liberation of platelets. We studied the in vitro effects of glycosaminoglycans (GAGs) which are abundant in the bone marrow extracellular matrices, on the morphogenesis of murine megakaryocytes and compared them with those of thrombopoietic cytokines. Heparin, heparan sulfate, chondroitin-6 sulfate, and dermatan sulfate promoted the formation of megakaryocytic processes. Hyaluronic acid failed to support this phenomenon, suggesting that sulfated GAGs in extracellular matrices are involved in the morphogenesis of megakaryocytes. Sulfated GAGs began to act on megakaryocytes with a higher ploidy (16N–32N) from 6 to 24 h after incubation, whereas neither rhIL-6 nor rhIL-11 affected this early phase. Our findings indicate that sulfated GAGs promote the morphogenesis of murine megakaryocytes and participate in thrombopoiesis in a different manner from that of cytokines such as rhIL-6 and rhIL-11.     

Emperipolesis of marrow cells within megakaryocytes in the bone marrow of sublethally irradiated mice

The incidence of megakaryocytic emperipolesis was studied in the bone marrow of normal and X-irradiated mice. Two groups of mice received total body irradiation with a single dose of 5 Gy and one of the two groups had been treated with a radioprotective drug, ethiofos (WR-2721), before irradiation. Mice from a third group remained unexposed to irradiation and served as controls. The Wright-Giemsa stained bone marrow smears were analyzed every 5 days during a 30-day period, starting 1 day after irradiation. The number of megakaryocytes exhibiting the phenomenon was determined and expressed as an average value for every experimental group. The frequency of megakaryocytic emperipolesis was less than 15% of megakaryocytes from control smears but increased to 34% in mice that had only been irradiated and to 43% when mice were treated with WR-2721 before irradiation. In the last case, i.e., irradiation and treatment with a radioprotective drug, a positive correlation between the macrocytic megakaryocytes and elevated emperipolesis was noted. Under light microscopy, there were no signs of phagocytosis; engulfed cells remained unaltered with their normal structure intact. Granulocytic, erythroid, and lymphoid cells appeared to be the most frequent marrow cells engulfed by mature megakaryocytes. The number of incorporated cells in one megakaryocyte ranged from 1 to 3, though occasionally more than 6 were seen in macrocytic megakaryocytes. Based on our findings and on a review of the associated literature, we believe emperipolesis is an interesting cellular phenomenon related to the fast passage of marrow cells across the marrow-blood barrier, especially through the cytoplasm of megakaryocytes in response to an increased demand for cell delivery. The high demand for cell delivery which occurs after irradiation may cause certain mature bone marrow cells to take a transmegakaryocyte path to enter the circulation of the blood. Irradiation seems to have an immediate effect (observed after 24 h) on emperipolesis, suggesting that a humoral factor is involved in the pathogenesis.     

Minor dysplastic changes are frequently observed in the bone marrow aspirate in elderly patients without haematological disease.

Bone marrow aspirates were obtained by sternal puncture prior to sternotomy in 54 volunteers (40 males and 14 females) aged 60 years or more. All underwent surgery for cardiological diseases and had normal blood counts, without any haematological abnormalities. Quantitative examination of these bone marrow aspirates yielded reference ranges for each cell type similar to those obtained in younger adults. However, qualitative analysis revealed certain discrepancies: dysplastic changes were observed frequently, mainly in megakaryocytes and erythroblasts, with a normal growth pattern of haematopoietic progenitor cells. A low proportion of macrophages and mast cells were noted in 30% of the bone marrow aspirates. Lymphoid aggregates, seen in 13% of these samples, were generally of moderate size, contained few mast cells and were non-clonal on immunophenotypic analysis.     

Minor dysplastic changes are frequently observed in the bone marrow aspirate in elderly patients without haematological disease

Bone marrow aspirates were obtained by sternal puncture prior to sternotomy in 54 volunteers (40 males and 14 females) aged 60 years or more. All underwent surgery for cardiological diseases and had normal blood counts, without any haematological abnormalities. Quantitative examination of these bone marrow aspirates yielded reference ranges for each cell type similar to those obtained in younger adults. However, qualitative analysis revealed certain discrepancies: dysplastic changes were observed frequently, mainly in megakaryocytes and erythroblasts, with a normal growth pattern of haematopoietic progenitor cells. A low proportion of macrophages and mast cells were noted in 30% of the bone marrow aspirates. Lymphoid aggregates, seen in 13% of these samples, were generally of moderate size, contained few mast cells and were non‐clonal on immunophenotypic analysis.     

Circulating Megakaryocytes and Platelet Release in the Lung

1. Megakaryocytes were demonstrated in central venous blood of each of23 patients who underwent cardiac catheterization. Cell counts ranged from0.7 to 5.9 megakaryocytes per ml. of blood; the equivalent of one-third ofthese cells were considered to contain a full complement of cytoplasm. It hasbecome evident that megakaryocytes are normal constituents of blood.

2. In an attempt to quantify megakaryocyte migration from the bone marrow it was calculated that from 20-50 per cent of the mature megakaryocytepopulation enters the blood and ultimately reaches the lungs. The possibilitythat all megakaryocytes migrate from the marrow is not precluded with certainty by these studies.

3. It was estimated that from 7-17 per cent of the body’s platelets are released in the pulmonary capillaries. If all megakaryocytes migrate from thebone marrow, then as much as 33 per cent of the platelet population is delivered to the blood in the lungs.

Submitted on January 15, 1965 Accepted on March 13, 1965     

Circulating megakaryocytes: Delivery of large numbers of intact,mature megakaryocytes to the lungs

Abstract: To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 × 10⁶ intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 × 10⁶ partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.     

Giant platelet granules in a child with the Chediak-Higashi syndrome.

Previous ultrastructural investigation have not identified abnormal lysosomes in platelets obtained from humans or animals with the Chediak-Higashi Syndrome. We report here a patient whose megakaryocytes and platelets were found to contain giant granules when viewed by light and electron microscopy. The granules measured up to 1.5 micrometer in diameter, contained either homogeneous or heterogeneous material, were acid phosphatase positive, and were present in approximately 30% of bone marrow megakaryocytes and 5% of circulating platelets. A decrease was observed in serotonin containing dense granules, serotonin uptake and serotonin release as reported previously. Microtubules in platelets and megakaryocytes were intact and no other morphologic abnormalities were identified. No clinical evidence of bleeding was observed in this patient and platelet counts have been normal. The lack of giant platelet lysosomes in other reported cases of Chediak-Higashi Syndrome attests to significant heterogeneity in this disease with a spectrum of clinical and laboratory findings.     

Platelet-derived growth factor is decreased in patients with myeloproliferative disorders

Platelet-derived growth factor (PDGF) has been suggested to play some role in the pathogenesis of myelofibrosis frequently encountered in patients with myeloproliferative disorders (MPD). In this study we measured PDGF activity and PF4 content in circulating platelets of patients with MPD. Both factors were lower than those of normal controls. PDGF activity in patients with myelofibrosis was slightly lower than in those without fibrosis. However, when adjusted to whole blood volume, there was a positive correlation between platelet count and PDGF activity per ml whole blood. Nevertheless, no correlation was found between activity and grade of bone marrow fibrosis. These results may support the idea that an abnormal release of PDGF occurs from platelets or megakaryocytes in the bone marrow environment, resulting in the stimulation of fibroblast proliferation, and hence, the occurrence of myelofibrosis in patients with MPD.     

Reticulated platelet counts in patients undergoing autologous bone marrow transplantation: An aid in assessing marrow recovery

Thiazole orange (TO) is a fluorescent dye that is commonly used for flow cytometric measurement of erythrocytic reticulocytes. This technique has also been validated for counting “reticulated” platelets, as a measure of bone marrow thrombopoietic activity. Patients with an established diagnosis of idiopathic thrombocytopenic purpura (ITP) have been reported to have a three- to five-fold increase in the percent of circulating reticulated platelets. The current study was conducted to determine if platelet reticulocyte counts predicted recovery of bone marrow thrombopoietic activity following autologous bone marrow transplantation. We found an increase in the percent of circulating reticulated platelets preceding recovery of the platelet count; then, as the patients' platelet counts rose, the percent reticulated platelets fell. In patients with prolonged thrombocytopenia, a persistent increase in the proportion of reticulated platelets suggested a consumptive process, as opposed to failure of engraftment. Moderate transfusion of platelet concentrates did not interfere with the ability of the platelet reticulocyte measurements to detect increased thrombopoietic activity. Platelet transfusions did obscure any decrease in the proportion of reticulated platelets. Our results show that platelet reticulocyte counts could be useful in monitoring the recovery of marrow function following autologous bone marrow transplantation. © 1994 Wiley-Liss, Inc.     

Effect of hypertransfusion on bone marrow regeneration in sublethally irradiated mice. II. Enhanced recovery of megakaryocytes and platelets

Hypertransfusion can enhance myeloid recovery after bone marrow depletion, but its influence on thrombopoietic recovery has not been previously defined. We have studied the pattern of platelet and megakaryocyte recovery in mice hypertransfused after receiving 350 rad whole body irradiation. The platelet counts of the hypertransfused group showing an initial fall due to hemodilution in the expanded blood volume and then fell to a lower nadir than that of the control mice. The rate of platelet recovery was more rapid in the hypertransfused mice. Bone marrow megakaryocyte concentrations in both groups showed a degenerative phase, abortive rise, and regenerative phase. The decrease in megakaryocytes was the same in both groups. The hypertransfused mice showed a greater abortive rise in megakaryocyte concentration preceded by the appearance of a greater number of large megakaryocytes in the bone marrow. However, the most striking effect of hypertransfusion was on megakaryocyte recovery. Although the time of onset of recovery was not different, the rate of recovery was approximately twice as rapid in the hypertransfused group. Administration of daily erythropoietin to hypertransfused mice abolished this more rapid recovery. Thus, the presence of a simultaneous demand for erythroid precursors does affect the rate of megakaryocyte regeneration. Just as the more rapid recovery of granulopoiesis following hypertransfusion may be clinically beneficial, the more rapid reconstitution of thrombopoiesis may also offer clinical advantage in some circcumstances.     

In vivo stimulation of megakaryocytopoiesis by recombinant murine granulocyte-macrophage colony-stimulating factor

Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was injected in mice, and the effects on bone marrow, splenic megakaryocytes, megakaryocyte precursors (megakaryocyte colony-forming units [CFU-Meg]) were evaluated. In mice injected three times a day for 6 days with 12,000 to 120,000 U rGM-CSF, no significant modification of both platelet levels and mean platelet volume was observed, while there was a twofold increase in blood neutrophils. However, the rate of platelet production, as assessed by the measurement of 75selenomethionine incorporation into blood platelets, was On the contrary, administration of up to 384,000 U rGM-CSF two times a day for 2 days, as for a typical "thrombopoietin assay," failed to modify platelet production. A significant dose-related increase in the number of splenic megakaryocytes occurred in mice receiving 60,000 to 120,000 U rGM-CSF, while a slight increase in the number of bone marrow megakaryocytes was observed in mice injected with 120,000 U rGM-CSF. The proportion of bone marrow megakaryocytes with a size less than 18 microns and greater than 35 microns resulted significantly higher in mice receiving rGM-CSF in comparison with controls; an increase in the percentage of splenic megakaryocytes greater than 35 microns was also observed. A statistically significant increase in the total spleen content of CFU-Meg was observed after administration of 90,000 and 120,000 U rGM-CSF three times a day for 6 days, while no effect on bone marrow CFU-Meg was recorded, irrespective of the dose delivered. Finally, 24 hours after a single intravenous injection of rGM-CSF, there was a significant increase in the proportion of CFU-Meg in S-phase, with the splenic progenitors being more sensitive than bone marrow-derived CFU-Meg. These data indicate that rGM-CSF has in vivo megakaryocyte stimulatory activity, and are consistent with previous in vitro observations. However, an effective stimulation of megakaryocytopoiesis in vivo, bringing about an increase in the levels of blood platelets, may require interaction of rGM-CSF with other cytokines.     

Biosynthetic origin and functional significance of murine platelet factor V

Factor V (FV), a central regulatory protein in hemostasis, is distributed into distinct plasma and platelet compartments. Although platelet FV is highly concentrated within the platelet alpha-granule, previous analysis of human bone marrow and liver transplant recipients has demonstrated that platelet FV in these individuals originates entirely from the uptake of plasma FV. In order to examine further the biosynthetic origins of the platelet and plasma FV pools, we performed bone marrow transplantations of Fv-null (Fv-/-) fetal liver cells (FLCs) into wild-type mice. Fractionation of whole blood from control mice demonstrated that approximately 14% of total blood FV activity is platelet-associated. Mice that received transplants of Fv-null FLCs displayed a high degree of engraftment and appeared grossly normal, with no evidence for spontaneous hemorrhage. Although total FV levels in Fv-null FLC recipients were only mildly decreased, the FV activity within the platelet compartment was reduced to less than 1% of that in normal mice. We conclude that the murine platelet FV compartment is derived exclusively from primary biosynthesis within cells of marrow origin, presumably megakaryocytes, and that an intact platelet FV pool is not required for protection from spontaneous hemorrhage or bleeding following minor trauma.     

Rosiglitazone induces decreases in bone mass and strength that are reminiscent of aged bone

Peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates both glucose metabolism and bone mass. Recent evidence suggests that the therapeutic modulation of PPARgamma activity with antidiabetic thiazolidinediones elicits unwanted effects on bone. In this study, the effects of rosiglitazone on the skeleton of growing (1 month), adult (6 month), and aged (24 month) C57BL/6 mice were determined. Aging was identified as a confounding factor for rosiglitazone-induced bone loss that correlated with the increased expression of PPARgamma in bone marrow mesenchymal stem cells. The bone of young growing mice was least affected, although a significant decrease in bone formation rate was noted. In both adult and aged animals, bone volume was significantly decreased by rosiglitazone. In adult animals, bone loss correlated with attenuated bone formation, whereas in aged animals, bone loss was associated with increased osteoclastogenesis, mediated by increased receptor activator of nuclear factor-kappaB ligand (RANKL) expression. PPARgamma activation led to changes in marrow structure and function such as a decrease in osteoblast number, an increase in marrow fat cells, an increase in osteoclast number, and a loss of the multipotential character of marrow mesenchymal stem cells. In conclusion, rosiglitazone induces changes in bone reminiscent of aged bone and appears to induce bone loss by altering the phenotype of marrow mesenchymal stem cells.