Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (～two-third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α-granules. © 1995 Wiley-Liss, Inc.     

The effect of hypercholesterolemia on guinea pig platelets, erythrocytes and megakaryocytes

This study has examined the effect of diet-induced hypercholesterolemia on guinea pig platelets, erythrocytes, megakaryocytes and plasma. The cholesterol/phospholipid ratios of plasma and erythrocytes began to increase after one day on the diet and increased steadily for two weeks and more slowly thereafter until 30 days. In contrast, the cholesterol/phospholipid ratio of platelets remained constant for 4-5 days, then increased until reaching a maximum of about 0.85 in two weeks. Thus, the time-course for increase of the cholesterol/phospholipid ratio is different for platelets than for erythrocytes and plasma. The increase in the cholesterol/phospholipid ratio of megakaryocytes was small and not dependent on the degree of increase in the plasma cholesterol/phospholipid ratio. The cholesterol esters of both platelets and megakaryocytes increased with time for two weeks. The increase in megakaryocyte cholesterol esters appeared to precede that of platelets. The protein content of platelets and megakaryocytes and average megakaryocyte size were increased. Normal platelets incubated in plasma from hypercholesterolemic guinea pigs did not accumulate excess cholesterol, but erythrocyte cholesterol increased 45% in 6 h under the same conditions. Cholesterol synthesis in megakaryocytes was depressed 50-80% by cholesterol feeding and by in vitro incubation of the cells in hypercholesterolemic plasma. The data suggest that the platelets and erythrocytes may accumulate excess cholesterol by different mechanisms. The effects of cholesterol feeding on megakaryocytes and the lag in accumulation of cholesterol in platelets relative to erythrocytes and plasma suggest that a defect in the megakaryocyte may be a primary determinant of accumulation of cholesterol in platelets.     

Cytochemical evidence of the origin of the dense tubular system in the mouse platelet

Summary Glucose-6-phosphatase (G6Pase) was used as a marker enzyme for the endoplasmic reticulum in mouse megakaryocytes and platelets. G6Pase activity was localized in the dense tubular system of the platelets. Enzyme activity was also observed in the nuclear envelope, and in the rough endoplasmic reticulum of the megakaryocytes. However, the Golgi apparatus of the megakaryocyte was never involved. The present study has added new cytochemical evidence for the hypothesis that the dense tubular system of the platelet originates from the endoplasmic reticulum of the megakaryocyte.     

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy and Platelet Generation

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.     

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.     

Wistar Furth Rat Megakaryocytes Lack Dense Compartments and Intercellular Plaques,Membranous Structures Rich in Cytoskeletal Proteins

Wistar Furth (WF) rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF rat megakaryocytes to those of rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administration, have revealed a previously unrecognized membrane structure in normal rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF rats. The second WF rat megakaryocyte abnormality is the absence of cytoplasmic dense compartments, another specialized membranous structure that is continuous with the megakaryocyte demarcation membrane system. Both the intercellular plaques and dense compartments of Wistar rat megakaryocytes were found to be rich in cytoskeletal proteins including actin, α-actinin, talin, and vinculin as indicated by ultrastructural immunogold labeling. We hypothesize that an abnormality in cytoskeletal protein function may be responsible for the lack of these structures in the WF rat.     

Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system

Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.     

Characterization of human megakaryocytic colony formation in human plasma

We have analysed the contribution to megakaryocyte colony formation in methylcellulose made by human plasma, serum, media conditioned by phytohemagglutinin (PHA) stimulated leukocytes (PHA-LCM), erythropoietin (EPO) preparations, and platelets. The culture system was used as a bioassay for megakaryocyte colony stimulating activity (Meg-CSA) in plasma samples of patients with perturbed megakaryocytopoiesis. Preparations of heparinized platelet-poor plasma yielded the most consistent results. Platelet-poor plasma of normal subjects will at best facilitate the occasional growth of small megakaryocyte colonies. Colony frequency and size are reproducibly enhanced in the presence of PHA-LCM as a source of exogenous Meg-CSA. Commercially available EPO preparations may vary in their content of activities that influence megakaryocyte colony formation. Addition of these preparations to cultures that contain plasma and PHA-LCM usually does not enhance colony formation. In contrast to platelet-poor plasma, platelet rich plasma and serum are less supportive of megakaryocyte colony growth. It is suggested that this loss of activity may be related to the release of inhibitors by activated platelets or alternatively caused by absorption of activities by platelets. Plasma samples from patients with megakaryocytopoietic dysfunction may contain components that promote colony formation without addition of PHA-LCM or EPO. This phenomenon is consistently observed for patients with severe aplastic anemia and bone marrow transplant recipients after completion of their ablative preparative regimen.     

Megakaryocyte colony growth-supporting activities in human plasma: modification by platelets and platelet membranes

Human megakaryocyte colonies are grown in methylcellulose with platelet-poor plasma and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) as a source of megakaryocyte colony stimulating factor (MEG-CSF). The megakaryocyte colony growth-supporting activity in human plasma can be absorbed by intact platelets or degranulated platelet membranes. It was possible to recover the activity by solubilizing platelet membranes with cholic acid. Filtration of the solubilized platelet membrane preparations through a Sephadex G-100 column yielded at least two activity peaks. The molecular weight of these two activities differs from that of the growth-promoting activity in PHA-LCM.     

Ability of plasma from patients with immune thrombocytopenic purpura (ITP) to promote the growth of megakaryocyte progenitors from normal bone marrow.

The ability of plasma from ITP patients (before and after splenectomy) to support the growth of megakaryocyte progenitors was compared with that from healthy subjects. Plasma Factor Index-Megakaryocyte PFI-Mk (ITP) which expressed resultant colony growth was significantly lower before splenectomy, but it normalized after splenectomy. (PFI-Mk) (ITP) did not relate neither to megakaryocyte nor to platelet counts. A positive correlation has been observed between megakaryocyte and platelet numbers in healthy subjects and in ITP patients after splenectomy, but not before splenectomy. The proportion of immature megakaryocytes was markedly higher in ITP marrow before splenectomy. This study indicates, that in ITP apart from antibodies directed to platelets and megakarocytes a low plasma stimulatory activity affected megakaryocytopoiesis.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

Mechanisms of platelet production

The precise mechanism by which platelets are formed from megakaryocytes (MK) remains unclear, despite numerous studies which have been performed during this century. Models have been proposed that attempt to account for platelet formation from disruption of elongated processes of MK cytoplasm, designated proplatelets, or by fragmentation of MK cytoplasm. MK demarcation membranes are hypothesized by some investigators to delineate platelet territories in the MK cytoplasm, and by others to act as a membrane reservoir for MK process formation. Platelet production has been variously speculated to occur primarily in the bone marrow or lung. Each theory or model has attempted to elucidate the phenomenon of size heterogeneity of circulating platelets and the changes that occur under conditions of altered thrombopoiesis. In this article, we have analyzed and compared the characteristics of previously proposed models for platelet production and suggested additional techniques for future studies of thrombopoiesis.     

Properties of the demarcation membrane system in living rat megakaryocytes

The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracellular indicator displayed the extensive nature of the channels formed by the DMS throughout the extranuclear volume. Under whole-cell patch clamp, the DMS is electrophysiologically contiguous with the peripheral plasma membrane such that a single capacitative component can account for the biophysical properties of all surface-connected membranes in the majority of recordings. Megakaryocyte capacitances were in the range of 64-694 pF, equivalent to 500-5500 platelets (mean value 1850). Based upon calculations for a spherical geometry, the DMS results in a 4- to 14-fold (average 8.1-fold) increase in specific membrane capacitance expressed per unit spherical surface area. This indicates a level of plasma membrane invagination comparable with mammalian skeletal muscle. Whole-cell capacitance measurements and confocal imaging of membrane-impermeant fluorescent indicators therefore represent novel approaches to monitor the DMS during megakaryocytopoiesis and thrombopoiesis.     

Incorporation of a circulating protein into alpha granules of megakaryocytes

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.     

Expression of the fibrinogen genes in rat megakaryocytes

A variety of evidence suggests that megakaryocytes synthesize fibrinogen and comparative immunochemical and structural studies indicate that fibrinogen produced in or associated with megakaryocytes may be different than fibrinogen produced in the liver. Two studies have reported that the gamma' chain, which is produced from the gamma chain gene by alternative splicing, is absent from fibrinogen produced in the megakaryocyte. Since there is only a single gene for each of the three fibrinogen chains the reported structural differences suggest different mechanisms for production of hepatic and megakaryocytic fibrinogen. We have begun an investigation of the varying mechanisms for expression of the fibrinogen genes by examining the structure of fibrinogen mRNA's in the two tissues. Fibrinogen mRNA's of identical length are found in both liver and megakaryocytes. Furthermore, despite the reported absence of the gamma' chain in platelet-associated fibrinogen, we have used a probe specific for the alternative spliced region of the gamma' mRNA to clearly demonstrate this chain in megakaryocyte mRNA. These studies indicate that the gamma' mRNA is either not translated in platelets or that the gamma' chain is unable to associated with the alpha and beta chains to form a mature molecule.     

The relationship between megakaryocyte nuclear DNA content and gene expression

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

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.     

Multiple levels of regulation of megakaryocytopoiesis

A working hypothesis for the regulation of megakaryocytopoiesis is described on the basis of current data. The hypothesis proposes that in vivo megakaryocytes are generated by 1) the expansion of clonable progenitor cells into immature megakaryocytes by locally produced (and regulated) interleukin-3 (IL-3) and 2) the development and maturation of immature megakaryocytes by a dual system; by a lineage specific mechanism involving thrombopoietic stimuli in the steady state and thrombocytopenic conditions, and by a lineage nonspecific mechanism via IL-3 in damaged or reconstituting marrow. The hypothesis predicts that if IL-3 is a significant in vivo regulator of megakaryocyte formation and development, receptor for IL-3 should be present on megakaryocytes and may be vestigially on platelets. Small but significant levels of 125I IL-3 were found to bind to platelets from normal mice. The level of binding on platelets was found to be enhanced sevenfold from mice that had received high levels of irradiation followed by bone marrow transplantation. This contrasted with a twofold increase in the level of binding to platelets from mice made acutely thrombocytopenic with antiplatelet serum. The data suggest that IL-3 may be involved in the in vivo regulation of murine megakaryocytopoiesis and may be a significant factor in rebound thrombopoiesis following bone marrow damage.     

人类胚胎期和成年期巨核细胞的分子特征比较

为探究人类不同发育时期巨核细胞的分子特征,基于人类胚胎期卵黄囊、胎肝和成年骨髓巨核细胞的单细胞转录组测序数据,从分子特征、基因调控网络等方面分别对其分子差异进行生物信息学分析。结果表明,胚胎期巨核细胞具有较强的增殖特征,高表达细胞增殖相关的转录因子;而成年期巨核细胞具有较强的血小板生成特征,高表达与巨核细胞分化成熟相关的转录因子。研究结果为研究不同发育阶段巨核细胞及其子代血小板的功能差异提供了理论依据。     

Pulmonary platelet production: a physical analogue of mitosis?

Cellular reduplication is normally achieved by mitosis. In mammals, a system unique to cell biology has evolved that does not utilise this reproductive process. Platelets are derived from the cytoplasm of megakaryocytes but they are not produced by mitosis. At present, both the site and mechanism of platelet production are still debated. This article describes a production mechanism that retains the natural simplicity of binary division. It is predominantly physical. Megakaryocytes leave the bone marrow as whole cells or large cytoplasmic fragments. The cytoplasm of these large circulating cells undergoes a sequence of binary divisions at the dichotomous branches of the pulmonary microcirculation. The lungs trap the majority of megakaryocyte naked nuclei that are removed subsequently by phagocytosis. After the organisation of a microtubular cytoskeleton, the much smaller cytoplasmic fragments leave the pulmonary bed as circulating platelets.