Biomedical subjects

J M Massé

Publications and source records attributed to J M Massé.

At least 19 recordsLinked to original sources

PubMed2005

Molecular study of the hematopoietic zinc finger gene in three unrelated families with gray platelet syndrome.

Hematopoietic zinc finger (HZF) null mice have features reminiscent of patients with gray platelet syndrome (GPS), a rare inherited bleeding disorder. This similarity has suggested that HZF deregulation might be involved in the human disease. The sequence of the eight exons of the HZF gene as well as the study of its expression in blood samples from five patients belonging to three different families did not reveal any modifications when compared with healthy donors. This study indicates that HZF is unlikely to be responsible for GPS.

Blood Platelet Disorders↗

PubMed2001

Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion.

CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction. Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actin-disrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction.

Actins↗

PubMed2001

Identification of PML oncogenic domains (PODs) in human megakaryocytes.

Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures.

Antigens, Nuclear↗

PubMed2001

Newly recognized cellular abnormalities in the gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased alpha-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in alpha-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal alpha-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.

Alkaline Phosphatase↗

PubMed2001

Of mice and men: comparison of the ultrastructure of megakaryocytes and platelets.

OBJECTIVE: Mice provide an excellent model for studying platelet and megakaryocyte (Mk) biology in vivo. Given the increasing use of transgenic and knockout mice, it is important that any similarities and differences between murine and human platelet/Mk biology be well defined. Therefore the objective of this study was to compare and contrast in detail any significant morphological differences between Mks, platelets, and mechanisms of thrombopoiesis in humans and mice. METHODS: The distinctive structural and ultrastructural features of murine and human platelets and Mks are reviewed. Several platelet and Mk glycoproteins were also localized in murine cells by immunoelectron microscopy using polyclonal antibodies directed against human platelet proteins and compared to existing human data. Finally, the ultrastructure of maturing murine and human Mks in culture and bone marrow were examined in detail to facilitate a comparison of either in vivo or in vitro platelet production. RESULTS: Human and murine platelets exhibit significant but well-established morphological differences. Murine platelets are smaller and more numerous and display much greater granule heterogeneity than their human counterparts. Immunoelectron microscopy also demonstrated that murine platelet alpha-granules are highly compartmentalized. In fact, they are remarkably similar to human alpha-granules, with asymmetrical distribution of von Willebrand factor (vWF), and labeling of alpha(IIb)beta(3) and P-selectin (CD62P) in the granule limiting membrane. In vivo, murine but not human Mks are also consistently localized within the spleen. Subcellular events accompanying platelet formation and release by murine Mks are presented for the first time, and compared to human. Consistent differences were found in the pathway of redistribution of demarcation membranes preceding platelet formation, which may be important for the clarification of the mechanism of platelet release. CONCLUSION: Human and murine platelets and Mks display several characteristic ultrastructural differences (size, number, histological distribution, platelet shedding) which have been emphasized and analyzed in this report. Nevertheless, since there are also many close similarities (organelle and glycoprotein subcellular distribution) mice offer an excellent in vivo model to study various aspects of human Mk and platelet biology.

Animals↗

PubMed2001

The storage defects in grey platelet syndrome and alphadelta-storage pool deficiency affect alpha-granule factor V and multimerin storage without altering their proteolytic processing.

Among proteins stored in alpha-granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in alpha-granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (alphadelta-SPD) and grey platelet syndrome (GPS, alpha-SPD), two conditions known to impair alpha-granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in alphadelta-SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin-factor V complexes in plasma. Like normal platelets, GPS and alphadelta-SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and alphadelta-SPD platelets, indicating that these conditions preserve some aspects of normal alpha-granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.

Blood Platelets↗

PubMed1999

Erythroblastic synartesis: an auto-immune dyserythropoiesis.

Erythroblastic synartesis is a rare form of acquired dyserythropoiesis, first described by Breton-Gorius et al in 1973. This syndrome is characterized by the presence of septate-like membrane junctions and "glove finger" invaginations between erythroblasts, which are very tightly linked together. This phenomenon, responsible for ineffective erythropoiesis, leads to an isolated severe anemia with reticulocytopenia. In the following report, we describe 3 new cases of erythroblastic synartesis associated with dysimmunity and monoclonal gammapathy. In all cases, the diagnosis was suggested by characteristic morphological appearance of bone marrow smears, and further confirmed by electron microscopy. Ultrastructural examination of abnormal erythroblast clusters showed that these cells were closely approximated with characteristic intercellular membrane junctions. The pathogenesis of the dyserythropoiesis was modeled in vitro using crossed erythroblast cultures and immunoelectron microscopy: when cultured in the presence of autologous serum, the erythroblasts from the patients displayed synartesis, whereas these disappeared when cultured in normal serum. Moreover, synartesis of normal erythroblasts were induced by the patient IgG fraction. Immunogold labeling showed that the monoclonal IgG were detected in, and restricted to, the synartesis. A discrete monoclonal plasmacytosis was also found in the patient bone marrow. The adhesion receptor CD36 appeared to be concentrated in the junctions, suggesting that it might be involved in the synartesis. These experiments indicated that a monoclonal serum immunoglobulin (IgG in the present cases) directed at erythroblast membrane antigen was responsible for the erythroblast abnormalities. Specific therapy of the underlying lymphoproliferation was followed by complete remission of the anemia in these cases.

Adult↗

PubMed1999

Multimerin processing by cells with and without pathways for regulated protein secretion.

Multimerin is a massive, soluble, homomultimeric, factor V-binding protein found in platelet alpha-granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules that lack the secretory granule membrane protein P-selectin, and in culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin's posttranslational processing and storage, we expressed human endothelial cell prepromultimerin in a variety of cell lines, with and without pathways for regulated secretion. The recombinant multimerin produced by these different cells showed variations in its glycosylation, proteolytic processing, and multimer profile, and human embryonic kidney 293 cells recapitulated multimerin's normal processing for constitutive secretion by human endothelial cells. When multimerin was expressed in a neuroendocrine cell line capable of regulated protein secretion, it was efficiently targeted for regulated secretion. However, the multimerin stored in these cells was proteolyzed more extensively than normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in multimerin's posttranslational processing on its functions is not yet known. Multimerin's sorting and targeting for regulated secretion may be important for its functions and its association with factor V in secretion granules.

Blood Platelets↗

PubMed1999

Intracellular trafficking of the alphaIIbbeta3 receptor antagonist, abciximab, in normal and Glanzmann's disease megakaryocytes.

The alphaIIbbeta3 platelet receptor antagonist abciximab (c7E3Fab, ReoPro(R)) has proved to be effective in preventing arterial thrombosis. However, its binding capacity to the platelet precursors, megakaryocytes (MKs), which also express alphaIIbbeta3, is not known. The purpose of this study was to establish whether abciximab is able to react with alphaIIbbeta3 located on human MKs, and to follow its subsequent intracellular trafficking. MKs were grown from CD34+ progenitors from normal subjects and from a patient with type I Glanzmann's thrombasthenia, and abciximab was added at day 10 of culture (4 microgram/ml). Cells were fixed at day 12, cryosectioned, and immunolabelled for abciximab. Labelling was prominent on the MK plasma membrane; it also lined the demarcation membration system. Interestingly, alpha-granule membranes were labelled showing that the antibody was internalized and further stored into MK secretory granules. Abciximab was also strongly detected on and in newly-formed platelets. Glanzmann's disease MKs (which completely lacked alphaIIbbeta3) were consistently negative, confirming that the antibody fragment was specifically interacting with alphaIIbbeta3. In conclusion, this study demonstrated that abciximab: (i) binds MK plasma membrane and demarcation membranes, (ii) trafficks into alpha-granules, and (iii) is expressed on and in nascent platelets. These findings could be taken in account when monitoring anti-alphaIIbbeta3 receptor therapy.

Abciximab↗

PubMed1998

Studies of multimerin in human endothelial cells.

Multimerin is a novel, massive, soluble protein that resembles von Willebrand factor in its repeating, homomultimeric structure. Both proteins are expressed by megakaryocytes and endothelial cells and are stored in the region of platelet alpha-granules resembling Weibel-Palade bodies. These findings led us to study the distribution of multimerin within human endothelial cells. Multimerin was identified in vascular endothelium in situ. In cultured endothelial cells, multimerin was identified within round to rod-shaped, dense-core granules, some of which contained intragranular, longitudinally arranged tubules and resembled Weibel-Palade bodies. However, multimerin was found primarily in different structures than the Weibel-Palade body proteins von Willebrand factor and P-selectin. After stimulation with secretagogues, multimerin was observed to redistribute from intracellular structures to the external cellular membrane, without detectable accompanied secretion of multimerin into the culture media. In early passage endothelial cell cultures, multimerin was associated with extensive, fibrillary, extracellular matrix structures, in a different distribution than fibronectin. Although multimerin and von Willebrand factor are stored together in platelets, they are mainly found within different structures in endothelial cells, indicating that there are tissue-specific differences in the sorting of these soluble, multimeric proteins.

Blood Proteins↗

PubMed1998

Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency.

In normal platelet alpha-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal alpha-granular multimerin and factor V, and normal alpha-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, alpha-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal alpha-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWE The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.

Blood Platelets↗

PubMed1997

Platelet and megakaryocyte dense granules contain glycoproteins Ib and IIb-IIIa.

Platelets contain two main types of secretory organelles, the dense granules and the alpha-granules. P-selectin, a specific receptor for leukocytes that is present in the alpha-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the alpha-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti-Gp IIb-IIIa or anti-Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti-Gp IIb-IIIa or anti-Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additional evidence of similarities between the alpha-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of alpha-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.

Blood Platelets↗

PubMed1997

Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.

The site and mechanism of platelet production by bone marrow megakaryocytes (MKs) has been the subject of extensive studies, but is still a matter of controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also called megakaryocyte growth and development factor (MGDF) or thrombopoietin, has resulted in considerable progress in the understanding of the maturation of the MK lineage. To better understand the mechanism of platelet production, we examined the late stage of MK maturation by electron microscopy in cells cultured in the presence of Mpl-l. Human bone marrow CD34+ CD38+ cells, which contain late MK progenitors, were purified by flow cytometry and cultured in a serum-free liquid medium containing recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the majority of cultured cells were large MKs with lobulated polyploid nuclei. The MKs displayed a smooth surface with harmonious cytoplasmic maturation and abundant, regularly distributed demarcation membranes and alpha-granules, and even some dense granules. Interestingly, approximately 30% of the MKs observed displayed morphologic evidence of platelet production: at optical microscopy, MKs formed long filamentous cytoplasmic extensions (proplatelets) that fragmented into platelet-sized particles. Moreover, flow cytometric analysis of this cultured cell population showed GPIIb-positive particles of the size of platelets. Electron microscopic observation showed that MKs producing platelets displayed thin pseudopods on the surface, and that the channels of the demarcation membrane system were dilated, allowing long strands of cytoplasm to extend from the cell periphery. These cytoplasmic strands displayed beading with constrictions separating platelet-sized segments; the more distal to the cell core, the smaller the fragments were. They eventually detached from the cell core into the culture medium either occasionally still elongated or, more often, separated into individual platelets. Parallel longitudinal and perpendicular microtubules were visualized in the constricted regions of these cytoplasmic strips; immunogold study of tubulin localization confirmed this subcellular distribution. On both sides of the constricted areas, vacuoles were noted, the fusion of which might have led to the detachment of individual platelets. Finally, in close proximity to the platelet-forming MKs, numerous microparticles were shed. Although some of these particles might correspond to transverse sections of pseudopods, this did not seem to be the case, since they were rarely seen around thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow cytometry showed that apart from shed cytoplasmic fragments of platelet size, numerous smaller particles strongly labeled for CD41 were also released by mature MKs. In conclusion, this study describes the ultrastructure of human platelet production in cultured MKs, involving the formation of proplatelets and the shedding of microparticles.

Blood Platelets↗

PubMed1997

Studies of a second family with the Quebec platelet disorder: evidence that the degradation of the alpha-granule membrane and its soluble contents are not secondary to a defect in targeting proteins to alpha-granules.

We recently described a Quebec family with an autosomal dominant bleeding disorder characterized by mildly reduced-low normal platelet counts, an epinephrine aggregation defect, multimerin deficiency, and proteolytic degradation of several, soluble alpha-granular proteins. Similar clinical features led us to investigate a second family with an unexplained, autosomal dominant bleeding disorder. The affected individuals had reduced to normal platelet counts, absent platelet aggregation with epinephrine, and multimerin deficiency. Their platelet alpha-granular proteins factor V, thrombospondin, von Willebrand factor, fibrinogen, fibronectin, osteonectin, and P-selectin were proteolyzed and comigrated with the degradation products found in patients from the other family. However, their platelet albumin, IgG, external membrane glycoproteins, CD63 (a lysosomal and dense granular protein), calpain, and plasma von Willebrand factor were normal, indicating restriction in the proteins proteolyzed. Electron microscopy studies indicated preserved alpha-granular ultrastructure, despite degradation of soluble and membrane alpha-granular proteins. Immunoelectron microscopy studies of the patients' platelets indicated that fibrinogen, von Willebrand factor, P-selectin, multimerin, and factor V were within alpha-granules, with normal to reduced labeling for these proteins. Pathologic proteolysis of alpha-granular contents, rather than a defect in targeting proteins to alpha-granules, may be the cause of the protein degradation in the Quebec platelet disorder.

Adult↗

PubMed1996

Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and V.

We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes. Ultra-thin sections of the cells were labeled with antibodies directed against glycocalicin, GPIb, GPIX, and GPV. We have shown that a significant and reproducible labeling for the three GPs was associated with the alpha-granule membrane, accounting for approximately 10% of the total labeling. Furthermore, GPIb labeling appears Willebrand factor (vWF). After thrombin activation, vWF remained close to the limiting membrane of the open canalicular system (OCS), suggesting an early association of both receptor and ligand. Plasma membrane and alpha-granule labeling was virtually absent from the Bernard-Soulier platelets (characterized by a GPIb deficiency), thus proving the specificity of the reaction. In Gray platelets (storage granule deficiency syndrome), the small residual alpha-granules were also occasionally labeled for GPIb, GPIX, and GPIX. Cultured megakaryocytes that displayed the classical GPIb distribution, eg, demarcation and plasma membranes, exhibited also a discrete labeling associated to the alpha-granules. In conclusion, this study shows that, evenly for these three GPs, the alpha-granule membrane mirrors the plasma membrane composition. This might occur through an endocytotic process affecting each plasma membrane protein to a different extent and could have a physiologic relevance in further presentation of a receptor bound to its alpha-granule ligand to the platelet surface.

Bernard-Soulier Syndrome↗

PubMed1993

Effects of the recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells.

Using a liquid culture system and human CD34+ marrow cells, we examined the effects of recombinant interleukin (IL)-3, IL-6, stem cell factor (SCF), and leukemia inhibitory factor (LIF) on megakaryocyte (MK) growth, endoreplication, and maturation. MK proliferation, ploidy distribution, and volume were studied by flow cytometry. IL-3 was the only cytokine that, alone, induced a marked increase in MK proliferation. At a high CD34+ cell concentration, addition of IL-6, SCF, and LIF to IL-3--containing medium increased the number of MK (approximately 20%). At a low CD34+ cell concentration, IL-3 alone was a less potent inducer of MK growth, but IL-6, SCF, and their combination had a marked effect, increasing the number of MK by a factor 1.7, 2.9, and 4.4, respectively. These differences may be related to the endogenous release of cytokines in the culture. The effects of these cytokines were subsequently tested on a more mature type of MK progenitor (CD34+ cells isolated after 6 days of incubation in liquid culture). IL-3 remained the most potent cytokine, but IL-6 or SCF alone also increased MK number in comparison to unstimulated cultures. The ploidy distribution of MKs grown with IL-3 was not markedly changed by the addition of the other cytokines, with the exception of SCF, which induced a significant increase in the mean ploidy. However, in all cultures, glycoprotein (GP)IIIa+ 2N and 4N cells were present in large but variable numbers (35% to 75%). The number of these low-ploidy MKs directly correlated with MK proliferation. Therefore, we subsequently explored the absolute number of polyploid MK produced in culture. SCF, IL-6, or their combination, in association with IL-3, increased the number of polyploid MK up to fourfold. In addition, they improved the maturation of MK grown in the presence of IL-3, leading to the synthesis of demarcation membranes and platelet shedding. A similar effect of growth factors on the maturation of day 6 CD34+ cells was observed. We conclude that IL-6 and SCF have a broad range of activities on megakaryocytopoiesis, acting both on the early and late stages. However, the proliferative properties of these cytokines largely predominate in our cultures. Therefore, in the absence of a specific MK regulator, this study further extends the need for a combination of growth factors to maximize megakaryocytopoiesis.

Adult↗

PubMed1993

Effect of thrombin on maturing human megakaryocytes.

Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis formation and for megakaryopoiesis autocrine regulation.

Cell Count↗

PubMed1992

Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

Adult↗