Gray platelet syndrome: immunoelectron microscopic localization of fibrinogen and von Willebrand factor in platelets and megakaryocytes

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha- granule content.     

Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha- granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.     

Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) "big-cell" colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) "heterogeneous" colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.     

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.     

Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes

CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin- stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross- reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet activation.     

In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.     

Serum from patients with various thrombopoietic disorders alters terminal cytoplasmic maturation of human megakaryocytes in vitro

Human bone marrow was depleted of progenitors (CFU-MK), but enriched for recognizable megakaryocytes (MK), and placed in cultures with serum from either normal donors (NABS) or patients with primary (PTS) or secondary (STS) thrombocytosis, autoimmune thrombocytopenia (ATS) or aplastic anemia (AAS). Mean MK diameters shifted during the 3-4 days of incubation. Endomitotic figure were visible and mean ploidy increased slightly during cytoplasmic maturation, where decreases in immature cells (stages 1 and 2) were accompanied by increases in the mature MK (stages 3 and 4). Cytoplasmic maturation was faster in AAS, ATS and STS than PTS or NABS; mean size and ploidy were similar in all cultures. Recognizable MK were not forced to undergo additional endoreduplication in response to stimulation. Only AAS augmented MK colony formation, which indicated that at least two humoral factors can regulate megakaryocytopoiesis at separate levels, the progenitors and morphologically recognizable MK.     

Identification of primary lysosomes in human megakaryocytes and platelets

The presence of lysosomal enzymes in human platelets is well documented; the identity of the "lysosome," however, has been the subject of some disagreement. In order to determine the time of appearance and subcellular localization of two lysosomal enzymes in megakaryocytes (MK) and platelets, we examined normal human bone marrow and blood by electron microscopy and cytochemistry. Acid phosphatase (AcPase) was present in the Golgi region in the youngest recognizable MK, as well as in those with a considerable degree of cytoplasmic maturation. Heavy reaction product was usually confined to one or two Golgi-associated cisternae and coated vesicles; other Golgi cisternae were sometimes lightly reactive. In mature MK, reaction product was limited to vesicles of variable size, but smaller than alpha-granules. Another lysosomal enzyme, arylsulfatase (AS), was localized in similar small vesicles in MK of all stages; it could not be demonstrated in the Golgi complex. Vesicles containing AS were also found in about 25% of platelet profiles, whereas vesicles containing AcPase were found in only about 15% of platelet profiles. The alpha-granules of all MK and platelets examined were negative for both enzymes. We conclude that the enzyme-containing vesicles in these cells constitute the lysosomes and that they are distinct from other platelet organelles. Since there was no evidence that they had participated in any digestive event, we believe that they are primary lysosomes, whose contents are secreted during platelet aggregation and the release reaction.     

A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23 [see comments]

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha- granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.     

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.     

The gray platelet syndrome: clinical spectrum of the disease

The gray platelet syndrome (GPS) is a rare inherited disorder of the megakaryocyte (MK) lineage. Thrombocytopenia and enlarged platelets are associated with a specific absence of alpha-granules and their contents. GPS patients exhibit much heterogeneity both in bleeding severity and in their response to platelet function testing. A unique feature is that proteins endogenously synthesised by megakaryocytes (MK) or endocytosed by MK or platelets fail to enter into the secretable storage pools that characterise alpha-granules of normal platelets. Although the molecular basis of the disease is unknown, evidence suggests that alpha-granules simply fail to mature during MK differentiation. One result is a continued leakage of growth factors and cytokines into the marrow causing myelofibrosis. While for some patients platelet function may be only moderately affected, for others thrombin and/or collagen-induced platelet aggregation is markedly modified and an acquired lack of the GPVI collagen receptor has been reported. In this review, we document the clinical and molecular heterogeneity in GPS, a unique disease of the biogenesis of platelet alpha-granules and of the storage of growth factors and secretable proteins.     

Inverse immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human bone marrow megakaryocytes

The time of appearance and pattern of expression of several α-granule proteins, von Willebrand factor (VWF), fibrinogen and immunoglobulins (Ig) were examined and compared in human bone marrow megakaryocytes (MK) using an immunocytochemical approach. VWF is synthesized by immature MK, whereas it has been shown that fibrinogen is incorporated from the plasma into α-granules. The present study was undertaken in order to determine whether there are chronological and morphological differences in the expression of VWF and fibrinogen in vivo in human MK. Seven paraffin-embedded biopsies of normal human bone marrow were labelled with specific antibodies for VWF and for fibrinogen, detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method, and analysed by immunomorphometry. We found a clear, statistically significant, difference in the labelling pattern of VWF and fibrinogen. The expression of other endocytosed α-granule proteins, immunoglobulins G and A, was therefore studied in bone marrow MK from two patients with multiple myeloma, one with monoclonal IgG and one with monoclonal IgA. The immunostaining pattern was similar to that of fibrinogen and different from VWF, and characteristic of endocytosed α-granule proteins.
This study demonstrates that: (i) immunohistochemical staining of MK α-granules proteins distinguishes the peripheral cockade distribution pattern of endocytosed protein from the perinuclear pattern of endogenously synthesized proteins; (ii) VWF is present in human bone marrow MK when fibrinogen is not yet detectable; (iii) VWF synthesis ceases while fibrinogen is still being incorporated; (iv) immunoglobulins can be detected in MK cytoplasm, with a staining pattern resembling that of fibrinogen.     

Characterization of guinea pig megakaryocyte subpopulations at different phases of maturation prepared with a Celsep separation system

We introduce a new method for preparing subpopulations of guinea pig megakaryocytes (MK). MK, partially purified by a density gradient, were separated according to size by sedimentation, starting as a monolayer, in an albumin gradient at unit gravity. Twenty-two fractions were collected. Cells were cytocentrifuged, ploidy was assessed by microdensitometry, and small MK were identified with anti-von Willebrand factor (vWF) immunoglobulin. Immaturity was assessed by uptake of 3H thymidine and synthesis of proteoglycans from 35S sulfate. About 88% of cells in fractions 2 through 18 were MK, of which 90% were viable. Fractions containing the largest cells were composed of 98% stage III and IV MK; fractions with the smallest cells contained up to 80% stage I and II MK. Six MK classes were isolated: immature cells, both stage I and II cells, at either the 8N, 16N or 32N ploidy class; mature cells, both stage III and IV cells, at either the 8N, 16N or 32N ploidy class. The fractions were pooled into three groups: (a) 8% of MK in group 1, fractions 2 through 11, were immature, and group 1 was composed of 92% of 16N and 32N mature classes; (b) 29% of MK in group 2, fractions 12 through 15, were immature, and group 2 was composed of 52% 16N mature, 24% 16N immature, and 13% 8N mature classes; 67% of MK in group 3, fractions 16 through 18, were immature, and group 3 contained 51% 8N immature, 14% 16N immature, and 18% mature 16N classes. The mean protein content of the three groups was 1.251, 0.624, and 0.284 mg/10(6) MK, respectively. Nine percent of cells in group 3 but no cells in group 1 took up large amounts of 3H thymidine. The synthesis of high-molecular-weight (high-mol-wt) proteoglycans in group 3 and synthesis of lower mol wt proteoglycans in groups 1 and 2 provided further evidence for differences in MK maturity. Thus, the method can isolate MK subpopulations that are viable and can be used to investigate the biochemical characteristics of MK at different phases of maturation.     

Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies

Immunogold staining was used to study the ultrastructural distribution of von Willebrand factor (vWF) in unstimulated platelets. vWF was detected in the alpha-granules with a specific eccentric distribution pattern opposite the nucleoids. Similar findings were obtained with a polyclonal antibody or a pool of monoclonal antibodies to human vWF. This labeling coincided with the presence of tubular structures located at the periphery of the alpha-granules. These structures were better visualized on platelets treated for standard electron microscopy: they formed a group of one to four tubules ranging from 200 A to 250 A in diameter. They closely resembled the internal tubular structures found in Weibel-Palade bodies, which are the storage organelles of vWF in endothelial cells.     

Effects of thrombocytopoiesis-stimulating factor on terminal cytoplasmic maturation of human megakaryocytes

Aplastic anemia serum (AAS) contains humoral factors that alter both proliferation and maturation of human megakaryocytes (MK). The ability of AAS to augment MK colony formation (colony-forming unit, CFU-MK) was neutralized by an antiserum against MK colony-stimulating factor (MK-CSF), a glycoprotein isolated from AAS. The adsorbed AAS still retained the ability to accelerate cytoplasmic maturation of recognizable MK. Similar experiments were done with thrombocytopoiesis-stimulating factor (TSF) and an anti-TSF antiserum to further define the activity in AAS responsible for accelerating cytoplasmic maturation. Bone marrow fractions enriched for recognizable human MK, but devoid of CFU-MK, were obtained by centrifugal elutriation and placed in short-term liquid cultures. MK progressed through identifiable maturation stages (1-4) more quickly in the presence of either TSF or AAS. TSF slightly enhanced the cloning efficiencies of CFU-MK, but did not alter the number of MK in individual colonies derived from non-adherent, low-density, T-cell-depleted bone marrow. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and crude AAS substantially augmented both MK colony formation and cells per colony. TSF also doubled the percent 35S incorporation into platelets of immunothrombocythemic mice, but stimulation was completely abolished by anti-TSF. Anti-TSF antiserum was then used to analyze the promotion of MK colony formation by cytokines. Cloning efficiencies of CFU-MK were reduced to baseline values when TSF was pretreated with anti-TSF; however, the MK colony-stimulating activity (MK-CSA) of GM-CSF, IL-3, or AAS was not altered by adsorption with anti-TSF. In contrast, the cytoplasmic maturation of recognizable MK was slower, and fewer mature stage-4 cells were present at days 1-3 in AAS adsorbed with anti-TSF than MK cultured in AAS treated with normal rabbit serum or untreated AAS. Therefore, TSF appears to be a major factor in AAS that accelerates terminal maturation of human MK. TSF primarily affects megakaryocytopoiesis by promoting MK maturation rather than enhancing CFU-MK proliferation.     

Development of human megakaryocytes: I. Hematopoietic progenitors (CD34+ bone marrow cells) are enriched with megakaryocytes expressing CD4

CD34 is expressed by essentially all human hematopoietic progenitors including cells of the megakaryocyte (MK) lineage. We have previously reported CD4 expression by some human MK (Blood 81:2,664, 1993). To study the role of maturation on CD4 expression by MK, we examined CD34+ bone marrow cells for their expression of CD41 (GPIIb-GPIIIa) and CD4 with specific monoclonal antibody (MoAb)-fluorochrome conjugates and for DNA polyploidization with propidium iodide or 7-aminoactinomycin D (7-AAD). Surprisingly, MK were at least 20-fold more common in the CD34+ progenitor pool (approximately 10%) than in the more mature CD34+ population (approximately 0.5%) of low density bone marrow cells. CD4 expression correlated with markers of immaturity in that CD4 was enriched among CD34+ cells, and the proportion of CD4+ MK declined with increasing ploidy. Almost all CD34+ polyploid ( > or = 8N) cells were CD4+. Despite these correlations with immaturity, CD34+CD4+ MK precursors were unable to produce MK colony-forming units (CFU-MK) when cultured under conditions that supported the growth of CFU-MK from CD34+CD4- MK lineage cells. MK became polyploid before the loss of either CD34 or CD4 expression. The presence of CD4 on these cells correlates with the onset of endomitotic reduplication and is associated with the loss of the ability of these cells to undergo normal mitotic division. The role of CD4 on immature MK as a differentiation antigen and/or receptor for the human immunodeficiency virus (HIV)-1 virus remains to be determined.     

Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.     

Developmental changes in human megakaryocyte ploidy

Megakaryocytes (MK) obtained from the differentiation of MK colony-forming units (CFU-MK) were grown from fetal liver, cord blood, and adult marrow in liquid culture containing aplastic plasma. Ploidy distribution was studied by a double-staining technique and flow cytometry and MK maturation by ultrastructural techniques. Cultured MK from fetuses and neonates were small sized (about 10 microns) in comparison to adult MK. They were mature cells that contained large membrane complexes as previously found in vivo. Only 2N and 4N MK were usually present in 8- to 10-week-old fetus cultures; 8N MK were detected at 20 weeks of gestation and in neonates. Higher ploidy classes were present in culture from adults but with a much lower frequency than in marrow. Therefore, a progressive shift to higher ploidy and an increase in MK size were observed simultaneously during development. Interleukin 3 (IL-3) increased MK proliferation as in adults but abrogated MK ploidization of 20-week-old fetus culture. The present results suggest that the changes occurring during ontogenesis are related to intrinsic MK modifications because no inhibitor of MK ploidization could be detected in fetal cultures.