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

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

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

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

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry.

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

Three stages of differentiation in mouse megakaryocyte progenitor cells (CFU-Meg).

Mouse megakaryocyte colonies developed in a fibrin clot culture system were morphologically classified into three types: immature homogeneous (IH), heterogeneous (H), and mature homogeneous (MH) colonies. The colony size (number of megakaryocytes per colony), the ploidy distribution of megakaryocytes in colonies, and the ratios of their progenitors (megakaryocyte colony-forming units, CFU-Meg) in the S-phase of the cell cycle were compared among the three types. Also, morphological changes in colonies were examined in time sequence. The mode of the number of megakaryocytes per colony on day 6 of culture was greater than 64, between 17 and 32, and between 4 and 8, in IH, H, and MH colonies, respectively. The mean DNA content of megakaryocytes was 3.1N, 4.2N, and 7.2N on day 6 of incubation, in IH, H, and MH colonies, respectively, these values being significantly different from each other (p less than 0.001). The mean proportion of CFU-Meg synthesizing DNA was 21.9%, 26.0%, and 48.6% for CFU-Meg forming IH, H, and MH colonies, respectively (p less than 0.01; IH- versus MH-CFU-Meg and H- versus MH-CFU-Meg). Successive observation of the colony morphology from days 5 to 11 of culture suggested a transformation of the colony types from IH colony to MH colony through H colony. These observations indicate that the three types of megakaryocyte colonies classified on the basis of their morphological features reflect various stages of differentiation of mouse CFU-Meg.

A monoclonal antibody to a megakaryoblast-like cell line detects a protein found in blood cells and in the epithelial cell lining of various rat tissues.

Megakaryoblasts of bone marrow differentiate into megakaryocytes that in turn are the source of blood platelets. We have raised monoclonal antibodies to a megakaryoblast-like cell line derived from rat bone marrow (RPM cells). One antibody (Mab 213) and the corresponding antigen has been characterized by Western blotting and immunohistochemistry. Biosynthetic labeling with [35S]methionine showed that this antigen is synthesized by the RPM cells. In Western blots the antibody recognized proteins of about 90 kDa and 160 kDa in Triton extracts of RPM cells, whereas it recognized proteins of about 160 kDa and 200 kDa in Triton extracts of rat platelets and one of about 200 kDa in Triton extracts of various rat tissues (kidney, lung, intestine, and heart). By immunohistochemistry, the antigen was localized to the apical part of the epithelium lining certain parts of kidney tubuli, bronchi and large intestine.

[A case of successfully treated cyclic amegakaryocytic thrombocytopenic purpura]. / Sikeresen kezelt ciklikus amegakaryocytás thrombocytopeniás purpura esete.

The case of a patient with cyclic, acquired, periodically amegakaryocytic thrombocytopenia is described. On the base of a seemingly typical megakaryocytic thrombocytopenia ITP was diagnosed at first. He did not respond to steroid, therefore splenectomy was performed. He relapsed and thereafter severe thrombocytopenia was observed, periodically, for 8-10 days in every five-six weeks. During the cytopenic periods recognizable megakaryocytes and precursors were totally absent from the bone marrow. After the haemorrhagic periods platelet counts elevated to normal or even higher levels and the marrow was plenty of megakaryocytes. Vincristine was unsuccessful, but cyclosporine administration for a longer time was not without success. The patient has a normal platelet count since one year. No similar case of cyclic amegakaryocytic thrombocytopenia was found in the literature.

Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system.

Megakaryocyte (MK) nuclear deoxyribonucleic acid (DNA) content was measured on a new image analysis system. Feulgen-stained bone marrow aspirate smears were analysed from 9 patients with thrombocytosis. Average optical density (OD) of stained nuclei was evaluated by pixel discrimination over 128 grey levels. Projected nuclear area was delineated manually. The product of these two parameters gave an index of nuclear DNA content. Neutrophils were used as 2N reference cells. Reproducibility of OD, nuclear area and their product was excellent for individual cells (CV less than 2.0% for MKs, less than 4.0% for neutrophils). The average CV for DNA content of 18 groups of 10 neutrophils was 5.5% (range 3.0-9.7%). A significant linear regression existed between MK nuclear area and DNA content (p much less than 0.001) for 627 MKs in essential thrombocythaemia (ET) and 346 MKs in reactive thrombocytosis (RT). Compared with RT, more 8N and 64N MKs were seen in ET (p less than 0.05). 128N MKs were unique to ET. Image analysis of MK ploidy may assist the clinical discrimination between primary and secondary thrombocytosis.

Regulation of megakaryocyte phenotype in human erythroleukemia cells.

Induction of human erythroleukemia (HEL) cells with nanomolar tumor-promoting phorbol myristate acetate (PMA) diesters results in the synchronous acquisition of multiple markers of the megakaryocyte phenotype. Induced cells markedly increase their content of cytoplasm and show features of morphological maturation. At the ultrastructural level, PMA-treated cells show increases in cytoplasm, nuclear lobulation and nucleolar content, and free ribosomes. Limited numbers of cells also express alpha-granules and nascent demarcation membrane systems. Functionally, PMA-stimulated HEL cells express increased amounts of the megakaryocyte/platelet proteins: glycoprotein IIb/IIIa, platelet factor 4, von Willebrand factor, glycoprotein Ib, and thrombospondin. No changes are observed in antigenic markers of the erythroid (glycophorin A) or macrophage lineages (MO-1 or MO-2). The increases in antigenic expression are rapid, reaching maximum levels within 3-4 d under serum-free conditions. Treatment with PMA also abruptly (within 1-2 d) inhibits cellular division in these cells. Washout studies indicate that phorbols exert their effect within 18-24 h, the approximate cell cycle time for these cells. Consistent with proliferative arrest, c-myc proto-oncogene transcripts begin to decline within 8 h of PMA treatment, although transcripts of c-myb are unaffected. Importantly, megakaryocyte differentiation is associated with endomitotic DNA synthesis (i.e., continued DNA synthesis in the absence of mitosis and cytokinesis), with HEL cells reaching a DNA content of 3-12 times that of unstimulated cells. Endomitosis is coordinately regulated with changes in antigenic expression and cell size such that those cells having the highest DNA content are the largest and also express the greatest levels of antigen.

Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages.

The nuclear factor GF-1 (also known as NF-E1, Eryf-1; refs 1-3 respectively) is important in regulation of the transcription of globin and other genes that are specifically expressed in erythroid cells. We have previously shown that GF-1 of both mouse and human origin is a 413-amino-acid polypeptide with two novel zinc-finger domains whose expression is restricted to erythroid cells. Using in situ hybridization of mouse bone marrow cells and northern blot analysis of purified cell populations and permanent cell lines, we show here that GF-1 is expressed in two other hematopoietic lineages, megakaryocytes and bone marrow-derived mast cells. Our findings are consistent with results from hematopoietic progenitor culture which suggest a relationship between erythroid, megakaryocytic and mast cell lineages, and imply that GF-1 is expressed in committed multipotential cells and their progeny. Hence, the mere presence of this transcription factor is unlikely to be sufficient to programme differentiation of a single haematopoietic lineage. GF-1 may regulate the transcription of not only erythroid genes, but also many genes characteristic of megakaryocytes and mast cells, or genes shared among these lineages.

Megakaryocytic and erythrocytic lineages share specific transcription factors.

Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines.

Human megakaryocytes. VII. Analysis of megakaryocytes for nuclear DNA content distribution in whole marrow cell suspensions by flow cytometry.

These studies were designed to quantitate and determine the DNA content distribution of human marrow megakaryocytes using whole bone marrow. Cellular DNA content within megakaryocytic cells was established by measuring propidium iodide staining in marrow cells expressing platelet glycoprotein IIb/IIIa (Gp IIb/IIIa). These studies were based on the development of a method for rapid analysis of whole marrow cell preparations by a dual fluorescent system. DNA values ranging from 2 to 64 C were observed in all samples tested, with 16 C corresponding to the modal ploidy class representing almost one-half of the cells containing Gp IIb/IIIa. The second most frequent ploidy class corresponded to 32 C, followed by 8 C, with 20% and 15%, respectively. Virtually all high-ploidy megakaryocytes (greater than or equal to 8 C) were of low density (less than 1.050 g/cm3), whereas 2 and 4 C megakaryocytes were evenly distributed between less than or equal to 1.050 and greater than 1.050 g/cm3 marrow cells. These studies conclusively establish DNA content distribution of normal human marrow megakaryocytes and provide a basis for the study of states of altered megakaryocytopoiesis.

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs.

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.

Partial purification and characterization of human megakaryocyte colony-stimulating factor (Meg-CSF).

Megakaryocyte colony-stimulating factor (Meg-CSF) in urinary extracts from patients with aplastic anemia was partially characterized and purified. Using Meg-CSF-enriched fractions, we established that the moiety has the following characteristics: 1) portions of the molecules having Meg-CSF activity have sialic acid, probably with a biantennary structure, and beta-galactose residues as the terminal and penultimate sugars; 2) disulfide residues are an essential chemical group of the molecule and are located on its surface; and 3) Meg-CSF activity is stable in n-propanol, but not in acetonitrile with trifluoroacetic acid. Partial purification of Meg-CSF by a four-step procedure of ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-sepharose chromatography, and high-resolution hydroxyapatite chromatography, yielded a concentrate with a 430- to 630-fold increase in specific activity. The partially purified Meg-CSF fractions stimulated both human and murine megakaryocytopoiesis in vitro (CFU-meg). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduced conditions, Meg-CSF activity was recovered in the 29-34 kDa molecular weight fractions. We have also shown that Meg-CSF, purified from the urine of aplastic anemia patients, stimulated murine megakaryocytopoiesis and platelet production in vivo. Final purification of human urinary Meg-CSF is currently in progress.

Location of sites of dopamine storage in megakaryocytes by autoradiography.

We have used quantitative electron microscope autoradiography to study the subcellular sites of 3H-dopamine uptake in mouse megakaryocytes after a single intraperitoneal injection. Autoradiographic grains were found to be associated almost exclusively with the vesicles of precursors of monoamine-storage organelles. The labeling intensity (radioactivity) of the demarcation membrane system which is continuous with the plasmalemma was also significantly greater than would be expected. On the other hand, radioactivity associated with the remaining sites in the cytoplasm was not significantly different from that expected of a random distribution. In order to compare 3H-dopamine uptake during cell maturation, light microscope autoradiographic studies were also done. Immature megakaryocytes were labeled slightly, but the number of silver grains increased significantly in maturing cells. Mature megakaryocytes were 2.7 times more radioactive than the maturing cells. Our results suggest that the megakaryocytes were able to accumulate dopamine from the early stages of cell maturation and to store dopamine in precursors of monoamine-storage organelles.

[Megakaryocyte ploidy in patients with myelodysplastic syndrome--by microcytofluorometry with DAPI staining after removal of Wright-Giemsa staining].

In the myelodysplastic syndrome (MDS) patients the ploidy distribution of megakaryocyte DNA was rarely reported. We applied DAPI (4',6-diamidino-2-phenylindole) staining for measuring nuclear DNA content in megakaryocytes which have been morphologically identified on the Wright-Giemsa stained smear in 8 normal subjects and 12 MDS patients. Briefly, megakaryocytes morphologically examined on a Wright-Giemsa stained smear were photographed, and were located. We then removed the Wright-Giemsa stains by immersing it in 50% ethanol, 37 degrees C for 1 hour and 100% methanol, 37 degrees C for 1 hour. The DAPI staining was performed in DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris-EDTA-2 Na buffer solution and 0.01 mol 2-mercaptoethylamine hydrochloride were mixed at the ratio of 0.5: 98.5: 1.0) for more than 30 min. The amount of nuclear DNA in the megakaryocyte previously identified was measured by cytofluorometry. The population of megakaryocyte in the normal subjects was the largest in the 16N, and in the 10 cases of the 12 MDS patients was the largest in the 8N, in the other 2 cases was the largest in the 4N. These results represent the development of the megakaryocyte nucleus in the MDS patients may be disturbed.

[Microcytofluorometrical measurement of DNA content in immunologically identified micromegakaryocytes of human bone marrow].

We have developed a method to microfluorometrically determine the amount of DNA in immunologically identified micromegakaryocytes on bone marrow smears. Bone marrow smears were fixed with acetone-formalin buffer and immunostained with a monoclonal anti-GPIIb/IIIa antibody, followed by FITC conjugated anti-mouse IgG. After the smears were re-fixed with methanol, DAPI (4', 6-diamidino-2-phenylindole) staining was performed. Using an automatic Digital-Microfluorometer (Olympus MMSP-FR-II), megakaryocytes on the smears were identifies by the GPIIb/IIIa immunofluorescence and, after changing the barrier filters, their nuclear DNA content was measured by the intensity of DAPI fluorescence, which is proportional to the amount of DNA. Using this method, we found that the DNA histogram of the megakaryocytes from a patient with myelodysplastic syndrome showed a shift to small ploidy compared with normal controls. This method may be valuable in the measurement of the megakaryocyte DNA content.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

Elevated level of p60c-src in virus-transformed murine megakaryocytic cell lines.

Megakaryocytic cell lines derived from mouse bone marrow cells transformed by the Myeloproliferative Leukemia Virus (MPLV) contain elevated levels of p60c-src. Northern blot analysis revealed the presence of a 4 kb normal sized c-src transcript only in MPLV-transformed megakaryocytic cell lines containing a high percentage of acetylcholinesterase positive cells (AChE+ greater than 10%), but not in MPLV-transformed erythroblastic or myeloblastic cell lines. The p60c-src protein was identified in lysates from in vivo labelled cells and in in vitro labelled membrane extracts by immunoblotting analysis and by immunoprecipitations with specific anti-src antibodies. In dimethylsulfoxide (DMSO) treated cells, the number of AChE+ cells increased together with p60c-src kinase activity indicating a possible correlation between p60c-src expression/activity and megakaryocytic differentiation.

[Megakaryoblastic crisis of chronic myelogenous leukemia cytological and clinical studies in three cases].

Diagnostic significance of the megakaryocyte markers and clinical findings were evaluated in three cases with chronic myelogenous leukemia in megakaryoblastic crisis. Platelet peroxidase (PPO), glycoprotein IIb/IIIa, Ib, von Willebrand factor antigen (vWF: Ag) and demarcation membrane system (DMS) were examined as the megakaryocyte markers. Blast phenotypes were as follows: PPO- IIb/IIIa+ vWF: Ag+ DMS+ in Case 1, PPO+ IIb/IIIa +/- Ib- vWF: Ag +/- in Case 2 and PPO+ IIb/IIIa+ vWF: Ag +/- DMS +/- in Case 3 (-: 0% +/-: less than 10% +: greater than or equal to 10%). In Cases 1 and 3, no markers other than those for the megakaryocyte lineage were detected, but myeloperoxidase-positive blasts coexisted with PPO-positive megakaryoblasts in Case 2. Megakaryoblast phenotypes and involvement of other lineages were much different in each case. Therefore, marker study for cytological diagnosis should be performed in consideration of lineage heterogeneity. As to the clinical findings, no clear features common to the three cases were present. However, multiple osteolytic lesions were demonstrated on bone survey in Case 1 and considered to be caused by the proliferation of megakaryoblasts.

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.