Combining counterflow centrifugal elutriation and glycoprotein Ib-dependent purification of human megakaryocytes: efficacy and selectivity

To estimate the efficacy of human megakaryocyte purification techniques, mixtures of known numbers of megakaryocytes with a known ploidy range and of bone marrow or peripheral blood mononuclear cells were made. These artificial bone marrow samples were submitted to either a counterflow centrifugal elutriation or Percoll density separation followed by the glycoprotein Ib-dependent agglutination procedure. Also crude bone marrow samples were submitted to counterflow centrifugal elutriation directly followed by a glycoprotein Ib-dependent agglutination. The counterflow centrifugal elutriation resulted in a mean megakaryocyte recovery of 81% (mean 81% +/- 2.3). Purification by glycoprotein Ib-dependent agglutination after either a Percoll density separation or counterflow centrifugal elutriation resulted in a recovery of 61% (mean 61% +/- 15%) and 81% (mean 81% +/- 6) respectively. Purity of the resulting material was 87% (mean 87% +/- 11) and 83% (mean 83% +/- 5) respectively. The various isolation procedures did not affect the ploidy distribution of megakaryocytes greater than or equal to 8N. Counterflow centrifugal elutriation was preferred as the preparing step before glycoprotein Ib-dependent agglutination because of the lower variability in recovery and purity of megakaryocyte populations. When large numbers of rather pure and mature megakaryocytes are required, counterflow centrifugal elutriation followed by the glycoprotein Ib-dependent agglutination is a relatively simple method to purify human megakaryocytes without an appreciable loss in ploidy class greater than or equal to 8N.     

Botrocetin agglutination of rat megakaryocytes: a rapid method for megakaryocyte isolation.

Botrocetin isolated from the venom of Bothrops jaracara has been shown by others to induce binding of von Willebrand Factor to glycoprotein Ib and thereby produce platelet agglutination in a wide range of animal species. We have found that botrocetin also facilitates the agglutination of megakaryocytes and have used this property to develop a method to isolate megakaryocytes from rat bone marrow. When botrocetin is added to a mixture of rat bone marrow, rat platelets, and rat plasma, coagglutination of megakaryocytes and platelets occurs. The agglutinated complexes, containing > 95% of the megakaryocytes, may then be separated from the remaining marrow cells by filtration. Megakaryocytes account for 39% (range 30%-48%) of the isolated cells and 83% (range 77%-88%) of the isolated cell mass. This method allows the virtually complete removal of megakaryocytes from bone marrow as well as their isolation to a high degree of purity. It should provide a useful, inexpensive, general method for the rapid isolation of megakaryocytes from multiple, small marrow samples from a wide range of species.     

Isolation of human megakaryocytes by immunomagnetic beads

A simple method was developed to purify human megakaryocytes to homogeneity from normal bone marrow aspirates. An initial separation of marrow between 1.020 and 1.050 g/ml. Percoll density cut was used to enrich megakaryocytes. After washing, the cells were suspended with immunomagnetic beads which were coated with sheep anti-mouse IgG antibody and treated with anti-human glycoprotein (GP) IIb/IIIa monoclonal antibody, or the cells were treated with human platelet GP IIb/IIIa monoclonal antibody and suspended with the immunomagnetic beads which were coated with sheep anti-mouse IgG antibody. Megakaryocytes were selectively separated using a magnet. All of the isolated cells were morphologically recognizable megakaryocytes. 1.5-3.1 x 10(4) megakaryocytes were obtained from 1.7-4.5 x 10(8) bone marrow nuleated cells. These cells were all positive in immunoenzymatic staining for GP IIb/IIIa. Megakaryocytes obtained by this method responded to recombinant human GM-CSF (rhGM-CSF) showing an increased 3H-thymidine (3H-dT) incorporation. These data show that this method is useful for obtaining pure megakaryocyte populations which can be submitted to comprehensive biological studies.     

Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.     

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

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

Comparative analyses of megakaryocytes derived from cord blood and bone marrow

Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.     

Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes

The localization of three known alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fg) has been studied in human megakaryocytes (MK) by immunofluorescence and immunoelectron microscopy. For this study, highly purified populations of MK were prepared from human bone marrow either by counterflow centrifugal elutriation or by cell culture from normal subjects and from two patients with megakaryoblastic leukemia. In normal bone marrow immature MK, TSP, and vWF were observed in the Golgi-associated vesicles and in small immature alpha-granules; in mature MK, they were found in the matrix of the mature large alpha-granules. Surprisingly, Fg was detected neither in the Golgi area, nor in the small precursors of alpha-granules; it was only found in the mature alpha-granules but this labeling was generally weaker than in blood platelets. In order to confirm these differences between the expression of Fg and vWF or TSP additional studies were performed on cultured maturing MK: immunofluorescent and ultrastructural immunogold labeling confirmed that vWF appeared early in the maturation while the same immature MK were negative for Fg. In the late maturation stage, the three proteins were detected in the alpha-granules. In order to know whether Fg was lately synthesized or endocytosed from the outside medium, normal MK were grown in the presence of either normal or afibrinogenemic plasma, and normal serum. Fg was detected only in the alpha-granules of MK grown in normal plasma. Similar results were observed with malignant MK, whose maturation was independent of the culture conditions. In conclusion, this study brings immunocytochemical evidence that vWF and TSP are synthesized by immature MK, whereas Fg appears later in the MK alpha-granules and its expression is dependent of the presence of an exogenous Fg source.     

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

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

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO(2)) than the core of the BM where stem and progenitor cells reside, we investigated whether pO(2) influences Mk maturation. Mks were generated from CD34(+) cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O(2). At day 15, CD41(+) Mk expansion in 20% and 5% O(2) cultures was 85-fold and 31-fold respectively. Twenty percent O(2) cultures also had higher levels of high ploidy (> or = 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O(2) cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O(2) promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41(+) Mks, indicating that pO(2) directly affects Mks. The change from 20% to 5% O(2) on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34(+) cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.     

Megakaryocyte apoptosis in immune thrombocytopenia

The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.     

Thromboxane synthesis in megakaryocytes isolated by centrifugal elutriation

A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C- arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.     

Exosome mediated differentiation of megakaryocytes: a study on TLR mediated effects

Journal of Thrombosis and Thrombolysis - Megakaryocytes are large polyploid bone marrow cells whose function is to produce circulatory platelets. Megakaryocytes are also known to release...     

Intact Megakaryocytes in the Venous Blood as a Marker for Thrombopoiesis

A total of 110 children, aged 0–15 years, were investigated for circulating megakaryocytes in cubital venous blood using the saponin-haemolysis leucoconcentration technique. The average number of megakaryocytes decreased from 17.8 per ml blood in the first year of life to 5.5 after the 6th year, which is the same value as in adult humans. The intact thrombo-cytogenic megakaryocyte value decreases from about 40 % in the first year of life to only a few percent (< 5 %) after the 10th year, the same value as in adult humans. There was a significantly higher number of megakaryocytes in children aged 0–6 years than in those aged 7–15 years. We concluded that occurrence of intact megakaryocytes ≥ 25 % in the venous blood is a sign of a normal thrombopoietic activity in the bone marrow, and the percentage of intact megakaryocytes in cubital venous blood reflects the decrease in thrombopoiesis in the bone marrow of fingers, hands and forearms during childhood. The decline in thrombopoietic activity is concentrated in three periods: 1–3, 6–7 and 10–11 years. An occurrence of intact megakaryocytes > 5 % in venous blood draining organs or bone marrow is a sign of some thrombopoietic activity.     

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Background

Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia.

Design and Methods

To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells.

Results

The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ^−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ^−/− megakaryocytes could be differentiated in culture from Lin⁻ fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ^−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ^−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ^−/− proplatelet buds in cultured and circulating platelets.

Conclusions

Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.     

Human megakaryocytes. III. Characterization in myeloproliferative disorders

Abnormal proliferation of the megakaryocytic line was observed in the marrow tissue from patients with myeloproliferative disorders. Megakaryocytes were identified by immunofluorescence using distinct platelet protein markers. Plasma factor VIII antigen (factor VIII:AGN) and platelet glycoproteins IIb and IIIa were detected in normal mature and early megakaryocytes, as well as in a morphologically heterogeneous population of low density marrow cells regarded as atypical megakaryocytes. Atypical megakaryocytes were defined as oval/round 14- 35-micron diameter blast-like mononuclear/multinucleated cells bearing platelet protein markers with distinct morphological features, including cytoplasmic vacuolation, variable nuclear/cytoplasmic ratios, and variable cytoplasmic granulation. Atypical megakaryocytes were observed in most chronic myelogenous leukemia (CML) patients and in two patients with polycythemia vera, representing between 60 and 1,840 cells/10(4) cells (less than 1.050 g Percoll/cu cm). No atypical megakaryocytes were found in (a) 20 normal controls, (b) two patients with essential thrombocythemia, (c) a patient with thrombocytosis secondary to acute bleeding, and (d) in two patients with CML. Atypical megakaryocytes appear to represent a single-cell population, as demonstrated by a series of double immunofluorescence assays using combinations of five different antiplatelet protein sera. There was a statistically significant correlation between the frequency of atypical megakaryocytes and the presence of immature forms of myeloid cells in blood. Analyses of Fc IgG receptors conducted with two different immunofluorescence systems have demonstrated that phenotypic similarities existed between atypical megakaryocytes and myeloproliferative platelet proteins and differentiation markers on megakaryocytes are useful in elucidating the pathophysiologic alterations occurring in the megakaryocytic compartment in patients with myeloproliferative disorders.     

Expression of CD4 by human megakaryocytes.

The CD4 antigen, which serves as the receptor for human immunodeficiency virus type 1 (HIV-1) on T cells, has been detected on human megakaryocytes. Recent evidence of impaired thrombopoiesis in HIV-1-related thrombocytopenia suggested that these cells could be directly infected by the virus and prompted a search for a receptor on megakaryocytes of normal subjects that could permit entry of HIV-1. Bone marrow specimens from uninfected normal control subjects were centrifuged over Ficoll-Hypaque (1.077 g/ml) and analyzed by three-color analysis with a flow cytometer utilizing monoclonal antibodies against CD4 and a glycoprotein present on the surface of megakaryocytes and platelets (GPIIb/IIIa; CD41), as well as 7-aminoactinomycin D, a stain for DNA. Cells presumed to be megakaryocytes were identified by having a DNA content greater than tetraploid and staining brightly with anti-CD41. Approximately 0.4% of the nucleated cells of the marrow met these criteria. Twenty-five percent of these megakaryocytes stained as brightly as CD4+ T cells. Several clones of antibody recognizing different epitopes of the CD4 molecule gave similar results. Platelets were CD4-. Staining of megakaryocytes with anti-CD4 was confirmed by direct microscopic examination of Percoll-gradient-enriched megakaryocytes employing two-color (CD4-phycoerythrin and CD41-fluorescein) immunofluorescence analysis and phase-contrast microscopy. The proportion of double-labeled cells among 112 phase-contrast-identifiable megakaryocytes from five bone marrow specimens varied between 20% and 26% with a mean and SD of 22% +/- 2.5%. Thus some human megakaryocytes express CD4 on their surface that should be capable of binding the HIV-1 gp120 envelope protein. This could serve as a portal of entry for HIV-1.     

Endocytosis of exogenous factor V by ex‐vivo differentiated megakaryocytes from patients with severe parahaemophilia

Although human megakaryocytes can synthesize factor V (FV), platelet FV derives largely from endocytosis of plasma FV. Recently, it has been shown that plasma transfusions can replenish the platelet FV pool in parahaemophilic patients. Here we corroborate this finding by showing FV endocytosis by ex vivo differentiated megakaryocytes derived from patients with inherited parahaemophilia. Mononuclear stem cells isolated from peripheral blood of healthy subjects and of three patients with severe parahaemophilia were cultured in the presence of thrombopoietin and interleukin‐3 and differentiated into CD41‐positive polynucleated megakaryocytes. Exogenous purified FV was added to the culture medium to evaluate FV endocytosis. Immunofluorescence staining revealed abundant FV expression in megakaryocytes derived from healthy donors, but no FV expression in those derived from patients with severe parahaemophilia. However, after the addition of purified FV to the culture medium, megakaryocytes from parahaemophilia patients became positive upon FV immunostaining, suggesting endocytosis of exogenous FV. Endocytosed FV retained factor Xa‐co‐factor activity as assessed by a prothrombin time‐based functional test in megakaryocyte lysates. Addition of exogenous FV to culture medium can restore the FV content of megakaryocytes derived from patients with severe FV defects. This rescue mechanism can have important clinical implications in the management of parahaemophilia patients.     

Incomplete antigenic cross-reactivity between platelets and megakaryocytes: relevance to ITP

Immune thrombocytopenias are usually associated with normal or increased numbers of megakaryocytes in the marrow. Therefore, the mechanism(s) responsible for the destruction of circulating platelets may not affect megakaryocytes in the same way. One of the possibilities which could account for the differential effect on the cells would be the development of antibodies to components of platelet membranes which are not exposed on the surface of all megakaryocytes. To investigate this possibility, a rabbit antiserum specific for mouse platelets was tested against fresh and cultured mouse megakaryocytes by indirect immunofluorescence. This antiserum cross-reacted with 46% of fresh murine megakaryocytes and 54% of cultured megakaryocytes. Phase- contrast microscopy revealed the reacting megakaryocytes to be fully granulated with irregular contours and in the process of releasing platelets. Nonreactive megakaryocytes demonstrated smooth contours and lacked morphological evidence of thrombocytopoiesis. Electron microscopy showed that only in megakaryocytes (MK) with an irregular contour had the demarcation membrane system (DMS) reached continuity with the plasma membrane. Ultrastructural analysis of megakaryocytes from patients with ITP showed approximately 25% to 50% of megakaryocytes without evidence of injury, whereas 50% to 75% had extensive damage. In undamaged cells, platelet territories had not yet reached the peripheral zone. The DMS of damaged megakaryocytes opened to the exterior elaborating platelets. The observations suggested that some platelet antibodies react only with megakaryocytes which have reached the stage of thrombocytopoiesis. Relevant target antigens may not be exposed on all megakaryocytes before cytoplasmic fragmentation occurs.     

Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics

We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.