Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.     

In Vivo Platelet Production from Mature Megakaryocytes: Does Platelet Release Occur via Proplatelets?

Although platelets are universally accepted to be born from megakaryocytes (MKs), the mechanism by which platelets are formed and released from MKs in vivo remains controversial. One theory, known as the proplatelet theory, postulates that platelets are released from proplatelet processes protruding from MKs into sinusoids located in the bone marrow hematopoietic compartment. Proplatelet formation (PPF) has been observed in in vitro experiments involving detailed analyses of related molecular events. PPF has also been used as a marker of MK maturation. However, PPF is suggested to be a nonphysiological phenomenon. On the other hand, transmission electron microscopy (TEM) analyses have revealed platelet formation via explosive fragmentation of MK cytoplasm in bone marrow and lung capillaries prepared by immersion fixation. Moreover, TEM and scanning electron microscopy studies of liquid-cultured MKs kept in suspension show that platelet formation occurs without PPF. Rather, an explosive and global fragmentation of the MK cytoplasm composed of platelet territories has been reported as the mechanism of platelet formation. In addition, in vivo and ex vivo observations of platelet release from MKs with phase-contrast microscopy strongly support the explosive-fragmentation theory. With all observations taken into account, PPF may not be a prerequisite for platelet release from MKs under real-life conditions. In this review, a new "protoplatelet" concept is proposed to support the explosive-fragmentation theory. Additionally, the role of the lungs in platelet production is reviewed and discussed.     

Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic beta-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by beta-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.     

In vitro culture of murine megakaryocytes from fetal liver-derived hematopoietic stem cells

Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000–20,000 MKs per liver. Approximately, 20–50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation.     

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbβ3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.     

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.     

Fraxiparin, a low-molecular-weight heparin, stimulates megakaryocytopoiesis in vitro and in vivo in mice

Summary. The effect of a low-molecular-weight heparin, faxiparin (Nadroparinŕ;), on murine megakaryocytopoiesis in vitro and in vivo was studied in comparison with unfractionated heparin. The addition of fraxiparin at 1–20 IU/ml into plasma clot cultures but not serum-free agar culture significantly enhanced MK colony growth. Furthermore, fraxiparin was found to potentiate the stimulating activity of aplastic anaemia serum (AAS) but not stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo), on MK colony growth in vitro , and to neutralize the inhibitory effect of platelet factor 4 (PF4) in vitro and in vivo . Fraxiparin also acted synergistically with heparin confactor II and antithrombin III to promote megakaryocyte colony formation. Intraperitoneal administration of fraxiparin twice daily for 4d at 0.1–25IU/injection increased in mice the level of blood platelet counts and the number of single MKs and CFU-MK in bone marrow. These data demonstrate that fraxiparin is able to positively regulate megakaryocytopoiesis.     

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2–3) and extensive emperipolesis, with many (36–65%) MKs containing 2–4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.     

Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1(low) mice

Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1(tm2Sho) or GATA-1(low) mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1(low) mutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1(low) mice. The block in maturation includes failure to properly organize alpha granules because von Willebrand factor is barely detectable in mutant MKs, and P-selectin, although normally expressed, is found frequently associated with the demarcation membrane system (DMS) instead of within granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in GATA-1(low) platelets. Mutant MKs are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process to establish contact with MKs by fusing their membrane with those of the DMS. As a result, 16% (in spleen) to 34% (in marrow) of GATA-1(low) MKs contain 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1(low) MKs have morphologic features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathologic neutrophil emperipolesis may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenvironment.     

Absence of GPIbalpha is responsible for aberrant membrane development during megakaryocyte maturation: ultrastructural study using a transgenic model

OBJECTIVE: The glycoprotein Ib/IX/V complex (GPIb-IX-V) mediates platelet attachment to von Willebrand factor in exposed subendothelium. Molecular defects in the genes for GPIbalpha, GPIbbeta, and GPIX give rise to the Bernard-Soulier syndrome, in which thrombocytopenia and giant platelets suggest that this receptor also is involved in platelet production. To study how giant platelets are produced in vivo, we used a model of GPIbalpha-deficient mice (GPIbalpha(null)) and mice rescued with the human GPIbalpha transgene (GPIbalpha(null;hTg)). MATERIALS AND METHODS: Using electron microscopy and immunogold labeling, we examined megakaryocytopoiesis in the bone marrow of these mice and developed a method to quantify the membranes of megakaryocytes (MK) and proplatelets by computer analysis. RESULTS: Abnormal membrane development in the perinuclear zone was found in immature MK of GPIbalpha(null) mice. This led to a poorly developed demarcation membrane system and other ultrastructural changes. As a result, well-organized platelet territories were rarely seen within the cytoplasm of mature MK. Membrane quantification confirmed that MK of GPIbalpha(null) mice had a reduced internal membrane pool. Whereas these MK normally crossed the endothelial barrier, their migration was accompanied by the production of unusually large MK fragments or proplatelets in the vascular sinus with an approximately 50% decrease in internal membrane content compared to wild-type. In the rescued GPIbalpha(null;hTg) model, GPIbalpha was normally localized in MK, and there was a total correction of the ultrastructural defects. CONCLUSIONS: GPIbalpha is essential for membrane development and distribution in maturing MK. Its absence leads to abnormal partitioning of the membrane systems and abnormal proplatelet production.     

Romiplostim administration shows reduced megakaryocyte response-capacity and increased myelofibrosis in a mouse model of MYH9-RD

Macrothrombocytopenia in MYH9-related disease (MYH9-RD) results from defects in nonmuscular myosin-IIA function. Thrombopoietin receptor agonists (eltrombopag; romiplostim) seem to improve hemostasis, but little is known about their biologic effects in MYH9-RD. We administered romiplostim to Myh9(-/-) mice (100 μg/kg, every 3 days, during 1 month). MKs increased to similar numbers in Myh9(-/-) and wild-type (WT) mice (with an increase in immature MKs), but Myh9(-/-) platelet count response was much less (2.5-fold vs 8-fold increase). A strong increase in MK nuclei emboli in the lung, in WT and Myh9(-/-) mice, indicates increased transmigration of MKs from the BM. Prolonged (but not acute) treatment with romiplostim decreased expression of GPIb-IX-V complex and GPVI, but not of GPIIbIIIa, and bleeding time increased in WT mice. Microcirculation was not altered by the increased number of large platelets in any of the assessed organs, but in Myh9(-/-) mice a much stronger increase in BM reticulin fibers was present after 4 weeks of romiplostim treatment vs WT mice. These data further encourage short-term use of thrombopoietic agents in patients with MYH9-RDs; however, myelofibrosis has to be considered as a potential severe adverse effect during longer treatment. Reduction of GPIbIX/GPVI expression by romiplostim requires further studies.     

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.     

The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34(+) cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1alpha (SDF-1alpha), and actin normally polymerized after SDF-1alpha or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.     

Type IIB Tampa: a variant of von Willebrand disease with chronic thrombocytopenia, circulating platelet aggregates, and spontaneous platelet aggregation

Type IIB von Willebrand disease is characterized by enhanced ristocetin- induced platelet aggregation and absence of large von Willebrand factor multimers from plasma. An alteration of the von Willebrand factor molecule resulting in increased reactivity with platelets appears to be the basis for these abnormalities. We have now identified a new variant of type IIB von Willebrand disease in a family in which the four affected members also have chronic thrombocytopenia, in vivo platelet aggregate formation, and spontaneous platelet aggregation in vitro. In spite of repeatedly prolonged bleeding times and persistent thrombocytopenia, their bleeding diathesis is only moderate.     

The WAVE2/Abi1 complex differentially regulates megakaryocyte development and spreading: implications for platelet biogenesis and spreading machinery

Actin polymerization is crucial in throm-bopoiesis, platelet adhesion, and mega-karyocyte (MK) and platelet spreading. The Wiskott-Aldrich syndrome protein (WASp) homolog WAVE functions downstream of Rac and plays a pivotal role in lamellipodia formation. While MKs and platelets principally express WAVE1 and WAVE2, which are associated with Abi1, the physiologic significance of WAVE isoforms remains undefined. We generated WAVE2(-/-) embryonic stem (ES) cells because WAVE2-null mice die by embryonic day (E) 12.5. We found that while WAVE2(-/-) ES cells differentiated into immature MKs on OP9 stroma, they were severely impaired in terminal differentiation and in platelet production. WAVE2(-/-) MKs exhibited a defect in peripheral lamellipodia on fibrinogen even with phorbol 12-myristate 13-acetate (PMA) costimulation, indicating a requirement of WAVE2 for integrin alpha(IIb)beta(3)-mediated full spreading. MKs in which expression of Abi1 was reduced by small interfering RNA (siRNA) exhibited striking similarity to WAVE2(-/-) MKs in maturation and spreading. Interestingly, the knockdown of IRSp53, a Rac effector that preferentially binds to WAVE2, impaired the development of lamellipodia without affecting proplatelet production. In contrast, thrombopoiesis in vivo and platelet spreading on fibrinogen in vitro were intact in WAVE1-null mice. These observations clarify indispensable roles for the WAVE2/Abi1 complex in alpha(IIb)beta(3)-mediated lamellipodia by MKs and platelets through Rac and IRSp53, and additionally in thrombopoiesis independent of Rac and IRSp53.     

A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice

During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1-deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1-deficient MKs in response to a gradient of stromal cell-derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1(-/-) MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be explained by an increase in integrin activation. The defect of migration and polarization was confirmed in vivo with demonstration of altered spatial localization of MKs within the bone marrow in PECAM-1-deficient mice, following immune-induced thrombocytopenia. This study identifies a novel role for PECAM-1 in regulating MK migration and thrombopoiesis.     

Prothrombotic megakaryocyte and platelet changes in hypertension are reversed following treatment: a pilot study

Platelets are formed from, and their function determined by, bone marrow megakaryocytes (MK). Previous studies have found that hypertension is associated with accentuated platelet function and that some antihypertensive drug classes have antiplatelet activity. We measured MK ploidy (DNA content), size, granularity, and expression of the adhesion molecule glycoprotein (GP) IIIa, using flow cytometry and measures of platelet function, in 12 untreated hypertensive patients and 14 normotensive subjects. Eight hypertensive patients were then treated with losartan (50 mg daily), an angiotensin receptor antagonist that lowers blood pressure, and MK and platelet parameters re-measured after 6 weeks. Hypertensive patients had, as compared with matched normotensive subjects: increased MK ploidy (mean +/- SD) 22.9 +/- 2.2 N versus 20.8 +/- 1.6 N (2P = 0.009); increased platelet size, 10.67 +/- 1.03fl versus 9.26 +/- 0.72fl (2P < 0.001); increased platelet expression of GP IIIa, 108.6 +/- 22.5 versus 92.0 +/- 12.3 (2P = 0.036); and reduced platelet count, (207 +/- 52) x 10(9)/l versus (257 +/- 55) x 10(9)/l (2P = 0.026). Losartan significantly reduced MK ploidy, 22.6 +/- 2.2 N versus 21.4 +/- 1.9 N (2P = 0.006); MK size, 607 +/- 22 versus 579 +/- 16 (2P = 0.003); and lengthened cutaneous bleeding time, 424 +/- 86s versus 563 +/- 164s (2P = 0.011), in hypertensive patients. Losartan did not alter MK granularity or GP IIIa expression, or platelet count, size, mass, GP IIIa expression, or aggregation. The data suggest that platelet changes in hypertension may be secondary to changes in MKs, and that anti-hypertensive treatment can alter MKs and the function of platelets they produce. Since antihypertensive therapy reduces the risk of stroke and myocardial infarction, MKs are a novel therapeutic target for the prevention of vascular events.     

Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture

Cellular and molecular analysis of megakaryocytopoiesis has been hampered thus far by the lack of pure and abundant megakaryocyte (MK) cell populations. In this study, hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood, were induced to megakaryocytic differentiation/maturation in serum-free liquid suspension culture treated with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal maturation; (2) further addition of mpIL increased the MK purity level to 80% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell number (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphological evaluation indicated the presence of putative mononuclear MK precursors and then of mature polynucleated platelet- forming MKs, peaking at days 5 and 12, respectively. Membrane phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before mature MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature MKs. Furthermore, MKs synthesize and secrete low but significant amounts of both IL-6 and granulocyte- macrophage colony-stimulating factor. Comparative culture studies were performed on purified bone marrow CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no proliferation. In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of highly purified or "pure" megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis.