Thrombopoietin (Tpo) is a major regulator of megakaryopoiesis both in vivo and in vitro. Tpo initiates its biologic effects by binding to the Mpl receptor, which is a member of the hematopoietin receptor family. To define the Tpo binding characteristics of the Mpl receptor, we iodinated purified 70-kD recombinant human Tpo using the Bolton-Hunter reagent. Autoradiographic analysis of 125I-Tpo binding to normal human marrow mononuclear cells showed many grains specifically associated with megakaryocytes; there were no grains specifically associated with myeloblasts or erythroblasts. Equilibrium binding experiments with 125I-Tpo and normal human platelets showed a single class of high-affinity receptors (kd, 190 pmol/L) with approximately 30 Mpl receptors per platelet. Affinity cross-linking with 125I-Tpo showed that the Mpl receptor on platelets is of molecular weight ∼98 kD. Despite their sequence similarity, erythropoietin and Tpo did not cross-compete for binding to BaF3 cells engineered to coexpress Mpl receptor and erythropoietin receptor. Progeny of normal human burst-forming units-erythroid (BFU-E) contained Mpl receptor mRNA, and flow cytometric analysis showed the presence of Mpl receptor protein on the surface of these cells. These data indicate that display of the Mpl receptor is not limited to the megakaryocytic lineage, but also includes progeny of BFU-E. Like receptors for other hematopoietic cytokines, the binding affinity of the Mpl receptor for Tpo is high, with relatively few receptors displayed per cell. These results suggest that the effects of Tpo to speed red blood cell recovery after myelosuppressive therapy in vivo and to enhance colony-forming unit-erythroid generation in vitro may be mediated by direct interaction of Tpo and erythroid progenitor cells.

THROMBOPOIETIN (Tpo), the ligand for the Mpl receptor, plays a major role in the regulation of megakaryopoiesis.1 Tpo promotes both the proliferation of colony-forming units-megakaryocyte (CFU-Meg) and the cytoplasmic and nuclear maturation of megakaryocytes in vitro.2-6 Tpo also exerts a direct effect on platelets; exposure of platelets to Tpo in vitro potentiates the effects of platelet aggregatory agents.7,8 Experiments using highly enriched populations of hematopoietic stem cells that are capable of reconstituting hematopoiesis long-term in lethally irradiated hosts suggest that Tpo can act directly on these primitive cells as well.9 10 

Studies of knock-out mice engineered to lack either Mpl receptor or Tpo confirm the importance of Tpo in the regulation of megakaryopoiesis.11-13 These mice have platelet counts that are approximately 6% to 15% of normal as well as equally reduced numbers of megakaryocytes in the marrow. Conversely, preclinical studies show that treatment with Tpo increases the platelet count 5- to 10-fold in normal animals and speeds platelet recovery after chemoradiotherapy.8 14-17 

Tpo and erythropoietin (Epo) share a number of structural features. The N-terminal domain of Tpo has 22% identical and 25% conservative substitutions in comparison to the amino acid sequence of Epo, and the location of 3 of the 4 cysteine residues are conserved between these two molecules.18 The Mpl receptor and the Epo receptor, both members of the hematopoietin receptor superfamily, share 26% amino acid homology.19 There is a degree of functional overlap between these two cytokines as well. Tpo and Epo display synergistic effects on megakaryopoiesis (stimulation of CFU-Meg colony growth)2 and on erythropoiesis (generation of colony-forming unit-erythroid [CFU-E] and growth of burst-forming unit-erythroid [BFU-E] progeny)20-22 in vitro. Tpo amplifies the production of BFU-E from cellular populations containing long-term culture-initiating cells.23 Moreover, Tpo can stimulate BFU-E colony growth from liver cells of fetal mice engineered to lack the Epo receptor.24 Although treatment with Tpo does not increase the hematocrit of normal mice, it does increase reticulocyte production and accelerate red blood cell recovery in myelosuppressed mice, likely by synergizing with the high level of endogenous Epo found in these mice.20 

Binding of Tpo to the Mpl receptor triggers a number of events, including tyrosine phosphorylation of the receptor and activation of JAK2, TYK2, and Shc as well as dimerization and nuclear translocation of STAT proteins.25-31 Although the intracellular events that follow Tpo binding to the Mpl receptor have been the focus of intense investigation, less is known about the cellular distribution and Tpo binding characteristics of the cell surface Mpl receptor. Mpl receptor mRNA has been identified in normal human CD34+ cells, in megakaryocytic cell lines, and in blasts obtained from a portion of patients with acute myelogenous leukemia.32-35 The majority of myeloid and erythroid cell lines studied have been reported to lack Mpl receptor mRNA. However, Mpl receptor mRNA has been found in murine fetal liver BFU-E progeny.24 Flow cytometric analysis and Western blotting with an anti-Mpl receptor monoclonal antibody (MoAb) have documented antigenic display of the Mpl receptor protein on human megakaryocytic cell lines and on platelets35; and platelets can bind and metabolize Tpo.36 37 

In the present study, we sought to more fully define the Tpo binding characteristics and cellular distribution of the functional Mpl receptor on normal human hematopoietic cells. We also investigated whether the overlapping biologic effects of Tpo and Epo could be explained in part by cross-competition for receptor binding and whether BFU-E and their progeny exhibit Mpl receptors. The results showed that normal human platelets display a low number of high-affinity Mpl receptors and that Tpo and Epo synergy cannot be explained by cross-competition for receptor binding to cells that coexpress functional receptors for both cytokines. Normal human BFU-E progeny contain Mpl receptor mRNA and display the Mpl receptor on the cell surface, suggesting that the effects of Tpo on erythropoiesis may be direct.

Normal hematopoietic cells and cell lines.Human marrow cells were obtained by aspiration from the posterior iliac crest of healthy adults after obtaining informed consent. These studies were approved by the Human Subjects Division of the University of Washington (Seattle, WA). Marrow mononuclear cells were prepared by density centrifugation using Ficoll-Hypaque (Organon Teknika Corp, Durham, NC). Marrow mononuclear cells were used for autoradiographic analysis of 125I-Tpo binding.

To obtain BFU-E and colony-forming unit–granulocyte-macrophage (CFU-GM) progeny, the mononuclear cells were cultured at a concentration of 1 to 2 × 105 cells/mL in 1.4% methylcellulose (Dow, Midland, MI) in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 30% heat-inactivated fetal calf serum (FCS; Hyclone, Logan, UT), 1% bovine serum albumin (Reheis, Phoenix, AZ), 2 U/mL recombinant human Epo,38 and 50 ng/mL recombinant human stem cell factor (a gift from Dr K. Langley, Amgen, Inc, Thousand Oaks, CA). For CFU-GM progeny, the cultures also included 2 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; a gift from Dr K. Kaushansky, University of Washington). After 5 to 14 days of incubation at 37°C, in a humidified atmosphere containing 5% CO2 , BFU-E or CFU-GM progeny were plucked under the inverted microscope as previously described.39 To further define the proliferative potential and cellular composition of the population of cells thus obtained, a portion of these cells were replated in CFU-E colony assays39 in the presence of Epo (2 U/mL) plus recombinant human interleukin-3 (IL-3; 1 ng/mL; Genzyme Corp, Boston, MA). The BFU-E progeny were also labeled with phycoerythrin (PE)-conjugated anti-glycophorin A antibody (Dako, Carpinteria, CA), as previously described,22 and analyzed by fluorescence microscopy (Zeiss Universal; Zeiss, Thornwood, NY).

Peripheral blood from healthy adult volunteers was drawn into a syringe containing 15% acid-citrate-dextrose anticoagulant. Platelets were isolated by centrifuging the blood at 250g for 10 minutes at room temperature. An aliquot of each platelet preparation was sent to the clinical laboratory at the University of Washington Medical Center, where the number and purity of the platelets were quantitated using a Abbott Cell-Dyn 3500 (Santa Clara, CA). All of the platelet preparations used for Scatchard analysis of 125I-Tpo binding or affinity cross-linking contained more than 99% platelets and less than 1% white blood cells. To assess the activation status of the platelets thus isolated, display of CD62P (P-selectin) on the platelet surface was examined.40 The platelets were incubated with biotinylated anti-CD61 MoAb (20 μg/mL; Southern Biotechnology Associates, Birmingham, AL) for 30 minutes at 4°C and then with a mixture of avidin-PE (1:100 final dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) plus either anti-CD62P MoAb directly conjugated to fluorescein isothiocyanate (FITC; 1:10 final dilution; PharMingen, San Diego, CA) or control mouse IgG1κ directly conjugated to FITC (1:10 final dilution; PharMingen) for 30 minutes at 4°C and analyzed with Coulter Epics Elite flow cytometer (Coulter, Miami, FL). Of the CD61+ cells, 8.5% displayed CD62P on the cell surface, which is within the range previously reported for normal human platelets.41 

The HEL and K562 human erythroleukemia cell lines were maintained in IMDM supplemented with 10% FCS. The growth factor-dependent murine lymphoblastoid cell line BaF3 engineered to express human Mpl receptor (BaF3-MplR)14 was maintained in IMDM supplemented with 10% FCS and 100 U/mL recombinant human Tpo (a gift from Dr D. Foster, ZymoGenetics Inc, Seattle, WA). BaF3 cells that coexpressed the mouse Epo receptor and human Mpl receptor were generated by electroporation of BaF-MplR cells with a plasmid vector that expresses the Epo receptor (pXM/mEpoR; kindly provided by Dr A. D'Andrea, Dana-Farber Cancer Institute, Boston, MA) and selection in 2 U/mL Epo. A cell line that proliferates in the presence of either human Epo or human Tpo was obtained by limiting dilution.

Binding experiments with 125I-Tpo.Purified recombinant human Tpo, expressed in BHK cells, was iodinated using the Bolton-Hunter reagent (Dupont/NEN, Boston, MA). Briefly, Tpo was incubated with the Bolton-Hunter reagent in 100 mmol/L phosphate buffer (pH 8.5) for 16 hours at 4°C and then passed over a Sephadex G 75-120 column previously equilibrated with 100 mmol/L phosphate-buffered saline (pH 7.4) containing 0.5% gelatin (Sigma Chemical Co, St Louis, MO). The radiologic specific activity of 125I-Tpo was determined by self-displacement analysis42 and was generally approximately 1,000 Ci/mmol. Parallel displacement curves were obtained, showing that 125I-Tpo and unlabeled Tpo exhibited similar receptor binding affinity.

Autoradiographic analysis of 125I-Tpo binding to normal human marrow mononuclear cells and to BaF3-MplR cells was performed by incubating 125I-Tpo (0.6 nmol/L) with the cells in binding buffer for 1 hour at 37°C.39 The binding buffer consisted of RPMI 1640 (MA Bioproducts, Walkersville, MD) supplemented with 50 mmol/L HEPES (pH 7.4), 0.5% gelatin, 0.1% azide, and 10 μg/mL cytochalasin B. Parallel samples were incubated with 125I-Tpo plus a 100-fold excess of unlabeled Tpo. At the conclusion of the incubation, the cell suspension was gently layered on percoll (density, 1.028 g/mL) and centrifuged for 1 minute at 4°C in a Beckman Microfuge 11 (Beckman, Palo Alto, CA) to separate cell-associated 125I-Tpo from free 125I-Tpo. Cytopreparations were made using a Shandon Southern Cytospin (Shandon Co, Pittsburgh, PA). The samples were exposed to photographic emulsion for 3 weeks at 4°C and developed as previously described.39 43 Grain counts were performed on approximately 30 cells in each sample. Specific binding was defined as the number of grains overlaying the cells in the presence of 125I-Tpo minus the number of grains overlaying the cells in the presence of 125I-Tpo plus unlabeled Tpo.

To determine whether 125I-Tpo and 125I-Epo could cross compete for receptor binding, BaF3 cells coexpressing functional Mpl and Epo receptors (1.2 × 106 cells) were incubated with 125I-Tpo (0.3 nmol/L) in the presence of a 100-fold molar excess of unlabeled Tpo or unlabeled recombinant human Epo for 1 hour at 37°C. In parallel, the BaF3-MplR-EpoR cells were incubated with 125I-Epo (0.3 nmol/L; Amersham, Arlington Heights, IL) with or without a 100-fold excess of unlabeled Tpo or unlabeled Epo for 1 hour at 37°C. The binding experiments were performed in triplicate. At the conclusion of the incubation, the cells were sedimented through phthalate oil as previously described39 and both cell-associated and free radiolabeled ligand were quantitated using a Packard Cobra II Gamma Counter (Packard Instrument Co, Downers Grove, IL).

To quantitate the binding affinity and number of Mpl receptors per cell, the cells were incubated in 125I-Tpo (30 pmol/L to 1.2 nmol/L) with or without a 100-fold excess of unlabeled Tpo in binding buffer that contained azide and cytochalasin B to inhibit internalization of the ligand for 1 hour at 37°C.39,44 For binding experiments with platelets, the binding buffer also contained 15% acid-citrate-dextrose to prevent platelet clumping. In one experiment, 125I-Tpo binding experiments with platelets were conducted overnight at 4°C and for 1 hour at 37°C in parallel. To determine whether partial receptor occupancy by Tpo acquired in vivo would alter the number of Mpl receptors detected by Scatchard analysis of 125I-Tpo binding, samples of platelets were incubated in IMDM supplemented with 10% FCS and 10% acid-citrate-dextrose for 90 minutes at 37°C to permit internalization and degradation of surface bound Tpo before quantitation of Mpl receptor number. All binding experiments were performed in triplicate, and the data were analyzed using the Ligand program.45 

Affinity cross-linking.Normal human platelets (1 × 109) or BaF3-MplR cells (2 ×107) were incubated with 125I-Tpo (0.6 nmol/L) with or without excess unlabeled Tpo in binding buffer for 1 hour at 37°C and then cross-linked with 1 mmol/L bis (sulfosuccinimidyl suberate) (BS3; Pierce Chemical Co, Rockford, IL) for 30 minutes at 22°C, as previously described.44 A parallel experiment was performed in the absence of BS3. The cells were lysed in 1% Triton X-100, 20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 10 mmol/L EDTA, 10% glycerol, 1 mmol/L phenylmethyl sulfonyl fluoride, 10 μg/mL leupeptin, 0.5 mg/mL aprotinin, and 10 μg/mL pepstatin. For some experiments, the cell lysates were directly suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing dithiothreitol, boiled for 3 minutes, and analyzed on a 6% polyacrylamide gel. In experiments designed to determine whether the cross-linked complexes contained Mpl receptor, the cell lysates were immunoprecipitated with affinity-purified anti-Mpl receptor polyclonal antibody (ZymoGenetics, Inc) or with control rabbit IgG and analyzed by SDS-PAGE. Molecular weight markers (range, 14 to 340 kD [Boehringer Mannheim, Indianapolis, IN] or 49.5 to 205 kD [BioRad, Hercules, CA]) were used.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Mpl receptor mRNA.Total cellular RNA was prepared from HEL and from K562 cells using the RNAgents Total RNA Isolation System (Promega, Madison, WI). Whole cell lysates from CD34+ cells isolated from human bone marrow and from day-5, day-7, and day-10 colonies derived from BFU-E progeny and from day-12 CFU-GM progeny were made by lysing the cells in a buffer containing 0.8% Igepal (Sigma), 5 mmol/L dithiothreitol (BioRad), and 30 U RNAisin (Promega). cDNA was made from HEL and K562 cell RNA, and from lysates from 5 × 104 CD34+ marrow cells and from lysates from BFU-E progeny using the Superscript Preamplification System (GIBCO-BRL, Gaithersburg, MD). Oligo dT primers were used to prime first-strand synthesis for all reactions. PCR amplification was performed using Pfu polymerase (Stratgene, La Jolla, CA) with an Ericomp Thermocycler (Ericomp Inc, San Diego, CA). To increase the sensitivity of detection, 5 μL of the product obtained after an initial 30 cycles of PCR was used in a second PCR amplification step using the same conditions. Amplification of the membrane-proximal extracellular region and the transmembrane domain of human mpl was performed using a sense primer with the sequence 5′-GACCCTGGAGCTGCGCCCGCGATCTCGCTA-3′ and an antisense primer with the sequence 5′-TCGAGTCAATGCCTCAGTCTCCTGTAGTGC-3′. The primers used for amplification of mpl were selected to span the boundary between exons 9 and 10 and result in a 238-bp PCR product from mRNA and a 490-bp PCR product from genomic DNA. Amplification of GAPDH cDNA was performed using a sense primer with the sequence 5′-CCATGGAGAAGGCTGGGG-3′ and an antisense primer with the sequence 5′-CAAAGTTGTCATGGATGACC-3′. Full-length human mpl in the expression vector PHZ-1 (ZymoGenetics, Inc) was used as a control for detection of the human mpl sequence.46 Amplified PCR products were analyzed by gel electrophoresis.

Flow cytometric analysis of Mpl receptor display on BFU-E and CFU-GM progeny.Colonies containing progeny of normal human BFU-E and CFU-GM were plucked as described above and were incubated with affinity-purified rabbit anti-Mpl receptor antibody (50 μg/mL; ZymoGenetics, Inc) or with purified control rabbit IgG (50 μg/mL; Jackson ImmunoResearch Laboratories) and then with mouse monoclonal anti-CD16 antibody to block nonspecific Fc receptors (1:10 final dilution; Dako) and FITC-conjugated goat antirabbit IgG (1:20 final dilution; Southern Biotechnology Associates) and were analyzed by flow cytometry. To determine whether the population of BFU-E progeny contained megakaryocytes, the cells were labeled as previously described22 with the Tab anti-CD41 MoAb47 (a gift from Dr R. McEver, University of Oklahoma, Oklahoma City, OK) or with mouse IgG1 control antibody (Caltag Laboratories, South San Francisco, CA) and then with FITC-conjugated goat antimouse IgG1 (American Quaalex, La Miranda, CA). All incubations were for 30 minutes at 4°C. HEL cells were used as a positive control for CD41 expression.

Autoradiographic analysis of 125I-Tpo binding to normal human bone marrow cells showed many grains associated with megakaryocytes (Fig 1). These grains were absent when the cells were incubated with 125I-Tpo in the presence of excess unlabeled Tpo. Grain counts showed that there were an average of approximately 1,300 grains per megakaryocyte in the presence of 125I-Tpo, and 200 grains per megakaryocyte in the presence of 125I-Tpo plus excess unlabeled Tpo. Thus, there were approximately 1,100 specifically bound grains per megakaryocyte. There was no detectable specific binding of 125I-Tpo to morphologically recognizable erythroblasts or myeloblasts. Parallel autoradiographic analysis of 125I-Tpo binding to the murine lymphoblastoid cell line BaF3 transfected with human Mpl receptor (BaF3-MplR cells) showed an average of 10 specifically bound grains per cell.

Fig. 1.

Autoradiographic analysis of 125I-Tpo binding to normal human bone marrow mononuclear cells. Original magnification × 100.

Fig. 1.

Autoradiographic analysis of 125I-Tpo binding to normal human bone marrow mononuclear cells. Original magnification × 100.

Close modal

Platelets obtained from normal human peripheral blood displayed a single class of high-affinity Mpl receptors, with a kd of 100 to 200 pmol/L (Fig 2). In this experiment, there were 25 receptors per platelet, with a binding affinity of approximately 110 pmol/L. In four additional experiments, the number of receptors ranged from 20 to 40 per platelet. When all five experiments were analyzed together using the ligand program,45 the kd was 190 pmol/L and the number of receptors per platelet was 30 ± 4 (mean ± SEM). The kd was 200 pmol/L when the equilibrium binding experiment was performed at 4°C, similar to results obtained at 37°C. When the platelets were preincubated for 90 minutes at 37°C to permit internalization and metabolism of Tpo36 acquired in vivo, before performing the equilibrium binding experiments, both the number of Mpl receptors detected (30 receptors per platelet) and the binding affinity were similar to results of a parallel experiment performed without preincubation of the platelets. This result suggests that partial Mpl receptor occupancy with Tpo is not altering the results of Scatchard analysis of Mpl receptor display on normal human platelets. BaF3-MplR cells exhibit a Tpo binding affinity of approximately 120 pmol/L, similar to that of the native receptor found in human platelets, and 480 ± 100 receptors per cell (mean ± SEM of 4 experiments, data not shown). No binding of 125I-Tpo to human marrow mononuclear cells was detected (N = 3 experiments). Because megakaryocytes comprise only a small portion of the heterogeneous population of marrow mononuclear cells (<0.5%), these data reinforce the results of autoradiography.

Fig. 2.

Binding of 125I-Tpo to normal human platelets. The platelets (1.0 × 108) were incubated with 125I-Tpo (30 pmol/L to 1.2 nmol/L) without (——) or with (⋅⋅⋅) or a 100-fold excess of unlabeled Tpo for 1 hour at 37°C (A). The results represent the mean of triplicate determinations. The data were analyzed using the ligand program (B). Four additional independent experiments gave similar results.

Fig. 2.

Binding of 125I-Tpo to normal human platelets. The platelets (1.0 × 108) were incubated with 125I-Tpo (30 pmol/L to 1.2 nmol/L) without (——) or with (⋅⋅⋅) or a 100-fold excess of unlabeled Tpo for 1 hour at 37°C (A). The results represent the mean of triplicate determinations. The data were analyzed using the ligand program (B). Four additional independent experiments gave similar results.

Close modal

Because of the structural similarity of Tpo and Epo, as well as the partial functional overlap between these two cytokines, we investigated whether Tpo and Epo could cross-compete for binding to BaF3 cells that coexpress functional human Mpl receptor and functional human Epo receptor (Fig 3). The BaF3-MplR-EpoR cells exhibit a proliferative response to both Tpo and Epo (data not shown). 125I-Tpo binding is specifically competed by unlabeled Tpo but not by unlabeled Epo (Fig 3). Similarly, 125I-Epo binding is inhibited by unlabeled Epo but not by an equivalent molar quantity of unlabeled Tpo. These results indicate that Tpo and Epo do not cross-compete for receptor binding to the BaF3-MplR-EpoR cells. Similar results were obtained in one experiment using HEL cells that coexpress native Mpl receptor and Epo receptor but do not exhibit a proliferative response to these cytokines.

Fig. 3.

Binding of 125I-Tpo or 125I-Epo to BaF3-MplR-EpoR cells. The cells (1.2 × 106) were incubated with 125I-Tpo (0.3 nmol/L) or 125I-Epo (0.3 nmol/L) in the presence or absence of a 100-fold excess of unlabeled Tpo or Epo for 1 hour at 37°C. The data represent the mean ± SEM of triplicate determinations from one of three similar experiments.

Fig. 3.

Binding of 125I-Tpo or 125I-Epo to BaF3-MplR-EpoR cells. The cells (1.2 × 106) were incubated with 125I-Tpo (0.3 nmol/L) or 125I-Epo (0.3 nmol/L) in the presence or absence of a 100-fold excess of unlabeled Tpo or Epo for 1 hour at 37°C. The data represent the mean ± SEM of triplicate determinations from one of three similar experiments.

Close modal

Affinity cross-linking of 125I-Tpo to its receptor on BaF3-MplR cells (Fig 4A) or on normal human platelets (Fig 4B) showed a prominent cross-linked complex. This radiolabeled complex was not seen when affinity cross-linking was performed in the presence of excess unlabeled Tpo or when the cross-linker was omitted. Immunoprecipitation with an affinity-purified polyclonal anti-Mpl receptor antibody confirmed that the cross-linked complex contained Mpl receptor protein (data not shown). After subtracting the contribution of the BHK-expressed 125I-Tpo in the cross-linked complex, the molecular weight of the functional form of Mpl receptor on normal human platelets is estimated to be 98 ± 2 kD (mean ± SEM of 6 experiments). No dimers of the 125I-Tpo-Mpl receptor complex were evident in this or in five independent cross-linking experiments with normal human platelets (Fig 4).

Fig. 4.

Affinity cross-linking with 125I-Tpo. BaF3-MplR cells (A) or normal human platelets (B) were incubated with 125I-Tpo (0.6 nmol/L) in the presence or absence of a 100-fold excess of unlabeled Tpo and cross-linked with 1 mmol/L BS3. Cell lysates were analyzed by SDS-PAGE and autoradiography. The upper band represents the major 125I-Tpo-Mpl receptor complex and the lower band is free 125I-Tpo. Lane 1, 125I-Tpo; lane 2, 125I-Tpo plus unlabeled Tpo. Molecular weight markers are indicated.

Fig. 4.

Affinity cross-linking with 125I-Tpo. BaF3-MplR cells (A) or normal human platelets (B) were incubated with 125I-Tpo (0.6 nmol/L) in the presence or absence of a 100-fold excess of unlabeled Tpo and cross-linked with 1 mmol/L BS3. Cell lysates were analyzed by SDS-PAGE and autoradiography. The upper band represents the major 125I-Tpo-Mpl receptor complex and the lower band is free 125I-Tpo. Lane 1, 125I-Tpo; lane 2, 125I-Tpo plus unlabeled Tpo. Molecular weight markers are indicated.

Close modal

To investigate Mpl receptor display during differentiation of normal human erythroid progenitor cells, progeny of BFU-E plucked after 5 to 10 days of culture and progeny of CFU-GM plucked after 12 days of culture were analyzed for Mpl receptor mRNA by RT-PCR, with CD34+ human marrow mononuclear cells, HEL cells, K562 cells, and cloned human Mpl cDNA serving as positive controls. The day-5, -7, and -10 BFU-E progeny contained Mpl receptor mRNA (Fig 5); the day-12 CFU-GM progeny also contained Mpl receptor mRNA (data not shown). The day-5 and day-7 BFU-E progeny were mainly large pale early erythroblasts, whereas the day-10 BFU-E progeny included a population of smaller well-hemoglobinized late erythroblasts. More than 80% of the day-10 BFU-E progeny expressed glycophorin A. Replating experiments showed that the day-5 BFU-E progeny contained approximately 33% CFU-E, whereas the day-7 BFU-E progeny contained approximately 27% CFU-E and the day-10 BFU-E progeny contained approximately 2% CFU-E. Two additional experiments gave similar results. Direct binding experiments failed to show reproducible 125I-Tpo binding to progeny of peripheral blood BFU-E plucked at day 7 through day 12 (N = 10 experiments, data not shown).

Fig. 5.

RT-PCR analysis of Mpl receptor mRNA. RT-PCR was performed to detect the presence of c-mpl or GAPDH mRNA in HEL cells (lanes 4 and 5), K562 cells (lanes 6 and 7), CD34+ human marrow cells (lanes 8 and 9), and colonies derived from day-5 (lanes 10 and 11), day-7 (lanes 12 and 13), and day-10 (lanes 14 and 15) BFU-E progeny. The primers used for amplification were specific for the extracellular domain of Mpl (lanes 3, 4, 6, 8, 10, 12, and 14) or for GAPDH (lanes 2, 5, 7, 9, 11, 13, and 15). Cloned human mpl cDNA was used as a positive control (lane 3). Negative controls were performed using no input cDNA (lanes 1 and 2).

Fig. 5.

RT-PCR analysis of Mpl receptor mRNA. RT-PCR was performed to detect the presence of c-mpl or GAPDH mRNA in HEL cells (lanes 4 and 5), K562 cells (lanes 6 and 7), CD34+ human marrow cells (lanes 8 and 9), and colonies derived from day-5 (lanes 10 and 11), day-7 (lanes 12 and 13), and day-10 (lanes 14 and 15) BFU-E progeny. The primers used for amplification were specific for the extracellular domain of Mpl (lanes 3, 4, 6, 8, 10, 12, and 14) or for GAPDH (lanes 2, 5, 7, 9, 11, 13, and 15). Cloned human mpl cDNA was used as a positive control (lane 3). Negative controls were performed using no input cDNA (lanes 1 and 2).

Close modal

To determine whether the Mpl receptor protein was displayed on the surface of BFU-E and CFU-GM progeny, the cells were labeled with an affinity-purified rabbit anti-Mpl receptor antibody or with purified control rabbit IgG and analyzed by flow cytometry (Fig 6). Cell surface expression of Mpl receptor protein was evident on the BFU-E progeny but not on the CFU-GM progeny. Three additional independent experiments with plucked BFU-E progeny gave similar results. These findings were verified by direct examination of the labeled cells by fluorescence microscopy. The majority of cells obtained from BFU-E progeny showed faint positivity with the anti-Mpl receptor antibody but not with the control antibody. The BFU-E progeny did not express the megakaryocytic marker CD41 (glycoprotein Iib/IIIa; Fig 7), suggesting that the finding of Mpl receptor expression was not due to the presence of megakaryocytes or platelets in this population of cells.

Fig. 6.

Flow cytometric analysis of Mpl receptor display on progeny of BFU-E and CFU-GM. Day-11 BFU-E and CFU-GM progeny labeled with control rabbit IgG (gray profile) or with affinity-purified rabbit anti-Mpl receptor antibody (dark profile) were analyzed by flow cytometry.

Fig. 6.

Flow cytometric analysis of Mpl receptor display on progeny of BFU-E and CFU-GM. Day-11 BFU-E and CFU-GM progeny labeled with control rabbit IgG (gray profile) or with affinity-purified rabbit anti-Mpl receptor antibody (dark profile) were analyzed by flow cytometry.

Close modal
Fig. 7.

Flow cytometric analysis of CD41 expression on progeny of BFU-E and on HEL cells. Day-11 BFU-E progeny and HEL cells labeled with control mouse IgG1 antibody (gray profile) or with the Tab anti-CD41 MoAb (dark profile) were analyzed by flow cytometry.

Fig. 7.

Flow cytometric analysis of CD41 expression on progeny of BFU-E and on HEL cells. Day-11 BFU-E progeny and HEL cells labeled with control mouse IgG1 antibody (gray profile) or with the Tab anti-CD41 MoAb (dark profile) were analyzed by flow cytometry.

Close modal

Our objective was to more fully define the cellular distribution and binding characteristics of the Mpl receptor on normal human hematopoietic cells. Among morphologically recognizable cells in the bone marrow, only megakaryocytes display 125I-Tpo binding that is detectable by autoradiography. Myeloid and erythroid precursor cells lacked detectable 125I-Tpo binding. This contrasts with findings with other cytokines that can stimulate megakaryopoiesis in vitro, including IL-3, Epo, and stem cell factor. 125I-Epo binding to both normal human megakaryocytes and erythroid precursor cells can be readily detected.48 Likewise, 125I-stem cell factor binds not only to megakaryocytes but also to both myeloid and erythroid precursor cells.49 The Mpl receptor shows a more restricted cellular distribution among morphologically identified hematopoietic precursor cells in normal human bone marrow than the receptors for these other cytokines.

Normal human platelets bind 125I-Tpo with high affinity. The binding affinity for Tpo, approximately 100 to 200 pmol/L, is similar to that found for other hematopoietic cytokines, including Epo, stem cell factor, IL-3, and GM-CSF. Murine platelets have been reported to bind 125I-murine Tpo with an affinity of 560 pmol/L and to display 220 Mpl receptors per platelet.37 However, the equilibrium binding experiments with the murine platelets in the latter study were performed under conditions that permitted internalization of the 125I-Tpo.37 Thus differences in species and in experimental techniques could contribute to these differing results. Tpo circulates in normal human plasma at a concentration of approximately 85 pg/mL.50 Tpo half-life is prolonged in Mpl receptor knock-out mice,37 reinforcing an earlier suggestion that platelets may regulate the level of Tpo in the circulation by binding and metabolizing the cytokine.3,36 The present results suggest that platelets would exhibit 1% to 2% receptor occupancy at the level of Tpo normally found in the circulation. Because a rich assortment of 125I-Tpo grains was seen on megakaryocytes, these cells may also contribute substantially to the metabolism of Tpo. Although we have not been able to obtain sufficient numbers of purified normal human megakaryocytes for Scatchard analysis of Mpl receptor number and binding affinity, autoradiographic analysis of 125I-Tpo binding showed that megakaryocytes display approximately 100-fold more grains per cell than do BaF3-MplR cells studied in parallel. The BaF3-MplR cells exhibit approximately 480 Mpl receptors per cell. Megakaryocyte metabolism of Tpo may explain the observation that circulating Tpo levels are not elevated in NF-E2 knock-out mice that fail to produce platelets51 or in patients with idiopathic thrombocytopenic purpura.52 

The Mpl receptor protein is found on the surface of at least a portion of normal human BFU-E isolated from peripheral blood or bone marrow.22 Debili et al35 used the M1 anti-Mpl receptor MoAb to examine receptor display on normal human bone marrow cells and concluded that Mpl receptor display is limited to late megakaryocytic progenitor cells, megakaryocytes, and platelets. However, the CD34+ M1+ fraction of marrow cells also contained late erythroid colony-forming cells that gave rise to erythroblasts after 8 days in culture,35 consistent with the present finding of Mpl receptor mRNA and cell surface expression of Mpl receptor protein in progeny of normal human BFU-E and with the report of Mpl receptor mRNA in murine fetal liver BFU-E progeny.24 

Tpo acts synergistically with Epo to increase CFU-E generation in liquid culture20 and expands both BFU-E and CFU-E numbers in myelosuppressed mice in vivo.20,53 Additionally, Tpo acts directly on progeny of BFU-E plucked after 5 to 6 days of culture to promote erythroid colony growth.21 Taken together with the cellular distribution of the Mpl receptor, these data suggest that the effects of Tpo on erythropoiesis are mediated by Tpo binding to erythroid progenitor cells. Tpo can also act in synergy with stem cell factor to promote CFU-GM colony growth.54 The observations that the number of hematopoietic progenitor cells from multiple lineages are reduced in Mpl receptor knock-out mice12,55 and that Tpo can increase the fraction of primitive hematopoietic stem cells entering the cell cycle10 also suggest that Tpo may act on hematopoietic cells at an earlier stage than previously appreciated.

A multitude of isoforms of human and mouse Mpl receptor has been identified. The Mpl P and Mpl K isoforms of the human Mpl receptor are identical in the extracellular domain but diverge in the intracellular domain after a common 9 amino acid juxtamembrane sequence.19 Both transmembrane and soluble forms of murine Mpl receptor have been found.56,57 A naturally occurring isoform of the murine Mpl receptor that contains a 180-bp deletion in the extracellular domain and fails to bind the 70-kD form of Tpo has also been described.58 The functional significance of these isoforms is unknown at present. Although flow cytometric analysis showed the presence of Mpl receptor protein on BFU-E progeny, 125I-Tpo binding to these cells could not be demonstrated. This finding could reflect the low number of Mpl receptors displayed per cell or the presence of an alternative isoform of Mpl receptor on BFU-E progeny. Binding of Tpo triggers tyrosine phosphorylation of a ∼95-kD form of murine Mpl receptor.25 The present studies of affinity cross-linking to normal human platelets show that this form of the Mpl receptor is also capable of binding Tpo.

Despite the degree of both structural and functional overlap between Tpo and Epo, these cytokines did not cross-compete for binding to cells engineered to express functional forms of the Mpl receptor and the Epo receptor. This suggests that there is not a shared receptor subunit that is present in limiting quantity, analogous to the common chain of the IL-3 receptor, GM-CSF receptor, and IL-5 receptor,59 that might explain some of the functional overlap between Tpo and Epo. Whether there is intracellular cross talk between the Tpo and Epo receptors, as has recently been shown for the Epo and c-kit receptors,60 remains to be determined. Because Tpo can promote BFU-E colony growth from liver cells of fetal mice that lack the Epo receptor, the presence of the Epo receptor is not an absolute requirement for Tpo-induced signal transduction.24 

We thank Drs C. Hart and D. Foster for providing Tpo and anti-Mpl receptor antibody, Dr K. Langley for providing stem cell factor, and Z. Sisk and A. Dimaunahan for manuscript preparation.

Supported by National Institutes of Health (NIH) Grants No. DK44194, CA31615, and DK49855 and by an American Cancer Society Faculty Research Award to V.C.B. A portion of this work was conducted in the Clinical Research Facility at the University of Washington, supported by NIH/National Center for Research Resources Grant No. M01 RR00037.

Address reprint requests to Virginia C. Broudy, MD, University of Washington, Division of Hematology, Box 357710, Seattle, WA 98195.

1
Kaushansky
K
Thrombopoietin: The primary regulator of platelet production.
Blood
86
1995
419
2
Broudy
VC
Lin
N
Kaushansky
K
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.
Blood
85
1995
1719
3
Kuter
DJ
Beeler
DL
Rosenberg
RD
The purification of megapoietin: A physiological regulator of megakaryocyte growth and platelet production.
Proc Natl Acad Sci USA
91
1994
11104
4
Nichol
JL
Hokom
MM
Hornkohl
A
Sheridan
WP
Ohashi
H
Kato
T
Li
YS
Bartley
TD
Choi
E
Bogenberger
J
Skrine
JD
Knudten
A
Chen
J
Trail
G
Sleeman
L
Cole
S
Grampp
Hunt P
Megakaryocyte growth and development factor. Analysis of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.
J Clin Invest
95
1995
2973
5
Debili
N
Wendling
F
Katz
A
Guichard
J
Breton-Gorius
J
Hunt
P
Vainchenker
W
The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86
1995
2516
6
Banu
N
Wang
JF
Deng
B
Groopman
JE
Avraham
H
Modulation of megakaryocytopoiesis by thrombopoietin: The c-Mpl ligand.
Blood
86
1995
1331
7
Chen
J
Herceg-Harjacek
L
Groopman
JE
Grabarek
J
Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin.
Blood
86
1995
4054
8
Harker
LA
Hunt
P
Marzek
UM
Kelly
AB
Tomer
A
Hanson
SR
Stead
RB
Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates.
Blood
87
1996
1833
9
Ku
H
Kaushansky
K
Ogawa
A
Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early-acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.
Blood
87
1996
4544
10
Sitnicka
E
Lin
N
Priestley
GV
Fox
N
Broudy
VC
Wolf
NS
Kaushansky
K
The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells.
Blood
87
1996
4998
11
Gurney
AL
Carver-Moore
de Sauvage
FJ
Moore
MW
Thrombocytopenia in c-mpl-deficient mice.
Science
265
1994
1445
12
Alexander
WS
Roberts
AW
Nicola
NA
Li
R
Metcalf
D
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietin receptor c-Mpl.
Blood
87
1996
2162
13
de Sauvage
EJ
Carver-Moore
K
Luoh
S-M
Ryan
A
Dowd
M
Eaton
DL
Moore
MW
Physiologic regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.
J Exp Med
183
1996
651
14
Lok
S
Kaushansky
K
Holly
RD
Kuijper
JL
Lofton-Day
CE
Oort
PJ
Grant
FJ
Helpel
MD
Burkhead
SK
Kramer
JM
Bell
LA
Sprecher
A
Blumberg
H
Johnson
R
Prunkard
D
Ching
AFT
Mathewes
SL
Bailey
MC
Forstrom
JW
Buddle
MM
Osborn
SG
Evans
SJ
Sheppard
PO
Presnell
SR
O'Hara
PJ
Hagen
FS
Roth
GJ
Foster
DC
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369
1994
565
15
Farese
AM
Hunt
P
Boone
T
MacVittie
TJ
Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates.
Blood
86
1995
54
16
Ulich
TR
del Castillo
J
Yin
S
Swift
S
Padilla
D
Senaldi
G
Bennett
L
Shutter
J
Bogenberger
J
Sun
D
Samal
B
Shimamoto
G
Lee
R
Steinbrink
R
Boone
T
Sheridan
WT
Hunt
P
Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice.
Blood
86
1995
971
17
Hokom
MM
Lacey
D
Kinstler
OB
Choi
E
Kaufman
S
Faust
J
Rowan
C
Dwyer
E
Nichol
JL
Grasel
T
Wilson
J
Steinbrink
R
Hecht
R
Winters
D
Boone
T
Hunt
P
Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.
Blood
86
1995
4486
18
de Sauvage
FJ
Hass
PE
Spencer
SD
Malloy
BE
Gurney
AL
Spencer
SA
Darbonne
WC
Henzel
WJ
Wong
SC
Kuang
W-J
Oles
KJ
Hultgren
B
Solberg
LA Jr
Goeddel
DV
Eaton
DL
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369
1994
533
19
Vigon
I
Mornon
JP
Cocault
L
Mitjavila
MT
Tambourin
P
Gisselbrecht
S
Souyri
M
Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: Identification of a member of the hematopoietic growth factor receptor superfamily.
Proc Natl Acad Sci USA
89
1992
5640
20
Kaushansky
K
Broudy
VC
Grossmann
A
Humes
J
Lin
N
Ren
HP
Bailey
MC
Papayannopoulou
Forstrom JW
Sprugel
KH
Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.
J Clin Invest
96
1995
1683
21
Kobayashi
M
Laver
JH
Kato
T
Miyazaki
H
Ogawa
M
Recombinant human thrombopoietin (mpl ligand) enhances proliferation of erythroid progenitors.
Blood
86
1995
2494
22
Papayannopoulou
T
Brice
M
Farrer
D
Kaushansky
K
Insights into the cellular mechanisms of erythropoietin-thrombopoietin synergy.
Exp Hematol
24
1996
660
23
Petzer
AL
Zandstra
PW
Piret
JM
Eaves
CJ
Differential cytokine effects on primitive (CD34+CD38−) human hematopoietic cells: Novel responses to Flt3-ligand and thrombopoietin.
J Exp Med
183
1996
2551
24
Kieran
MW
Perkins
AC
Orkin
SH
Zon
LI
Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.
Proc Natl Acad Sci USA
93
1996
9126
25
Drachman
JG
Griffin
JD
Kaushansky
K
The c-mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, shc, and c-Mpl.
J Biol Chem
270
1995
4979
26
Tortolani
PJ
Johnston
JA
Bacon
CM
McVicar
DW
Shimosaka
A
Linnekin
D
Longo
DL
O'Shea
JJ
Thrombopoietin induces tyrosine phosphorylation and activation of the Janus kinase, JAK2.
Blood
85
1995
3444
27
Gurney
AL
Wong
SC
Henzel
WJ
de Sauvage
FJ
Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.
Proc Natl Acad Sci USA
92
1995
5292
28
Miyakawa
Y
Oda
A
Druker
BJ
Kato
T
Miyazaki
H
Handa
M
Ikeda
Y
Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets.
Blood
86
1995
23
29
Sattler
M
Durstin
MA
Frank
DA
Okua
K
Kaushansky
K
Salgia
R
Griffin
JD
The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases.
Exp Hematol
23
1995
1040
30
Morella
KK
Bruno
E
Kumaki
S
Lai
C-F
Fu
J
Wang
H-M
Murray
L
Hoffman
R
Timour
M
Bénit
L
Gisselbrecht
S
Zhuang
H
Wojchowski
DM
Baumann
H
Gearing
DP
Signal transduction by the receptors for thrombopoietin (c-mpl) and interleukin-3 in hematopoietic and nonhematopoietic cells.
Blood
86
1995
557
31
Miyakawa
Y
Oda
A
Druker
BJ
Miyazaki
H
Handa
M
Ohashi
H
Ikeda
Y
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets.
Blood
87
1996
439
32
Methia
N
Louache
F
Vainchenker
W
Wendling
F
Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis.
Blood
82
1993
1395
33
Vigon
I
Dreyfus
F
Melle
J
Viguié
F
Ribrag
V
Cocault
L
Souyri
M
Gisselbrecht
S
Expression of the c-mpl proto-oncogene in human hematologic malignancies.
Blood
82
1993
877
34
Matsumura
I
Kanakura
Y
Kato
T
Ikeda
H
Ishikawa
J
Horikawa
Y
Hashimoto
K
Moriyama
Y
Tsujimura
T
Nishiura
T
Miyazaki
H
Matsuzawa
Y
Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.
Blood
86
1995
703
35
Debili
N
Wendling
F
Cosman
D
Titeux
M
Florindo
C
Dusanter-Fourt
I
Schooley
K
Methia
N
Charon
M
Nador
R
Bettaieb
A
Vainchenker
W
The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.
Blood
85
1995
391
36
Kuter
DJ
Rosenberg
RD
The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit.
Blood
85
1995
2720
37
Fielder
PJ
Gurney
AL
Stefanich
E
Marian
M
Moore
MW
Carver-Moore
K
de Sauvage
FJ
Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets.
Blood
87
1996
2154
38
Broudy
VC
Tait
JF
Powell
JS
Recombinant human erythropoietin: Purification and analysis of carbohydrate linkage.
Arch Biochem Biophys
265
1988
329
39
Broudy
VC
Lin
N
Brice
M
Nakamoto
B
Papayannopoulou
T
Erythropoietin receptor characteristics on primary human erythroid cells.
Blood
77
1991
2583
40
Michelson
AD
Flow cytometry: A clinical test of platelet function.
Blood
87
1996
4925
41
Wun
T
Paglieroni
TG
Lachant
NA
Desmopressin stimulates the expression of P-selectin on human platelets in vitro.
J Lab Clin Med
125
1995
401
42
Calvo
JC
Radicella
JP
Charreau
EH
Measurement of specific radioactivities in labeled hormones by self-displacement analysis.
Biochem J
212
1983
259
43
Broudy
VC
Lin
N
Zsebo
KM
Birkett
NC
Smith
KA
Bernstein
ID
Papayannopoulou
T
Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor.
Blood
79
1992
338
44
Broudy
VC
Morgan
DA
Lin
N
Zsebo
K
Jacobsen
FW
Papayannopoulou
T
Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor.
Blood
82
1993
436
45
Munson
PJ
Rodbard
D
LIGAND: A versatile computerized approach for characterization of ligand-binding systems.
Anal Biochem
107
1980
220
46
Foster
DC
Sprecher
CA
Grant
FJ
Kramer
JM
Kuijper
JL
Holly
RD
Whitmore
TE
Heipel
MD
Bell
LA
Ching
AFT
McGrane
V
Hart
C
O'Hara
PJ
Lok
S
Human thrombopoietin: Gene structure, cDNA sequence, expression, and chromosomal localization.
Proc Natl Acad Sci USA
91
1994
13023
47
McEver
RP
Baenziger
NL
Majerus
PW
Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody.
J Clin Invest
66
1980
1311
48
Fraser
JK
Lin
F-K
Berridge
MV
Expression of high affinity receptors for erythropoietin on human bone marrow cells and on the human erythroleukemic cell line, HEL.
Exp Hematol
16
1988
836
49
Broudy
VC
Smith
FO
Lin
N
Zsebo
KM
Egrie
J
Bernstein
ID
Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor.
Blood
80
1992
60
50
Nichol J, Hornkohl A, Selesi D, Wyres M, Hunt P: TPO levels in plasma of patients with thrombocytopenia or thrombocytosis. Blood 86:371a, 1995 (abstr, suppl 1)
51
Shivdasani
RA
Rosenblatt
MF
Zucker-Franklin
D
Jackson
CW
Hunt
P
Saris
CJM
Orkin
SH
Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.
Cell
81
1995
695
52
Emmons
RVB
Reid
DM
Cohen
RL
Meng
G
Young
NS
Dunbar
CE
Shulman
NR
Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction.
Blood
87
1996
4068
53
Kaushansky
K
Lin
N
Grossmann
A
Humes
J
Sprugel
KH
Broudy
VC
Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice.
Exp Hematol
24
1996
265
54
Kobayashi
M
Laver
JH
Kato
T
Miyazaki
H
Ogawa
M
Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3.
Blood
88
1996
429
55
Carver-Moore
K
Broxmeyer
HE
Luoh
S-M
Cooper
S
Peng
J
Burstein
SA
Moore
MW
de Sauvage
FJ
Low levels of erythroid and myeloid progenitors in thrombopoietin- and c-mpl-deficient mice.
Blood
88
1996
803
56
Vigon
I
Florindo
C
Fichelson
S
Guenet
J-L
Mattei
M-G
Souyri
M
Cosman
D
Gisselbrecht
S
Characterization of the murine Mpl proto-oncogene, a member of the hematopoietic cytokine receptor family: Molecular cloning, chromosomal location and evidence for a function in cell growth.
Oncogene
8
1993
2607
57
Skoda
RC
Seldin
DC
Chiang
M-K
Peichel
CL
Vogt
TF
Leder
P
Murine c-mpl: A member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.
EMBO J
12
1993
2645
58
Lofton-Day C, Buddle M, Berry J, Lok S: Differential binding of murine thrombopoietin to two isoforms of murine C-MPL. Blood 86:594a, 1995 (abstr, suppl 1)
59
Miyajima
A
Mui
AL-F
Ogorochi
T
Sakamaki
K
Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5.
Blood
82
1993
1960
60
Wu
H
Klingmüller
Besmer P
Lodish
HF
Interaction of the erythropoietin and stem-cell-factor receptors.
Nature
377
1995
242
Sign in via your Institution