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Thrombopoiesis: Capturing the unicorn
THROMBOPOIESIS
JEROME E. GROOPMAN
Capturing the unicorn The elusive factor that stimulates megakaryocytes to produce platelets has at last been found; as well as its physiological interest, this factor thrombopoietin - may be of considerable therapeutic importance. Platelets, the anucleate cell fragments that are crucial to clotting reactions, are spawned into the circulation by megakaryocytes, which in term are generated from pluripotent hematopoietic stem cells in a multi-step process known as thrombopoiesis (Fig. 1). This particular pathway of cell maturation has been a difficult one to characterize, partly because of the technical difficulties of isolating bone marrow megakaryocytes and their progenitors in sufficient numbers, and also because of the lack of good in vitro model systems for thrombopoiesis. By analogy with erythropoiesis, it was expected that there would be a specific hematopoietic growth factor that stimulates thrombopoiesis, but the search for such a factor seemed to many researchers in the field to be as productive as hunting for a unicorn. Indeed, serious doubts were raised as to the existence of a distinct thrombopoietin, in part because several pleiotropic cytokines - interleukin-6, interleukin-ll1, interleukin-13, granulocyte-macrophage colony stimulating factor and interleukin-3 - were found to potentiate megakaryocytopoiesis and thrombopoiesis both in vitro and in vivo [1]. What was required to capture this unicorn turned out to be the right trap, molded specifically to fit the hoof of the beast - namely, the thrombopoietin receptor. The story began four years ago, when Souyri and coworkers [2] reported that a murine retrovirus that induces myeloproliferative leukemia (known thus as MPL virus) encodes a cell-surface protein that can immortalize hematopoietic progenitor cells. This protein turned out to be similar in sequence to members of the cytokine
receptor family, including the granulocyte colony stimulating factor and erythropoietin receptors. A cellular homolog of this viral protein, c-Mpl, was identified and found to have an expression pattern notable for its high degree of restriction - to megakaryocytes, platelets and CD34 + bone marrow cells [3]. This discovery launched a series of intense and concerted efforts by a number of research teams in industry and academia to identify the ligand for c-Mpl, hoping that it would prove to be the elusive thrombopoietin. The molecule has recently been snared, and as it passes from the realm of mythology to reality, there is almost breathless expectation in the hematology community as its traits are characterized. Two different strategies were taken to identify thrombopoietin. de Sauvage et al. [4] and Bartley et al. [5] inde.pendently used c-Mpl to purify its ligand from the plasma of irradiated pigs and irradiated dogs, respectively. They obtained partial amino-acid sequence data for the purified ligand - now known to be thrombopoietin and this allowed them to identify clones of the corresponding genomic DNA; using the pig and dog oligonucleotides as probes, both groups were able to identify homologous human genomic and cDNA clones. Lok et al. [6] and Kaushansky et al. [7] took a different approach - they mutagenized growth-dependent murine hematopoietic cell lines expressing the murine c-Mpl gene, and then selected for autonomously growing clones. One of the clones selected in this way turned out to produce thrombopoietin as an autocrine factor that sustained their growth in vitro. It was then a relatively straightforward matter to isolate the cDNA for murine
Fig. 1. Platelets and erythrocytes are both generated by multi-step processes - thrombopoiesis and erythropoiesis, respectively - from a common pluripotent progenitor cell, in turn derived from a bone-marrow stem cell. Although a growth factor for erythropoiesis has been known for some time, a similar factor for thrombopoiesis has only recently been identified (see text). 1016
© Current Biology 1994, Vol 4 No 11
DISPATCH thrombopoietin, by expression cloning using cDNA libraries prepared from the selected cell line. In vitro studies had previously suggested that c-Mpl is important in megakaryocytopoiesis (the production of new megakaryocytes). The inhibition of c-Mpl mRNA expression using antisense oligodeoxynucleotides was found to cause a significant decrease in the in vitro generation of human megakaryocytic colonies (CFU-MK) from CD34+ marrow progenitors [8]. No reduction in in vitro erythropoiesis or myelopoiesis was seen in these antisense-treated bone marrow progenitor cultures. Wendling et al. [9] recently extended these initial studies, and demonstrated that soluble, recombinant c-Mpl can remove all the megakaryocyte-colony-stimulating and thrombopoietic activities in the plasma of irradiated mice, rats, dogs and pigs. The involvement of c-Mpl in thrombopoiesis was recently confirmed when 'knock-out' mice lacking a functional c-Mpl gene were generated. These mice were found to have dramatically reduced numbers of circulating platelets and bone marrow megakaryocytes [10]. A compensatory increase in thrombopoietin activity was observed in the plasma of these knock-out mice. It is of note that the knock-out mice still generate megakaryocytes and platelets at levels sufficient to prevent spontaneous hemorrhage, suggesting that there are alternative growth factors that are capable of supporting some degree of thrombopoiesis. No data are yet available on the response of these knock-out mice to inflammatory cytokines such as interleukin-6 or interleukin-11, which have been reported to augment platelet production when administered exogenously. The molecular structure of thrombopoietin The human thrombopoietin cDNA cloned by de Sauvage et al. [4] includes an open reading frame encoding a protein of 353 amino acids. Processing at a putative cleavage site between residues 21 and 22 of the protein product would yield a mature polypeptide of 332 amino acids (38 kD), with an amino-terminal sequence identical to that of the Mpl ligand purified by this group from aplastic pig serum. This amino-terminal sequence is also strikingly similar to the equivalent region of erythropoietin, including the presence of three conserved cysteines. The carboxy-terminal half of the thrombopoietin sequence shows no similarity to any known protein, but it includes a number of potential N-glycosylation sites, and in view of this, the ability of the factor to bind to lectin-affinity columns and the presence of high-molecular-weight species of approximately 60 kD with thrombopoietin activity, de Sauvage et al. suggested that mature thrombopoietin is likely to be a glycosylated protein. The sites of synthesis of human thrombopoietin have not been fully defined, but low levels of a 1.8 kilobase (kb) transcript were detected in both fetal and adult liver [4]. The cloning of the cDNA for mouse thrombopoietin showed that the predicted mature protein has a similar
two domain structure to its human homologue, with a predicted proteolytic cleavage site at a dibasic junction between the amino- and carboxy-terminal domains. The expression pattern in mouse tissues reported by Lok et al. [6] differed from that seen by de Sauvage et al. [4] with the human probe on Northern blots: hybridization of a murine thrombopoietin cDNA probe to low-abundance 1.8 and 5 kb mRNAs was detected in a variety of mouse tissues and organs. In vitro activities of thrombopoietin Taken together, the data on human and mouse recombinant thrombopoietin indicate that it functions primarily as a differentiation factor, with limited ability alone to promote megakaryocyte progenitor growth [4-7]. The generation of megakaryocyte colonies (CFU-MK) in semi-solid matrices, reflecting megakaryocyte progenitor proliferation and maturation, was most potently stimulated when thrombopoietin was used in combination with other, early-acting growth factors such as interleukin-3 or the c-kit ligand. In liquid culture, thrombopoietin augments terminal differentiation of megakaryocytes, as demonstrated by a significant increase in their DNA content (polyploidy) as well as expression of the platelet-specific differentiation antigens gplb and gpIIb/IIIa. Starting with human CD34+ marrow cells, Bartley and coworkers [5] noted that, after eight days of thrombopoietin treatment in liquid culture, -40% of the cells were morphologically mature megakaryocytes expressing platelet-specific glycoproteins. No significant effects on erythropoiesis or myelopoiesis in vitro were noted in these studies. These in vitro data were extended to in vivo studies of rodents treated with recombinant thrombopoietin: striking increases in circulating platelet numbers were noted in the treated animals, as well as massive generation of mature megakaryocytes in the bone marrow [4,6,7]. Thus, thrombopoietin stimulated megakaryocyte progenitor growth and differentiation in both in vitro and in vivo assays. The identification of a distinct thrombopoietin provides some symmetry in the pattern of lineage-specific hematopoietic growth factors (Fig. 1). As noted above, the sequence of thrombopoietin is similar to that of the erythrocyte-differentiation factor erythropoietin, and the in vitro and in vivo effects of the two factors are similar. Like the unicorn and the horse, the observer most readily identifies the features that are shared by the two factors, but the exciting traits are ultimately those that are different. Many important unknown issues concerning thrombopoietin should be resolved in the upcoming months, including its site of synthesis, the physiological regulation of its production, the modulation of expression of its receptor, the nature of its signal transduction pathway, and the structure-function relationships of the native protein. Beyond these physiological issues, there is intense interest in identifying mutations in thrombopoietin or c-Mpl genes associated with inherited or acquired - such as neoplasias hematological diseases.
1017
1018
Current Biology 1994, Vol 4 No 11 The roles of previously identified pleiotropic cytokines, such as interleukin-6 and interleukin- 1, in the regulation of megakaryocytopoiesis are unlikely to be physiological. Platelet counts often rise during inflammatory conditions, as a non-specific response, and it is likely that inflammatory cytokines mediate this phenomenon as part of the host response. One may conceive of two basic pathways of thrombopoiesis: a physiological one mediated by thrombopoietin, and an alternative one mediated by non-specific inflammatory cytokines. How these two pathways may be exploited therapeutically in the context of thrombopoiesis suppressed by radiation or chemotherapy needs to be addressed in animal models, in which these growth factors are administered alone and in combination. Although it appears that thrombopoietin is the physiological regulator of thrombopoiesis, which drives megakaryocyte differentiation and ultimately the spawning of platelets, it is still unclear whether it also functions as the primary growth factor for megakaryocyte progenitor commitment and proliferation. This biological question may be answered in the course of the clinical development of thrombopoietin. There is a great clinical need to boost platelet numbers independent of transfusion in thrombocytopenic states associated with chemotherapy, bone marrow transplantation and primary marrow failure disorders. If thrombopoietin proves to function pharmacologically like granulocyte colony stimulating factor in these conditions - boosting committed progenitor numbers and accelerating production of mature, functional cells - it will satisfy the pressing clinical need; if it resembles erythropoietin - which is already present at high concentrations in the plasma of such patients and has a limited effect when given in higher doses - then its utility for patients with these conditions may be more modest. The once elusive thrombopoietin will hopefully
fulfill its promise and emerge as an important member of our therapeutic armoury. References 1. Metcalf D: Blood: thrombopoietin - at last. Nature 1994, 369:519-520. 2. Souyri M, Vignon I, Penciolelli F, Heard M, Tambourin P, Wendling F: A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell 1990, 63:1137-1147. 3. Vignon 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 1992, 89:5640-5644. 4. de Sauvage F, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ et a.: Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994, 369:533-538. 5. Bartley TD, Bogenberger J, Hunt P, Li Y-S, Lu HS, Martin F, Chang MS, Samal B, Nichol JL, Swift S et al.: Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994, 77:1117-1124. 6. Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort P, Grant Fl, Heipel MD, Burkhead SK, Kramer JM et al.: Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994, 369:565-568. 7. Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FSet al.: Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 1994, 369:568-571. 8. Methia N, Louache F, Vainchenker W, Wendling F: Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993, 82:1395-1401. 9. Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W: c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994, 369:571-574. 10. Gurney AL, Carver-Moore K, de Sauvage F, Moore MW: Thrombocytopenia in c-mpl-deficient mice. Science 1994, 265:1445-1447.
Jerome E. Groopman, Harvard Medical School, Division of Hematology/Oncology, New England Deaconess Hospital, 110 Francis Street, Suite 4A, Boston, Massachusetts 02215, USA.
JEROME E. GROOPMAN
Capturing the unicorn The elusive factor that stimulates megakaryocytes to produce platelets has at last been found; as well as its physiological interest, this factor thrombopoietin - may be of considerable therapeutic importance. Platelets, the anucleate cell fragments that are crucial to clotting reactions, are spawned into the circulation by megakaryocytes, which in term are generated from pluripotent hematopoietic stem cells in a multi-step process known as thrombopoiesis (Fig. 1). This particular pathway of cell maturation has been a difficult one to characterize, partly because of the technical difficulties of isolating bone marrow megakaryocytes and their progenitors in sufficient numbers, and also because of the lack of good in vitro model systems for thrombopoiesis. By analogy with erythropoiesis, it was expected that there would be a specific hematopoietic growth factor that stimulates thrombopoiesis, but the search for such a factor seemed to many researchers in the field to be as productive as hunting for a unicorn. Indeed, serious doubts were raised as to the existence of a distinct thrombopoietin, in part because several pleiotropic cytokines - interleukin-6, interleukin-ll1, interleukin-13, granulocyte-macrophage colony stimulating factor and interleukin-3 - were found to potentiate megakaryocytopoiesis and thrombopoiesis both in vitro and in vivo [1]. What was required to capture this unicorn turned out to be the right trap, molded specifically to fit the hoof of the beast - namely, the thrombopoietin receptor. The story began four years ago, when Souyri and coworkers [2] reported that a murine retrovirus that induces myeloproliferative leukemia (known thus as MPL virus) encodes a cell-surface protein that can immortalize hematopoietic progenitor cells. This protein turned out to be similar in sequence to members of the cytokine
receptor family, including the granulocyte colony stimulating factor and erythropoietin receptors. A cellular homolog of this viral protein, c-Mpl, was identified and found to have an expression pattern notable for its high degree of restriction - to megakaryocytes, platelets and CD34 + bone marrow cells [3]. This discovery launched a series of intense and concerted efforts by a number of research teams in industry and academia to identify the ligand for c-Mpl, hoping that it would prove to be the elusive thrombopoietin. The molecule has recently been snared, and as it passes from the realm of mythology to reality, there is almost breathless expectation in the hematology community as its traits are characterized. Two different strategies were taken to identify thrombopoietin. de Sauvage et al. [4] and Bartley et al. [5] inde.pendently used c-Mpl to purify its ligand from the plasma of irradiated pigs and irradiated dogs, respectively. They obtained partial amino-acid sequence data for the purified ligand - now known to be thrombopoietin and this allowed them to identify clones of the corresponding genomic DNA; using the pig and dog oligonucleotides as probes, both groups were able to identify homologous human genomic and cDNA clones. Lok et al. [6] and Kaushansky et al. [7] took a different approach - they mutagenized growth-dependent murine hematopoietic cell lines expressing the murine c-Mpl gene, and then selected for autonomously growing clones. One of the clones selected in this way turned out to produce thrombopoietin as an autocrine factor that sustained their growth in vitro. It was then a relatively straightforward matter to isolate the cDNA for murine
Fig. 1. Platelets and erythrocytes are both generated by multi-step processes - thrombopoiesis and erythropoiesis, respectively - from a common pluripotent progenitor cell, in turn derived from a bone-marrow stem cell. Although a growth factor for erythropoiesis has been known for some time, a similar factor for thrombopoiesis has only recently been identified (see text). 1016
© Current Biology 1994, Vol 4 No 11
DISPATCH thrombopoietin, by expression cloning using cDNA libraries prepared from the selected cell line. In vitro studies had previously suggested that c-Mpl is important in megakaryocytopoiesis (the production of new megakaryocytes). The inhibition of c-Mpl mRNA expression using antisense oligodeoxynucleotides was found to cause a significant decrease in the in vitro generation of human megakaryocytic colonies (CFU-MK) from CD34+ marrow progenitors [8]. No reduction in in vitro erythropoiesis or myelopoiesis was seen in these antisense-treated bone marrow progenitor cultures. Wendling et al. [9] recently extended these initial studies, and demonstrated that soluble, recombinant c-Mpl can remove all the megakaryocyte-colony-stimulating and thrombopoietic activities in the plasma of irradiated mice, rats, dogs and pigs. The involvement of c-Mpl in thrombopoiesis was recently confirmed when 'knock-out' mice lacking a functional c-Mpl gene were generated. These mice were found to have dramatically reduced numbers of circulating platelets and bone marrow megakaryocytes [10]. A compensatory increase in thrombopoietin activity was observed in the plasma of these knock-out mice. It is of note that the knock-out mice still generate megakaryocytes and platelets at levels sufficient to prevent spontaneous hemorrhage, suggesting that there are alternative growth factors that are capable of supporting some degree of thrombopoiesis. No data are yet available on the response of these knock-out mice to inflammatory cytokines such as interleukin-6 or interleukin-11, which have been reported to augment platelet production when administered exogenously. The molecular structure of thrombopoietin The human thrombopoietin cDNA cloned by de Sauvage et al. [4] includes an open reading frame encoding a protein of 353 amino acids. Processing at a putative cleavage site between residues 21 and 22 of the protein product would yield a mature polypeptide of 332 amino acids (38 kD), with an amino-terminal sequence identical to that of the Mpl ligand purified by this group from aplastic pig serum. This amino-terminal sequence is also strikingly similar to the equivalent region of erythropoietin, including the presence of three conserved cysteines. The carboxy-terminal half of the thrombopoietin sequence shows no similarity to any known protein, but it includes a number of potential N-glycosylation sites, and in view of this, the ability of the factor to bind to lectin-affinity columns and the presence of high-molecular-weight species of approximately 60 kD with thrombopoietin activity, de Sauvage et al. suggested that mature thrombopoietin is likely to be a glycosylated protein. The sites of synthesis of human thrombopoietin have not been fully defined, but low levels of a 1.8 kilobase (kb) transcript were detected in both fetal and adult liver [4]. The cloning of the cDNA for mouse thrombopoietin showed that the predicted mature protein has a similar
two domain structure to its human homologue, with a predicted proteolytic cleavage site at a dibasic junction between the amino- and carboxy-terminal domains. The expression pattern in mouse tissues reported by Lok et al. [6] differed from that seen by de Sauvage et al. [4] with the human probe on Northern blots: hybridization of a murine thrombopoietin cDNA probe to low-abundance 1.8 and 5 kb mRNAs was detected in a variety of mouse tissues and organs. In vitro activities of thrombopoietin Taken together, the data on human and mouse recombinant thrombopoietin indicate that it functions primarily as a differentiation factor, with limited ability alone to promote megakaryocyte progenitor growth [4-7]. The generation of megakaryocyte colonies (CFU-MK) in semi-solid matrices, reflecting megakaryocyte progenitor proliferation and maturation, was most potently stimulated when thrombopoietin was used in combination with other, early-acting growth factors such as interleukin-3 or the c-kit ligand. In liquid culture, thrombopoietin augments terminal differentiation of megakaryocytes, as demonstrated by a significant increase in their DNA content (polyploidy) as well as expression of the platelet-specific differentiation antigens gplb and gpIIb/IIIa. Starting with human CD34+ marrow cells, Bartley and coworkers [5] noted that, after eight days of thrombopoietin treatment in liquid culture, -40% of the cells were morphologically mature megakaryocytes expressing platelet-specific glycoproteins. No significant effects on erythropoiesis or myelopoiesis in vitro were noted in these studies. These in vitro data were extended to in vivo studies of rodents treated with recombinant thrombopoietin: striking increases in circulating platelet numbers were noted in the treated animals, as well as massive generation of mature megakaryocytes in the bone marrow [4,6,7]. Thus, thrombopoietin stimulated megakaryocyte progenitor growth and differentiation in both in vitro and in vivo assays. The identification of a distinct thrombopoietin provides some symmetry in the pattern of lineage-specific hematopoietic growth factors (Fig. 1). As noted above, the sequence of thrombopoietin is similar to that of the erythrocyte-differentiation factor erythropoietin, and the in vitro and in vivo effects of the two factors are similar. Like the unicorn and the horse, the observer most readily identifies the features that are shared by the two factors, but the exciting traits are ultimately those that are different. Many important unknown issues concerning thrombopoietin should be resolved in the upcoming months, including its site of synthesis, the physiological regulation of its production, the modulation of expression of its receptor, the nature of its signal transduction pathway, and the structure-function relationships of the native protein. Beyond these physiological issues, there is intense interest in identifying mutations in thrombopoietin or c-Mpl genes associated with inherited or acquired - such as neoplasias hematological diseases.
1017
1018
Current Biology 1994, Vol 4 No 11 The roles of previously identified pleiotropic cytokines, such as interleukin-6 and interleukin- 1, in the regulation of megakaryocytopoiesis are unlikely to be physiological. Platelet counts often rise during inflammatory conditions, as a non-specific response, and it is likely that inflammatory cytokines mediate this phenomenon as part of the host response. One may conceive of two basic pathways of thrombopoiesis: a physiological one mediated by thrombopoietin, and an alternative one mediated by non-specific inflammatory cytokines. How these two pathways may be exploited therapeutically in the context of thrombopoiesis suppressed by radiation or chemotherapy needs to be addressed in animal models, in which these growth factors are administered alone and in combination. Although it appears that thrombopoietin is the physiological regulator of thrombopoiesis, which drives megakaryocyte differentiation and ultimately the spawning of platelets, it is still unclear whether it also functions as the primary growth factor for megakaryocyte progenitor commitment and proliferation. This biological question may be answered in the course of the clinical development of thrombopoietin. There is a great clinical need to boost platelet numbers independent of transfusion in thrombocytopenic states associated with chemotherapy, bone marrow transplantation and primary marrow failure disorders. If thrombopoietin proves to function pharmacologically like granulocyte colony stimulating factor in these conditions - boosting committed progenitor numbers and accelerating production of mature, functional cells - it will satisfy the pressing clinical need; if it resembles erythropoietin - which is already present at high concentrations in the plasma of such patients and has a limited effect when given in higher doses - then its utility for patients with these conditions may be more modest. The once elusive thrombopoietin will hopefully
fulfill its promise and emerge as an important member of our therapeutic armoury. References 1. Metcalf D: Blood: thrombopoietin - at last. Nature 1994, 369:519-520. 2. Souyri M, Vignon I, Penciolelli F, Heard M, Tambourin P, Wendling F: A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell 1990, 63:1137-1147. 3. Vignon 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 1992, 89:5640-5644. 4. de Sauvage F, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ et a.: Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994, 369:533-538. 5. Bartley TD, Bogenberger J, Hunt P, Li Y-S, Lu HS, Martin F, Chang MS, Samal B, Nichol JL, Swift S et al.: Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994, 77:1117-1124. 6. Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort P, Grant Fl, Heipel MD, Burkhead SK, Kramer JM et al.: Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994, 369:565-568. 7. Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FSet al.: Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 1994, 369:568-571. 8. Methia N, Louache F, Vainchenker W, Wendling F: Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993, 82:1395-1401. 9. Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W: c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994, 369:571-574. 10. Gurney AL, Carver-Moore K, de Sauvage F, Moore MW: Thrombocytopenia in c-mpl-deficient mice. Science 1994, 265:1445-1447.
Jerome E. Groopman, Harvard Medical School, Division of Hematology/Oncology, New England Deaconess Hospital, 110 Francis Street, Suite 4A, Boston, Massachusetts 02215, USA.