C-mpl Ligand Research Articles

A possible role for thrombopoietin and its receptor c-mpl in the pathobiology of essential thrombocythemia.

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder of unknown origin primarily involving the megakaryocyte/platelet lineage. In an attempt to evaluate the role of the receptor c-mpl and its ligand thrombopoietin (TPO) in ET, various studies have been undertaken, the results of which are discussed herein. Normal or even elevated TPO levels in ET despite an increased megakaryocyte/platelet mass suggest abnormal feedback mechanisms in the regulation of TPO production and consumption. Deregulated levels of TPO in the circulation of patients with ET may be the result of an abnormal TPO consumption by platelets and megakaryocytes. It has been shown that proto-oncogene c-mpl is involved in spontaneous megakaryopoiesis in myeloproliferative disorders. Evidence for a decisive role of deregulated TPO in ET also comes from observations in mice overexpressing a TPO transgene where increased TPO production has resulted in a fatal myeloproliferative syndrome. Future research will study quantitative and qualitative deficiencies of c-mpl, the kinetics of the interaction of c-mpl with its ligand (TPO), and the role of autocrine or paracrine production of c-mpl ligand in increasing megakaryopoiesis so as to further elucidate its pathobiologic significance in ET.

Elemental study of gene therapy with thrombopoietin

Thrombopoietin (TPO) is also referred to as the c-mpl ligand or megakaryocyte growth and development factor, inducing thrombopoiesis by proliferation of megakaryocyte progenitors and promoting endoreplicatton of mature megakaryocytes, t The identification of TPO, and the demonstration that administration to normal experimental animals with recombinant TPO results in a marked increase in platelets count, 2 has opened the possibility of using TPO to rescue patients from life-threatening thrombocytopenia with high dose chemotherapy and radiotherapy. But recombinant TPO is a foreign protein and has adverse effects on patients to different extent. This study is to establish a new TPO administration by gene therapeutic methods, i.e. that after transferred into animals, the TPO gene expresses functional TPO in vivo and maintains high platelet level.

An erythroid and megakaryocytic common precursor cell line (B1647) expressing both c-mpl and erythropoietin receptor (Epo-R) proliferates and modifies globin chain synthesis in response to megakaryocyte growth and development factor (MGDF) but not to erythropoietin (Epo).

A human megakaryocyte cell line (B1647) has been established from bone marrow cells obtained from a patient with acute myelogenous leukaemia (FAB M2). The cells were CD34-, CD33+, HLA-DR+, CD38+, and expressed the immunophenotypic markers of the megakaryocyte lineage (CD41 and von Willebrand factor). Moreover the cells expressed the c-mpl (thrombopoietin receptor) mRNA and protein. On the other hand, the B1647 cells also possessed erythroid lineage characteristics: the vast majority of cells were glycophorin positive, and about 10% of unstimulated cells stained with an anti-globin gamma chain MoAb. In addition, S1 protection analysis demonstrated expression of beta-globin mRNA, and Epo receptor (Epo-R) protein was detected by cytofluorimetric assay. Several growth factors, when tested alone or in combination, failed to influence the B1647 cell growth. A significant increase of cell proliferation was observed only after the addition, in serum-free culture, of recombinant human megakaryocyte growth development factor (MGDF), a recombinant c-mpl ligand encompassing the receptor-binding domain and identical to thrombopoietin (TPO), at concentrations ranging from 0.01 to 1 ng/ml. Interestingly, MGDF failed to induce megakaryocytic differentiation of the B1647 cells, but significantly increased the synthesis of the globin gamma-chain. B1647 cells could be a useful model for studying the biological effect of TPO on common megakaryocyte and erythroid progenitors.

Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: role of activation of very late antigen (VLA)-4 and VLA-5.

Thrombopoietin (Tpo), the ligand for c-mpl and a principal regulator of megakaryocytopoiesis and platelet production, has been demonstrated to stimulate the growth and differentiation of megakaryocyte as well as multipotent hemopoietic progenitor cells. In the present study we demonstrate that Tpo can stimulate the adhesion of the Mo7e progenitor cell line to fibronectin (Fn) as well as vascular cell adhesion molecule-1 through activation of very late antigen (VLA)-4 and VLA-5, adhesion molecules previously demonstrated to be involved in regulation of steady state hemopoiesis. Tpo-induced adhesion was concentration dependent, reached a maximum following 30 min, and appeared to be dependent on adenylate cyclase, and tyrosine kinase activity. Furthermore, second messenger inhibitors implicated essential and complimentary roles of phosphatidylinositol-3-kinase and protein kinase C in mediating Tpo-induced adhesion. The ability of Tpo to promote adhesion to fibronectin was comparable to that of IL-3, but less than that of stem cell factor. Unlike the ability of these cytokines to synergistically enhance growth of Mo7e as well as normal progenitor cells, no synergy was observed with regard to their ability to enhance adhesion. Finally, Tpo stimulated adhesion of primitive (CD34+ CD38-) human bone marrow cells to fibronectin, predominantly through activation of VLA-5, whereas no such effect could be observed on CD34+ CD38+ bone marrow cells. Thus, Tpo might play an important role in early hemopoiesis, at least in part through its ability to promote adhesion through activation of adhesion molecules on hemopoietic progenitor cells.

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture.

We examined the effects of recombinant human thrombopoietin (TPO, c-Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum-free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34+ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)-3, IL-6, stem cell factor, erythropoietin, granulocyte-macrophage colony-stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte-macrophage (GM) colonies, erythroid bursts (E), granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone-sorted CD34+ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum-free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long-term culture-initiating cells. These effects were completely abrogated by anti-TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.

Effects of thrombopoietin (c-mpl ligand) on growth of blast cells from patients with transient abnormal myelopoiesis and acute myeloblastic leukemia.

Thrombopoietin (TPO) is a ligand for c-mpl that promotes both proliferation and differentiation of megakaryocytes in vivo and in vitro. We investigated the expression of c-mpl transcripts and the effects of recombinant human TPO (rhTPO) on the proliferation and differentiation of human leukemic cell lines or fresh samples obtained from 32 patients with transient abnormal myelopoiesis (TAM) or acute myeloblastic leukemia (AML). Cells were cultured with TPO alone or combined with rh interleukin-3 (IL-3) or stem cell factor (SCF). Expression of c-mpl was verified in 6 of 13 cases tested. All but one of the cases that showed c-mpl expression responded to TPO. Blasts from all cases of TAM or French-American-British (FAB) subtype M7 showed growth responses to TPO with higher sensitivity than cells of other FAB subtypes and these responses were increased by addition of rhIL-3 or rhSCF in some cases. Responses of cells of other FAB subtypes varied. In addition, increased expression of platelet-specific surface antigens on MO7E cells after incubation with rhTPO was observed. These data suggest that TPO may be involved in the abnormal proliferation and differentiation of human leukemic cells, especially of M7 and TAM cells, considered to be of megakaryocytic lineage.

Serum thrombopoietin levels in patients undergoing autologous peripheral blood stem cell transplantation.

Recently, the ligand for c-mpl has been cloned and initial studies have shown it to be the platelet regulatory factor, thrombopoietin (TPO). To elucidate the role of TPO in the reconstitution of megakaryopoiesis and platelet production after stem cell transplantation, we measured serum TPO levels in nine patients undergoing autologous peripheral blood stem cell transplantation (PBSCT) and in healthy volunteers. Serum TPO levels significantly correlated with the degree of peripheral thrombocytopenia and a strong inverse correlation between serum TPO level and platelet count was observed (r = -0.700, P < 0.001). Serum TPO levels began to rise as the platelet count decreased after chemotherapy, TPO levels peaked at over 25.00 fmoles/ml between days 0 and 10; TPO levels then decreased gradually as the platelet count began to rise. One patient with multiple myeloma received purified CD34+ peripheral blood stem cells. No difference was observed in the kinetics of serum TPO levels between unfractionated and purified PBSCT. These observations suggest that TPO plays a critical role in the reconstitution of megakaryopoiesis and platelet production after PBSCT.

Characterization of the Human Thrombopoietin Gene Promoter: A POSSIBLE ROLE OF AN Ets TRANSCRIPTION FACTOR, E4TF1

Thrombopoietin (TPO), the ligand for c-Mpl, is a cytokine that regulates megakaryocyte growth and development. We have cloned the 5'-flanking region of the human TPO gene and analyzed its promoter activity. The human TPO gene promoter lacks a TATA box and directs transcription initiation at multiple sites over a 50-nucleotide region. Transient expression in a human liver cell line (PLC) of promoter fragment-luciferase reporter gene constructs containing a series of 5'-truncated sequences or site-directed mutations identified a sequence 5'-ACTTCCG-3' from -69 to -63 as a positive cis-acting element for high level expression of TPO gene. This sequence contains a core motif (C/A)GGA(A/T) for Ets family proteins in the noncoding strand. Gel mobility shift assays performed with nuclear protein from PLC cells identified a DNA binding protein(s) specific for the element. Anti-E4TF1-60(GABPalpha) or anti-E4TF1-53/47(GABPbeta) antibodies supershifted the complex in gel shift assay. Furthermore, co-expression of E4TF1-60 and E4TF1-53/47 squelched TPO gene expression in PLC and HepG2 cells. It is concluded that Ets family transcription factor E4TF1(GABPalpha/beta), an ubiquitously expressed protein, is required for high level expression of the TPO gene in liver.

Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand.

We studied the effects of stem cell factor (SCF) and flt3 ligand (FL) on the ex vivo expansion of human umbilical cord blood (CB)-derived CD34+ cells in combination with various cytokines, including interleukin (IL)-3, IL-6, IL-11, and c-Mpl ligand (thrombopoietin, TPO), in a short-term serum-free liquid suspension culture system. Among the two-factor combinations tested, SCF plus IL-3 most effectively expanded committed progenitor cells, including mixed colony-forming units (CFU-Mix). The expansion efficiency (EE) of FL for each progenitor was inferior to that of SCF in the presence of various cytokines, except TPO. IL-6 significantly increased the EE for granulocyte/macrophage colony-forming units (CFU-GM) obtained with SCF + IL-3 or FL + IL-3. Interestingly, TPO markedly augmented the EE for committed progenitors, including CFU-GM, erythroid burst-forming units (BFU-E), and CFU-Mix, in the presence of SCF + IL-3 or FL + IL-3. The combinations of SCF + IL-3 + TPO + IL-6 or IL-11 maximally stimulated the expansion of committed progenitors. The maximum EE for CFU-GM, BFU-E, and CFU-Mix was respectively 197-fold (day 14), 60-fold (day 7) and 51-fold (day 14). Other combinations of cytokines without IL-3 failed to expand effectively these committed progenitors. Our data demonstrate that it is possible to expand human CB-derived committed progenitors in vitro using SCF or FL with several other cytokines including TPO, and that IL-3 is the key cytokine promoting the expansion of human hematopoietic progenitors in the presence of SCF or FL.

Transgenic Mice Overexpressing Human c-mpl Ligand Exhibit Chronic Thrombocytosis and Display Enhanced Recovery From 5-Fluorouracil or Antiplatelet Serum Treatment

AbstractThe consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.

Transgenic Mice Overexpressing Human c-mpl Ligand Exhibit Chronic Thrombocytosis and Display Enhanced Recovery From 5-Fluorouracil or Antiplatelet Serum Treatment

The consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.

Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors

AbstractThrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.

Thrombospondin, a negative modulator of megakaryocytopoiesis

The effects of native thrombospondin (TSP), an 18 kd recombinant protein comprising residues 1–174 of TSP (TSP 1–174) with heparin-binding domain and a fusion protein comprising residues 559–669 of TSP (TSP 559–669) on murine hematopoiesis, were studied by using different in vitro culture systems. TSP by itself did not show an inhibitory effect on colony-forming unit-megakaryocyte (CFU-MK) growth in a serum-free agar system and on the growth of colony-forming unit-granulocyte and macrophage (CFU-GM) in a plasma clot system. It was, however, found that in the plasma clot culture system when using aplastic anemia serum as the source of thrombopoietin or C-Mpl ligand (TPO), TSP and TSP 1–174, but not TSP 559–669, were able to inhibit the growth of CFU-MK from unfractionated and lineage negative ( Lin-) bone marrow cells in a dose-dependent manner. A statistically significant suppression was seen at 1 μg/ml of TSP and 5 μg/ml of TSP 1–174. This inhibitory effect of TSP was further found in both the serum-free agar system and the plasma clot system without aplastic anemia serum, where megakaryocyte colony growth was stimulated by recombinant TPO, basic fibroblast growth factor (bFGF), or interleukin-3 (IL-3). In a methylcellulose system, where a combination of stem cell factor (SCF), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were used, TSP inhibited the growth of colony-forming unit-granulocyte-erythroblast, megakaryocyte, and macrophage (CFU-GEMM) but not CFU-GM and burst-forming unit-erythroblast (BFU-E). Interestingly, this inhibitory effect of TSP on megakaryocyte colony growth could be counteracted by Fraxiparin, a low-molecular-weight heparin. These results demonstrate that TSP is a negative modulator of megakaryocytopoiesis and suggest that its inhibitory effect is at least partially mediated by N-terminal heparin-binding domain.

A Single Injection of Pegylated Murine Megakaryocyte Growth and Development Factor (MGDF ) Into Mice Is Sufficient to Produce a Profound Stimulation of Megakaryocyte Frequency, Size, and Ploidization

AbstractDespite numerous studies investigating the action of c-mpl ligand, no reports have defined the in vivo changes in megakaryocytopoiesis in response to a single injection of this cytokine. Here we compare the kinetics of the megakaryocytopoietic response in C57Bl/6J mice administered 25 μg/kg or 250 μg/kg of pegylated (PEG) murine megakaryocyte growth and development factor (MGDF ) as a single intravenous injection. Megakaryocytes of mice treated with MGDF had normal ultrastructure, showing a typical distribution of the demarcation membrane system, α-granules, and other cytoplasmic organelles. Megakaryocyte ploidy, size, and frequency were markedly increased with both MGDF doses. Megakaryocyte ploidy was maximally increased from a modal value of 16N to 64N on day 3, with both doses of MGDF. Similarly, a comparable increase in megakaryocyte size occurred in the two MGDF groups. Increased megakaryocyte size was coupled to the increase in megakaryocyte ploidy, and no evidence for independent regulation of megakaryocyte size within individual ploidy classes was apparent. In contrast to megakaryocyte ploidy and size, the increase in megakaryocyte frequency was markedly different with the two doses of MGDF. The proportion of 2N and 4N cells was increased from a baseline of 0.035% to 0.430% by day 4 in mice treated with the higher dose of MGDF, but only to 0.175% in mice administered 25 μg/kg of MGDF. The marked increase in the pool of these immature megakaryocytes translated to a sustained elevation in the frequency of polyploid megakaryocytes (8N cells and greater). In contrast to the sustained increase in the frequency of polyploid cells, the level of polyploidization was downregulated on days 6 to 10, but normalized by day 14. We conclude that a single injection of MGDF is able to expand the megakaryocytic pool in a dose-dependent manner, which, with subsequent maturation, should lead to an increased rate of platelet production.

Role for C-MPL and its Ligand Thrombopoietin in Early Hematopoiesis

Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed.

Effect of thrombopoietin (c-Mpl ligand) alone and in combination with other hematopoietic growth factors on human megakaryocytopoiesis in serum-free cultures.

The effect of human recombinant (hr) thrombopoietin (TPO) on human megakaryocytopoiesis was studied in a serum-free system. hrTPO induced megakaryocyte colony formation by purified CD 34-positive cells and polyploidization of megakaryocytes by purified CD41a-positive cells. hrTPO gave rise to much smaller colonies which appeared at an earlier time compared to the use of human recombinant interleukin-3 (hrIL-3), suggesting that hrTPO predominantly affects the population of megakaryocyte progenitor cells in the late stage. hrIL-3 additively increased the hrTPO-induced megakaryocyte colony formation by CD34-positive cells. The hrTPO-induced megkaryocyte colony formation was also increased by the presence of hrIL-6, hrIL-11, human recombinant erythropoietin (hrEpo) or human recombinant stem cell factor (hrSCF), none of which stimulated megakaryocyte colony growth when added alone. The combined addition of hrTPO, hrIL-3 and hrSCF to CD34-positive cells markedly stimulated megakaryocyte colony formation and produced large numbers of megakaryocytes. hrTPO stimulated the polyploidization of CD34-positive cell-derived megakaryocytes in liquid culture. However, the addition of hrIL-6, hrIL-11 or hrEpo to hrTPO did not further enhance the hrTPO-induced polyploidization. These findings indicate that at the megakaryocyte progenitor cell level, the effect of hrTPO can be promoted by the presence of various hematopoietic growth factors involved in human megakaryocytopoiesis.

Thrombopoietin, But Not Erythropoietin, Directly Stimulates Multilineage Growth of Primitive Murine Bone Marrow Progenitor Cells in Synergy With Early Acting Cytokines: Distinct Interactions With the Ligands for c-kit and FLT3

Thrombopoietin (Tpo), the ligand for c-mpl, has been shown to be the principal regulator of megakaryocytopoiesis and platelet production. The ability of Tpo to potently stimulate the growth of committed megakaryocyte (Mk) progenitor cells has been studied in detail. Murine fetal liver cells, highly enriched in primitive progenitors, have been shown to express c-mpl, but little is known about the ability of Tpo to stimulate the growth and differentiation of primitive multipotent bone marrow (BM) progenitor cells. Here, we show that Tpo alone and in combination with early acting cytokines can stimulate the growth and multilineage differentiation of Lin- Sca-1+ BM progenitor cells. In particular, Tpo potently synergized with the ligands for c-kit (stem cell factor [SCF]) and flt3 (FL) to stimulate an increase in the number and size of clones formed from Lin- Sca-1+ progenitors. When cells were plated at 1 cell per well, the synergistic effect of Tpo was observed both in fetal calf serum-supplemented and serum-depleted medium and was decreased if the addition of Tpo to cultures was delayed for as little as 24 hours, suggesting that Tpo is acting directly on the primitive progenitors. Tpo added to SCF + erythropoietin (Epo)-supplemented methylcellulose cultures potently enhanced the formation of multilineage colonies containing granulocytes, macrophages, erythrocytes, and Mks. SCF potently enhanced Tpo-stimulated production of high-ploidy Mks from Lin- Sca-1+ progenitors, whereas the increased growth response obtained when combining Tpo with FL did not translate into increased Mk production. The ability of Tpo and SCF to synergistically enhance the growth of Lin- Sca-1+ progenitors was predominantly observed in the more primitive rhodamine 123(lo) fraction. Tpo also enhanced growth of Lin- Sca-1+ progenitors when combined with interleukin-3 (IL-3) and IL-11 but not with IL-12, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or Epo. Epo, which has high homology to Tpo, was unable to stimulate the growth of Lin- Sca-1+ progenitors alone or in combination with SCF or FL, suggesting that c-mpl is expressed on more primitive stages of progenitors than the Epo receptor. Thus, the present studies show the potent ability of Tpo to enhance the growth of primitive multipotent murine BM progenitors in combination with multiple early acting cytokines and documents its unique ability to synergize with SCF to enhance Mk production from such progenitors.

Recombinant human ligand for MPL, megakaryocyte growth and development factor (MGDF), stimulates thrombopoiesis in vivo in normal and myelosuppressed baboons.

Megakaryocyte growth and development factor (MGDF) is a ligand for c-mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5-fluorouracil (5-FU). rHuMGDF was administered to normal baboons as a single s.c. injection at doses of 1, 10, 25 and 50 micrograms/kg/day for 10 days and, as a control, heat-inactivated MGDF was administered at a dose of 10 micrograms/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat-inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7-, 3.4-, 5.1- and 4.0-fold above baseline in animals administered 1, 10, 25 and 50 micrograms/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 micrograms/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in the circulation. Following a single dose of 5-FU (120 mg/kg) animals were given either saline or pegylated (PEG) rHuMGDF (25 micrograms/kg/day) for 14 days. Platelet counts recovered to baseline by 13.8 +/- 1.8 days for PEG-rHuMGDF-treated baboons compared with 16.8 +/- 0.6 days for saline treated controls. Marrow biopsies revealed more rapid recovery of overall marrow cellularity and megakaryocytes in PEG-rHuMGDF-treated animals compared with controls. Thus, rHuMGDF specifically stimulates thrombopoiesis in normal and myelosuppressed baboons. rHuMGDF may be useful for stimulating thrombopoiesis in humans in clinical settings after myelosuppression.

Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice.

Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy-induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia; it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG-CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis.

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 micrograms/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 micrograms/kg/d resulted in platelet counts 5-fold above normal. However, doses > 60 micrograms/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.