ABSTRACT: After an almost 40-year search for a primary regulatory of platelet production, thrombopoietin has recently been purified and cloned. Thrombopoietin regulates all stages in the production of platelets by promoting both the proliferation of megakaryocyte progenitors and their maturation into platelet-producing megakaryocytes. In preclinical studies in normal mice and nonhuman primates, administration of thrombopoietin resulted in a rapid rise in platelet counts to levels previously unattainable with other thrombopoietic cytokines. In myelosuppressed animal models, use of thrombopoietin following chemotherapy, radiation, or stem-cell transplantation accelerated megakaryocyte and platelet recovery. Thrombopoietin has rapidly moved from the laboratory to the clinic in the last 3 years. Preliminary results of clinical trials using truncated or full-length forms of the molecule indicate that thrombopoietin is a powerful stimulus to the production of megakaryocytes and normal platelets in humans and enhances platelet recovery following chemotherapy. Although the peripheral effect is selective on platelet lineage, thrombopoietin mediates stimulatory effects on progenitors of multiple cell lineages at the bone marrow level and mobilizes progenitor cells into the peripheral blood. These biological effects suggest that thrombopoietin holds promise as a useful agent for the prevention and treatment of thrombocytopenia in cancer patients and for other disorders of thrombocytopenia. [ONCOLOGY 12(11):1597-1608, 1998]
Thrombocytopenia is a well-recognized problem that occurs in patients with malignancies. Although multiple etiologic factors of primary (eg, intrinsic bone marrow disease) or secondary (eg, tumor-induced coagulopathy, immune-mediated thrombocytopenia, hypersplenism-related platelet sequestration) origin can contribute to thrombocytopenia in cancer patients, the most common etiology of clinically significant thrombocytopenia is the increasing use of myelosuppressive chemotherapy.
Administration of antibiotics, transfusion of blood products, and modifications in chemotherapy doses have been the major means of combating chemotherapy-induced hematologic toxicity. Over the past decade, the clinical availability of hematopoietic growth factors, such as granulocyte colony-stimulating factor (G-CSF, filgrastim(Drug information on filgrastim) [Neupogen]), granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim(Drug information on sargramostim) [Leukine]), and erythropoietin(Drug information on erythropoietin) (Epogen, Procrit) have helped reduce clinical complications associated with granulocyte and erythroid cell lineages. Thrombocytopenia has been managed primarily by platelet transfusions and chemotherapy dose reductions.
Severe thrombocytopenia is a frequent clinical problem in the management of leukemia and pediatric malignancies, as well as in the setting of bone marrow or peripheral blood progenitor-cell transplantation. Although clinically significant thrombocytopenia does not commonly occur with most standard chemotherapeutic regimens used in the treatment of solid tumors, cumulative thrombocytopenia can be problematic with multiple cycles of certain chemotherapeutic regimens used in the treatment of lymphoma, sarcoma, breast cancer, ovarian cancer, and germ cell tumors (Table 1).[1-11] Furthermore, the concept that the dose intensity of chemotherapy may be important in improving overall outcome has led to the use of more aggressive, potentially curative regimens in patients with chemosensitive malignancies.
The use of peripheral blood progenitor cells has significantly reduced the duration of thrombocytopenia in patients receiving myeloablative or highly myelosuppressive therapy. However, 80% of patients undergoing autologous or allogeneic marrow transplantation continue to require transfusions 3 weeks after transplantation, as compared with 20% of patients receiving autologous peripheral blood progenitor cell transplants.
The use of platelet transfusions to manage severe thrombocytopenia has increased approproximately twofold in the United States from 1982 to 1992.[13,14] Advances in organ transplantation and cardiac surgery and the use of aggressive chemotherapy in patients with treatment- responsive malignancies have all contributed to this marked increase in the need for platelets and to rising health-care costs.
The total national direct medical cost of treating chemotherapy-induced thrombocytopenia was estimated to range from $146 to $233 million in 1993. In a recently conducted, observational study of platelet utilization in patients undergoing myeloablative treatment and stem-cell transplantation at 18 transplant centers in the United States and Canada, which used conservative cost figures for platelet transfusions, the average 60-day platelet cost per patient was estimated at $3,500 for autologous peripheral blood progenitor-cell support and $9,200 for allogeneic bone marrow transplantation (BMT).
Although platelet transfusions may decrease the risk of fatal bleeding complications, repeated use of platelets increases the risks of transmission of infectious diseases, transfusion reactions, alloimmunization, and graft-vs-host disease. All of these factors have motivated the search for an agent that could stimulate platelet production and prevent or ameliorate thrombocytopenia.
Platelets are small, anucleated cells that play an essential role in maintaining normal hemostasis. The normal circulating platelet count (range, 150 to 450 × 103/L) varies greatly between individuals but remains fairly constant in any one individual; there is minimum day-to-day variation in platelet count throughout life unless altered by a disease process or a change in physiologic condition.
The body maintains a constant circulating mass of platelets through the process of megakaryocytopoiesis. Peripheral blood platelets are derived from megakaryocytes, which are located primarily in the bone marrow. However, their large size and ability to attain a multilobular nucleus with a polyploid complement of DNA and abundant cytoplasm allow megakaryocytes to produce several thousand platelets per cell.
During megakaryocytopoiesis, pluripotent hematopoietic cells undergo commitment to the megakaryocyte lineage. The burst-forming unit-megakaryocyte (BFU-MK) is the most primitive megakaryocyte progenitor cell. The BFU-MKs take 21 days to develop in culture, and their immunophenotypic cell surface markers are CD34+, c-kit+ and HLA-DR-. The BFU-MKs differentiate and produce colony-forming unit-megakaryocytes (CFU-MKs), which take 12 days to appear in culture and carry cell surface markers CD34+, c-kit+, and HLA-DR+.
Colony-forming unit-megakaryocytes give rise to megakaryocytes (MKs), which lose their capacity to undergo mitosis but retain their capacity for endoreduplication. Megakaryocytes become large polyploid cells (up to 128N) with maturation of cytoplasm and acquisition of biochemical properties characteristic of functional platelets. Platelets are produced by transendothelial cytoplasmic fragmentation and have a life span of approximately 10 days.
The concept of a lineage-specific regulator involved in platelet development originated nearly 40 years ago. The term "thrombopoietin" was first used in 1958 to describe a humoral regulator present in thrombocytopenic plasma that restored platelet counts to a normal level after the onset of the thrombocytopenic state. Since then, many groups have attempted to isolate and purify thrombopoietin from thrombocytopenic plasma, serum, and other physiologic sources.
A major step in the search for thrombopoietin came in 1992 with the cloning of the cellular homolog of the viral oncogene, c-Mpl. This line of research began in 1986 with the identification of the murine retrovirus myeloproliferative leukemia virus (MPLV). The responsible viral oncogene, v-Mpl, was cloned in 1990 and the corresponding cellular proto-oncogene, c-Mpl, was identified in 1992. Sequence analysis of c-Mpl suggested homology to the genes encoding a family of hematopoietic growth factor receptors.
Subsequent studies established the link between c-Mpl and megakaryocytopoiesis. The expression of c-Mpl appeared to be restricted to CD34+ hematopoietic stem cells, megakaryocytes, and platelets. Furthermore, c-Mpl antisense oligonucleotides selectively inhibited megakaryocytic colony form- ation in vitro without affecting the growth of erythroid or granulocyte-macrophage colonies, indicating that a putative ligand for c-Mpl may be a lineage-specific regulator of megakaryocytopoiesis.
From that point on, the search for the c-Mpl ligand was aggressively pursued. In 1994, five different groups [24-28] using three distinct strategies reported on the identification, purification, and cloning of cDNA for the Mpl ligand. The observation that all of the megakaryocyte colony- and platelet-stimulating activity from thrombocytopenic plasma can be removed by recombinant c-Mpl further confirmed that this ligand is, indeed, thrombopoietin; the ligand was also named megakaryocyte growth and development factor (MGDF) and megapoietin by different investigators.
The human thrombopoietin gene is located on chromosome 3q27 and consists of seven exons joined to six introns.[28,30] The precursor protein product is made of 353 amino acids, and cleavage of a signal peptide at the amino terminus produces a mature peptide of 332 amino acids with two domains (Figure 1).
The amino terminal domain consists of 153 amino acids, including 4 cysteine(Drug information on cysteine) residues, and has 23% homology with erythropoietin. The similarity increases to 50% when conservative amino acids are taken into account.
The carboxy terminal domain consists of 179 amino acids and has several N-linked glycosylation sites.[17,31] Whereas the amino terminal domain is sufficient for the thrombopoietic effects of the molecule, the carboxy terminal domain is likely important in maintaining its circulating half-life.
The major site of thrombopoietin production is the liver (fetal and adult).[25,27,30] Other sites expressing thrombopoietin mRNA include bone marrow, kidney, brain, testes, and spleen.[17,25,27,30]
The c-Mpl receptor, the receptor for thrombopoietin, is a transmembrane protein with an N-terminal extracellular domain and a C-terminal intracellular domain. The receptor expression is largely restricted to the tissues that support hematopoiesis, ie, bone marrow cells, spleen, and fetal liver, and is mostly expressed on CD34+ cells, cells of megakaryocyte lineage, and platelets.[20,23,32] The gene for the c-Mpl receptor is located on mouse chromosome 4 and human chromosome 1p34.
The c-Mpl receptor binds to the ligand (thrombopoietin), which results in proliferation and differentiation of megakaryocyte progenitors. Activation of the c-Mpl receptor results in the activation of the JAK/STAT pathway and possibly the Ras signal transduction pathway. With receptor activation, there is an increase in megakaryocyte number, increased nuclear mass, increased ploidy, and cytoplasmic maturation and release of platelets.
In Vitro Properties
In vitro studies have demonstrated that thrombopoietin stimulates all stages of megakaryocytopoiesis.[26,31,35,36] It acts as a megakaryocyte colony-stimulating factor in the early stages of megakaryocytopoiesis to stimulate the proliferation of megakaryocytic progenitors and in later stages to promote maturation of megakaryocytes into large polyploid cells capable of producing platelets (Figure 2). In suspension cultures, thrombopoietin increases the size and ploidy of megakaryocytes and the expression of Ib and IIb/IIIa glycoproteins on the cell surface.[18,37,38] Thrombopoietin-induced megakaryocytes express increased formation of demarcation membranes, platelet-specific granules, and platelet territories.[39,40]
Studies have demonstrated that thrombopoietin is essential for full megakaryocyte maturation in cell cultures. In these studies, the soluble form of the Mpl receptor was used to neutralize all of thrombopoietin’s biological activity. The addition of this soluble receptor terminated megakaryocyte formation despite the presence of C-kit ligand (stem cell factor), interleukin-6 (IL-6), or inter-leukin-11 (IL-11). However, in the presence of interleukin-3 (IL-3), production of megakaryocytes decreased but did not cease entirely; in addition, megakaryocytes lacked demarcation membranes and platelet-specific granules and had low ploidy (4N) levels.
The effects of thrombopoietin are not limited to megakaryocytopoiesis. In vitro studies demonstrate that, in the presence of erythropoietin, thrombopoietin enhances the growth of erythroid progenitor cells.[41,42] In addition, thrombopoietin directly stimulates primitive hematopoietic stem cells in the presence of early-acting cytokines (IL-3 or C-kit ligand) and exhibits both proliferative and differentiative effects on these progenitor cells.[42-44]
Physiologic Role and Regulation
The role of thrombopoietin as a physiologic regulator of platelet production has been supported by the inverse relationship between the platelet count and serum thrombopoietin level in animal models of thrombocytopenia induced by antiplatelet-antiserum or by chemotherapy or radiation treatment, as well as in cancer patients following myeloablative chemotherapy and bone-marrow transplantation.[45-49]
The importance of the c-Mpl ligand and receptor system in physiologic regulation of platelet production in vivo is further demonstrated by the generation of c-Mpl and thrombopoietin knock-out mice. Mice genetically altered to be defective in c-Mpl or the thrombopoietin gene exhibit an 85% reduction in peripheral platelet count, as well as decreased bone marrow and splenic megakaryocytes.[50,51]
In addition, the lack of thrombopoietin or c-Mpl results in significant reductions in the levels of erythroid, myeloid, and multipotential progenitor cells. This indicates that thrombopoietin acts not only on cells committed to megakaryocyte lineage but also on primitive hematopoietic progenitor cells.[52,53]
As in other thrombocytopenic animal models, circulating thrombopoietin levels are elevated in c-mpl-/- mice.[50,54] However, there is no detectable difference between the c-mpl-/-and c-mpl+/+ mice with respect to thrombopoietin mRNA levels in any tissue. This suggests that transcriptional regulation of the thrombopoietin gene is not involved in the increased level of thrombopoietin observed during thrombocytopenia.
An alternative theory of thrombopoietin regulation states that circulating thrombopoietin levels are regulated by platelet mass. Recent studies support this hypothesis.[16,17,55] Injection of normal platelets into c-mpl-/- mice resulted in a decrease in plasma thrombopoietin level and normalization of platelet levels.[54,55] These findings suggest that thrombopoietin is constitutively synthesized and released into the circulation by the liver and/or kidney. Under normal circumstances, platelets clear most of the thrombopoietin from the circulation via binding to the c-Mpl receptors.[51,54,55]
In disorders of platelet production, such as aplastic anemia, or in patients undergoing chemotherapy or radiotherapy, plasma thrombopoietin levels are elevated. This is due most likely to a decrease in the clearance mechanism related to the low platelet mass. However, in conditions in which low platelet numbers are accompanied by normal or increased numbers of megakaryocytes (such as idiopathic thrombocytopenic purpura), circulating thrombopoietin levels remain normal since thrombopoietin binds to megakaryocytes via c-Mpl receptors.
In normal mice, administration of thrombopoietin causes a rapid, four- to sixfold rise in circulating platelet count, along with increases in number, size, and ploidy of bone marrow and splenic megakaryocytes.[27,35,36] The magnitude of platelet response to thrombopoietin is higher than has previously been achieved with other thrombopoietic cytokines. In addition, thrombopoietin also increases the number of burst-forming unit-erythroid (BFU-E) in the bone marrow and spleen.
Although, in normal animals, thrombopoietin exerts its effects exclusively on platelets, in the myelosuppressed murine model, administration of recombinant murine thrombopoietin accelerated not only platelet recovery but also red blood cell recovery.[57,59,60] Thrombopoietin-treated animals showed an increase in megakaryocytic as well as myeloid and erythroid progenitors in the bone marrow and spleen. Thus, the effect of thrombopoietin on multiple cell lineages at the progenitor-cell level, along with increased levels of other endogenous cytokines in a severely myelosuppressed model, may enhance recovery of other cell lineages.
In the myelosuppressed nonhuman primate model, thrombopoietin also ameliorated platelet nadirs following whole-body irradiation. These preclinical trials have shown major activity of this cytokine without significant toxicity.