Thrombopoietin  Biology and Clinical Applications

K Pavithran, DC Doval
Department of Medical Oncology, Rajiv Gandhi Cancer Institute and Research Centre, Rohini, New Delhi, India

Introduction

Thrombocytopenia is an important problem in clinical haematology/oncology for patients receiving intensive chemotherapy or bone marrow transplantation. The condition increases the risk of bleeding and often limits the dose of chemotherapeutic agents that may be given. Although platelet transfusion decreases the risk of haemorrhage, approximately 30% of transfusions result in complications such as transmission of viral diseases, febrile reactions, alloimmunisation, and sepsis,¹ facilitated by the need to store platelets at room temperature. Platelet transfusions are also expensive. The limitations of platelet transfusion have prompted the development of agents with the potential to stimulate platelet production, which can reduce or eliminate the need for transfusions.

Kelemen et al first used the term 'thrombopoietin' to describe the humoral substance responsible for increasing platelet production after the onset of thrombocytopenia.² Thrombopoietin (TPO), also referred to as c-Mpl ligand, mpl ligand, megapoietin, and megakaryocyte growth and development factor, is the most potent cytokine that physio-logically regulates platelet production.³

Megakaryocyte Development

The generation of platelets from the marrow progenitors is a complex process. Each day an adult produces 100 x 10⁹/L platelets, which can increase 10-fold in times of increased demand. All the formed elements of blood develop from haematopoietic stem cells through a series of cell divisions.⁴ This process of cellular proliferation and differentiation requires the support of several cytokines. Thrombopoietin, along with other cytokines, has many actions during mega-karyocyte development.⁵ One of the earliest identifiable megakaryocyte progenitors is the high proliferative potential colony forming unit-megakaryocyte (HPP CFU-MK). The next stages in the sequence involve burst forming unit-MK (BFU-MK), CFU-MK, promegakaryoblasts, and megakaryoblasts. Colony forming unit-megakaryocytes are initially mitotic cells but have the ability to stop cellular division (cytokinesis) while continuing to undergo DNA replication (endomitosis) to produce immature megakaryocytes that are polypoid and contain up to 64 times the normal amount of DNA. The immature megakaryocytes then develop into larger, mature megakaryocytes that shed platelets into bone marrow sinusoids.

Cloning and Characterisation of Thrombopoietin

In 1994, 5 separate groups of researchers purified or cloned cDNA for TPO.^6-10 Thrombopoietin is a glycoprotein consisting of 353 amino acids with a molecular weight of 30 KD. The gene for TPO is located on chromosome 3q27.¹¹ Structurally, TPO can be divided into 2 domains amino terminal and carboxyl terminal. The amino terminal domain binds to the c-Mpl receptor.⁶ The C-terminal is important for protein stability.

Two forms of thrombopoietin are currently being studied in clinical trials. One, termed recombinant human TPO (rhTPO), is a full-length polypeptide. The other, a truncated protein containing only the receptor-binding region, which has been chemically modified by the addition of poly-ethylene glycol (PEG), is termed PEG-conjugated recombinant human megakaryocyte growth and development factor (PEG-MGDF, also known as PEG-rhMGDF)). The polypeptide has 163 amino acids and is conjugated with polyethylene glycol on the N terminal by reductive alkylation. The bio-logical activities of both of these proteins are similar.¹²

Physiology

The primary sites of TPO production are the hepatocytes and sinusoidal endothelial cells in the liver. Lesser amounts of TPO are seen in the kidneys, brain, and testes. Thrombopoietin is synthesised and immediately released as and when required, as for erythropoietin. Following a fall in the platelet count, TPO levels maximally increase by 50% after 8 hours and peak at 24 hours.

Thrombopoietin Receptor

The Mpl receptor was discovered to be the product of the gene c-mpl, the normal homologue of the oncogene v-mpl. v-mpl is the transforming gene of the murine myeloproliferative leukaemia virus.¹³ The TPO receptor does not contain intrinsic tyrosine kinase activity. Ligand binding to c-Mpl is associated with the activation of the tyrosine kinase, JAK2, and the tyrosine phosphorylation of a number of molecular targets, including signal transducer and signal transduction activators of transcription proteins (activators of transcription), Shc adaptor protein, and the c-Mpl receptor itself.

Regulation of Thrombopoietin Expression

Circulating levels of TPO are inversely related to platelet mass. Platelets contain an avid TPO receptor that efficiently binds and removes TPO from circulation.¹⁴ Thus, normal or elevated levels of platelets inhibit the action of TPO on target cells (bone marrow) by binding to circulating TPO. This observation is clinically important, because:

• platelet transfusions may blunt the recovery of mega-karyocytes
• other cytokines or disorders may modify the constitutive hepatic production of TPO, similar to reduced erythro-poietin levels in renal disease
• disease-related abnormalities in the platelet's ability to clear TPO may alter TPO levels. For example, diminished clearance of TPO by abnormal platelets may account for the elevated platelet counts seen in myeloproliferative syndromes such as essential thrombocythaemia.

In conditions associated with marrow failure (aplastic anaemia), TPO levels are high whereas, in immune thrombocytopenic purpura, TPO levels are low.¹⁵ Thus, TPO levels may be used for the differentiation of thrombocytopenia due to bone marrow failure or increased destruction.

Cytokine Regulation

Thrombopoiesis is regulated by many cytokines in which the rate of platelet production responds to the number or mass of circulating platelets. Interleukin-3 (IL-3), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and steel factor (SCF, kit ligand) act at the progenitor cell stage, whereas IL-6 acts late in maturation. Thrombopoietin and IL-11 stimulate all stages of megakaryocytopoiesis, including the proliferation of pro-genitors and the development and complete maturation of polypoid megakaryocytes (Figure 1). In addition, TPO acts in synergy with erythropoietin to stimulate the growth of erythroid progenitor cells¹⁶ and, with IL-3 or SCF, TPO stimulates the proliferation and prolongs the survival of haematopoietic stem cells and all types of blood cell progenitors. Thrombo-poietin also has some role in regulating neutrophil activation. Thrombopoietin can sensitise platelets to various agonists,¹⁷ and may predispose them to thrombosis when therapeutically administered.

Pharmacological Properties of Thrombopoietin

Of the haematopoietic growth factors, TPO has the longest half-life of approximately 30 hours.¹⁸ Pegylation of TPO further increases the plasma half-life by 10-fold. Following systemic administration, the platelet count begins to increase after 3 to 5 days. The maximum elevation in platelet count occurs 2 to 3 weeks after commencement of treatment with TPO, since TPO acts by stimulating the production and maturation of megakaryocytes. The most common adverse events are disturbance of the gastrointestinal system and arthralgia. In therapeutic doses, MGDF does not have any effect on platelet function and TPO is being trialled for a variety of indications (Table 1).

Potential Side Effects of Thrombopoietin

Clinical studies show that TPO is well tolerated.¹⁹ Toxicities such as flu-like symptoms, fatigue, or major organ toxicities that occur with other cytokines have not been reported with TPO. Other side effects are listed in Table 2.

Clinical Trials of Thrombopoietin

Clinical trials of the clinical efficacy of TPO in various clinical settings, especially phase III studies, are relatively limited at the present time. The following text highlights the clinical situations where early studies have shown promising results.

Solid Tumour Chemotherapy

Clinical trials of PEG-rhMGDF or rhTPO in patients with cancer at risk for chemotherapy-induced severe thrombocytopenia showed that TPO is safe and, when given before chemotherapy, results in marked stimulation of platelet production. Initially, rhTPO was evaluated in patients with sarcoma receiving adriamycin and ifosphamide-based chemotherapy, where a single dose of TPO was given intravenously 3 weeks prior to the chemotherapy cycle. The first cycle of chemotherapy served as an internal control. Thrombocytopenia decreased in cycle 2 compared with cycle 1 for some patients, however, the nadir value of platelet count was not significantly different between the 2 cycles.¹⁸ Another study gave subcutaneous rhTPO to patients with gynaecological malignancies, followed by carboplatin 3 weeks later (cycle 2). The degree and duration of thrombocytopenia was reduced from 75% in cycle 1 (without TPO) to 25% in cycle 2 (with TPO).²⁰ Fanucchi et al, in a randomised controlled trial with PEG-rhMGDF for patients with lung cancer receiving carboplatin and paclitaxel, showed a dose-dependent increase in platelet count.²¹ Basser et al, in a randomised trial of patients with advanced cancer, demonstrated accelerated platelet recovery for recipients of PEG-rhMGDF.²²

Bone Marrow Transplantation

For patients undergoing autologous transplantation using bone marrow stem cells for breast cancer, administration of PEG-rhMGDF accelerated the increase in platelet count to 20 x 10⁹/L after 5 to 6 days, enabling a 48% reduction in the use of platelet transfusions as compared with placebo.²³ However, in the setting of autologous peripheral blood stem cells (PBSC) for breast cancer, administration of PEG-rhMGDF was not associated with any significant reduction in the duration of severe thrombocytopenia or the requirement for prophylactic platelet transfusions.²⁴

Delayed Recovery or Engraftment Failure

Approximately 10 to 20% of patients undergoing bone marrow transplantation do not recover their platelet count and remain transfusion-dependent beyond 30 days. A multicentre, phase I dose-escalation study of rhTPO for patients with persistent severe thrombocytopenia 20 x 10⁹/L for more than 35 days after HSCT failed to show any improvement in platelet count.²⁵

Mobilisation of Peripheral Blood Stem Cells

Thrombopoietin was found to be effective for mobilising autologous stem cells for transplantation in patients under-going intensive chemotherapy. Somlo et al showed that TPO and G-CSF were more effective than G-CSF alone for mobilising high numbers of CD34+ cells, resulting in fewer apheresis procedures being required.²⁶ After high-dose chemotherapy, patients with breast cancer receiving peripheral progenitor cells mobilised by TPO/G-CSF experienced somewhat faster haematopoietic recovery and received significantly fewer erythrocyte and platelet transfusions than those re-ceiving progenitors mobilised by G-CSF alone.

Ex Vivo Expansion of Haematopoietic Stem Cells

Cytokine-mediated ex vivo expansion of haematopoietic stem cells (HSCs) has been proposed as a means of increasing the number of HSCs for transplantation and several cytokines have been investigated for their effectiveness. The ability of TPO to promote expansion of HSCs and megakaryothrom-bocytopoiesis has made it widely used in preclinical ex vivo expansion of HSCs. Preliminary studies show that ex vivo expanded PBSC can provide rapid neutrophil recovery and can reduce the risk of tumour cell inoculation with autotransplants.^27,28 In addition, TPO may be valuable in situations where small stem cell doses are available, as in poor mobilisers of stem cells or when using umbilical cord stem cells.

Acute Leukaemia

As thrombocytopenia is routinely seen during induction treatment of acute myeloid leukaemia, TPO may be beneficial in this situation. However, expression of TPO receptors on leukaemic cells has been reported to be associated with poor prognosis and a lower response to chemotherapy.²⁹ CD7+ leukaemic blast cells express functional TPO receptors andproliferate in response to TPO.³⁰ Two recently reported randomised controlled trials show that PEG-rhMGDF was well tolerated by patients receiving induction and consolidation therapy for acute myeloid leukemia.^31,32 However, there was no effect on the duration of severe thrombocytopenia or the platelet transfusion requirement for these patients and there was no apparent stimulation of leukaemia.

Transfusion Medicine

Thrombopoietin may have an important role in transfusion medicine. In a randomised controlled trial, subcutaneous PEG-rhMGDF given to healthy donors in a single dose of 1 µg/kg or 3 µg/kg of body weight increased platelet counts to provide a median 3-fold greater apheresis platelets compared with untreated donors.³³ In another placebo controlled study, Goodnough et al showed that platelets collected from healthy donors following PEG-rhMGDF therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.³⁴

Conclusion

Thrombopoietin is unique among the haematopoietic cyto-kines because it is necessary both for terminal maturation and regulation of lineage-specific megakaryocytes, and for maintenance of the most primitive haematopoietic stem cells. Clinical use of the thrombopoietin molecules PEG-rhMGDF and rhTPO has been found to be promising for recovery of platelets after chemotherapy, mobilisation of stem cells, and collection of platelets by apheresis from normal donors. Studies are ongoing in non-chemotherapy settings such as immune thrombocytopenic purpura, neonatal thrombocytopenia, myelodysplastic syndrome, liver disease, and thrombocytopenia caused by human immunodeficiency virus infection. Fusion proteins of IL-3 with thrombopoietin and thrombopoietin peptide mimetics are currently in the early phases of development. Future large randomised clinical trials will help to establish the clinical safety and therapeutic uses of these thrombopoietic growth factors.

References

1. Heddle NM, Klama L, Singer J, et al. The role of the plasma from platelet concentrates in transfusion reactions. New Engl J Med 1994;331: 625-628.

2. Kelemen E, Cserhati I, Tanos B. Demonstration and some properties of human thrombopoietin in thrombocythaemic sera. Acta Haematol 1958;20: 350-355.

3. Wendling F, Cohen-Solal K, Villeval JL, et al. Mpl ligand or thrombopoietin: biological activities. Biotherapy 1998;10: 269-277.

4. Wendling F. Thrombopoietin: its role from early hematopoiesis to platelet production. Haematologica 1999;84: 158-166.

5. Harker LA. Physiology and clinical applications of platelet growth factors. Curr Opin Hematol 1996;6: 127-134.

6. Kuter DJ, Beeler DL, Rosenberg RD. The purification of mega-poietin: a physiological regulator of megakaryocyte growth and platelet production. Proc Natl Acad Sci USA 1994;91: 11104-11108.

7. de Sauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994;369: 533-538.

8. Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murinethrobopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369: 565-568.

9. Wendling F, Maraskovsky E, Debili NI, et al. CMpl is a humoral regulator of megakaryocytopoiesis. Nature 1994;369: 571-574.

10. Sohma Y, Akahori H, Seki N, et al. Molecular cloning and chromosomal localisation of the human thrombopoietin gene. FEBS Lett 1994;353: 57-61.

11. Foster DC, Sprecher CA, Grant FJ, et al. Human thrombopoietin: gene structure, chromosomal localisation and conserved alter-native splice forms of thrombopoietin. Blood 1995;85: 981-988.

12. Souryi M, Vigon I, Penciolelli JF, et al. A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell 1990;63: 1137-1147.

13. Vigon I, Mornon JP, Cocault L, et al. 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.

14. Fielder PJ, Gurney AL, Stefanich E, et al. Regulation of thrombo-poietin levels by c-mpl-mediated binding to platelets. Blood 1996;87: 2154-2161.

15. Espanol I, Hernandez A, Muniz-Diaz E, et al. Usefulness of thrombopoietin in the diagnosis of peripheral thrombocytopenias. Haematologica 1999;84: 608-613.

16. Kaushansky K, Broudy VC, Grossmann A, et al. Thrombopoietin expands erythroid progenitors increases red cell production and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest 1995;96: 1683-1687.

17. Chen J, Herceg-Harjacek L, Groopman JE, Grabarek J. Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin. Blood 1995;86: 4054-4062.

18. Vadhan-Raj S, Murray LJ, Bueso-Ramos C, et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Ann Intern Med 1997;126: 673-681.

19. Kaushansky K. Thrombopoietin. New Engl J Med 1998;339: 746-754.

20. Vadhan-Raj S, Verschraegen DF, Bueso-Ramos C, et al. Recombinant human thrombopoietin attenuates carboplatin-induced severe thrombocytopenia and need for severe platelet transfusions in patients with gynecologic cancer. Ann Intern Med 2000;132: 364-368.

21. Fanucchi M, Glaspy J, Crawford J, et al. Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor in platelet counts after chemotherapy for lung cancer. New Engl J Med 1997;336: 404-409.

22. Basser RL, Rasko JEJ, Clarke K, et al. Randomised, blinded, placebo controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer. Blood 1997;89: 3118-3128.

23. Beveridge R, Schuster M, Waller E, et al. Randomized, double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in breast cancer patients following autologous bone marrow transplantation (ABMT) [abstract]. Blood 1997;90 (Suppl 1): 580a.

24. Bolwell B, Vredenburgh J, Overmoyer B, et al. Phase 1 study of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in breast cancer patients after autologous peripheral blood progenitor cell (PBPC) transplantation. Bone Marrow Transplant 2000;26: 141-145.

25. Nash RA, Kurzrock R, DiPersio J, et al. A phase I trial of recombinant human thrombopoietin in patients with delayed platelet recovery after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2000;6: 25-34.

26. Somlo G, Sniecinski I, ter Veer A, et al. Recombinant human thrombopoietin in combination with granulocyte colony-stimulating factor enhances mobilization of peripheral blood progenitor cells, increases peripheral blood platelet concentration, and accelerates hematopoietic recovery following high-dose chemotherapy. Blood 1999;93: 2798-2806.

27. McNiece I, Jones R, Bearman SI, et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 2000;96:3001-3007.

28. Engelhardt M, Douville J, Behringer D, et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myelo-ablative chemotherapy. Bone Marrow Transplant 2001;27: 249-259.

29. Vigon I, Dreyfus F, Melle J, et al. Expression of the c-mpl proto-oncogene in human hematologic malignancies. Blood 1993;82: 877-883.

30. Tokunaga Y, Miyamoto T, Gondo H, et al. Effect of thrombo-poietin on acute myelogenous leukemia blasts. Leuk Lymphoma 2000;37: 27-37.

31. Schiffer CA, Miller K, Larson RA, et al. A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia. Blood 2000;95:2530-2535.

32. Archimbaud E, Ottmann OG, Yin JA, et al. A randomized, double-blind, placebo-controlled study with pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) as an adjunct to chemotherapy for adults with de novo acute myeloid leukemia. Blood 1999;94: 3694-3701.

33. Kuter DJ, Goodnough LT, Romo J, et al. Thrombopoietin therapy increases platelet yields in healthy platelet donors. Blood 2001;98: 1339-1345.

34. Goodnough LT, Kuter DJ, McCullough J, et al. Prophylactic platelet transfusions from healthy apheresis platelet donors undergoing treatment with thrombopoietin. Blood 2001;98: 1346-1351.