K. PavithranSee also Management of Venous Thromboembolism by Pavithran
Address for correspondence:
Dr.K.PavithranMD,DM(Med Oncology),Dept of Haematology
Medical College Hospital
Thrombocytopenia is an important clinical problem in the management of patients with cancer. It increases the risk of bleeding and often limits the dose of chemotherapeutic agents. The recovery of platelets after bone marrow transplantation or chemotherapy often occurs later (23 to 60 days after transplantation) than the recovery of other hematopoietic lineages. Even though platelet transfusion decreases the risk of hemorrhage, about 30 percent of transfusions result in complications like1 transmission of viral diseases, febrile reactions, alloimmunization and sepsis facilitated by the need to store platelet at room temperature. Platelet transfusions are also expensive.
The term "thrombopoietin" was first used by Kelemen in 1958 to describe the humoral substance responsible for increasing platelet production after the onset of thrombocytopenia2. Thrombopoietin (TPO), also referred to as c-MpI ligand, mpl ligand, megapoietin, and magakaryocyte growth and development factor, is the most potent cytokine that physiologically regulates platelet production3. TPO is a hormone constitutively produced by the liver and kidneys. Plasma levels of TPO are regulated through receptor-mediated uptake, internalization and catabolism. TPO has pleiotropic effects on hematopoiesis 4.
The generation of platelets from the marrow progenitors is a complex process. Each day the adult produce 1x1011 platelets a number that can increase to 10 fold in times of increased demand. All the formed elements are formed from hematopoietic stem cells, through a series of cell divisions5. This process of cellular proliferation and differentiation requires the support of several interleukins, colony-stimulating factors, and hormones. Thrombopoietin, along with other cytokines, has several actions during megakaryocyte development 6,7.
One of the earliest identifiable megakaryocyte progenitor is the high proliferative potential colony forming unit - megakaryocyte (HPP CFU-MK). Next stages in the sequentially are burst forming unit-MK (BFU-MK) and CFU-MK, promegakaryoblasts and megakaryoblasts. CFU-MK are initially mitotic cells but then 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 MK then develop into larger, mature MK that shed platelets into bone marrow sinusoids8,9.
Cloning and Characterization of Thrombopoietin
Thrombopoietin was cloned by five independent groups in 1994 10-14. It is a glycoprotein consisting of 353 amino acidsand molecular weight of 30 KD 15. Gene for thrombopoietin is located on chromosome 3q27 16. Structurally, TPO can be divided into two structural domains - amino (erythropoietin) domain and carbohydrate domain. The amino terminal 155 residues of thrombopoietin have 21 percent sequence identity and 46 percent overall sequence similarity with human erythropoietin. It is this domain that binds to the c-Mpl receptor 10. In contrast, the C-terminal 177 residues have no homology to any known proteins. Although deletion of this region does not affect the activity of the protein in vitro, it substantially reduces its bioavailability after parenteral administration 17.
Two forms thrombopoietin are being studied in clinical trials. One, termed recombinant human thrombopoietin (rTPO), 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 polyethylene glycol (PEG), is termed PEG-conjugated recombinant human megakaryocyte growth and development factor (peg-MGDF, also known as PEG-rHuMGDF)). The polypeptide has 163 amino acids and is conjugated with polyethylene glycol on the N terminal by reductive alkylation. The biologic activities of both of these proteins are similar 18.
The primary site of thrombopoietin production is the liver. Lesser amounts are seen in the kidneys, brain and testes. Thrombopoietin is synthesized and immediately released as and when required like erythropoiesis. Following a fall in the platelet count thrombopoietin levels rises half maximally by 8 hours and peak by 24 hours.
Mpl receptor was discovered as the product of the gene c-mpl, the normal homologue of the oncogene v-mpl. V-mpl is the transforming gene of murine myeloproliferative leukemia virus 19. The human TPO receptor was cloned in 1992 20. It is a member of the cytokine receptor superfamily. Using the c-Mpl receptor, the protein that bound to it (c-Mpl ligand was identified, purified sequenced and cloned. c-MPL RNA transcript can be detected in hematopoietic cell lines, peripheral blood, bone marrow and spleen and cells of the megakaryocytic lineage. 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 STAT proteins (activators of transcription), Shc adaptor protein and the c-Mpl receptor itself.
Regulation of thrombopoietin expression
Circulating levels of thrombopoietin are inversely related to platelet mass. Platelets contain an avid thrombopoietin receptor that efficiently binds and removes thrombopoietin from circulation 4. Thus normal or elevated levels of platelets inhibit inhibit the action of TPO on target cells (bone marrow) by binding to circulating TPO. This observation is clinically important because 1. Platelet transfusions may blunt the recovery of magakaryocytes 7 2. Other cytokines or disorders may modify the constitutive hepatic production of thrombopoietin, similar to reduced erythropoietin levels in renal disease 3. Small molecules could be developed to decrease the platelet's clearance of thrombopoietin and in turn stimulate platelet production 4. Disease related abnormalities in the platelet's ability to clear thrombopoietin may alter thrombopoietin levels. E.g. diminished clearance of thrombopoietin by abnormal platelets may account for the elevated platelet counts seen in myeloproliferative syndromes such as essential thrombocythemia. In conditions associated with marrow failure( e.g. aplastic anemia) thrombopoietin levels are high where as in ITP thrombopoietin levels are low 21. Thus TPO levels may be used for the differentiation of thrombocytopenia due to bone marrow failure or increased destruction.
Thrombopoiesis is regulated by many cytokines in which the rate of platelets production responds to the number or mass of circulating platelets. IL-3, G-CSF,GM-CSF and steel factor(SCF, kit ligand) acts in the progenitor cell stage. Where as IL-6 acts late in the maturation. TPO and IL-11 stimulate all stages of megakaryocytopoiesis, including the proliferation of progenitors and the development and complete maturation of polypoid megakaryocyte. In addition, thrombopoietin acts in synergy with erythropoietin to stimulate the growth of erythroid progenitor cells 22, and with interleukin-3 or steel factor it stimulates the proliferation and prolongs the survival of hematopoietic stem cells and all types of blood-cell progenitors. TPO have some role in regulating neutrophil activation also. Thrombopoietin can sensitise platelets to various agonists, 23 and may predispose to thrombosis when administered therapeutically.
Pharmacologic Properties of Thrombopoietin
Of the hematopoietic growth factors thrombopoietin has got the longest half-life i.e. 30hours. PEGylation of thrombopoietin further increases the plasma half-life by 10 fold. Following systemic administration, the platelet count begins to increase after 3-5 days. This is because thrombopoietin acts by stimulating the production and maturation of megakaryocytes 7. The most common adverse events were disturbances of the gastrointestinal system, and arthralgia. In therapeutic doses MGDF doesn't have any effect on platelet function.
Potential clinical uses of thrombopoietin
- Chemotherapy of solid tumors: both rhTPO and MDGF are effective in attenuating both the degree and duration of thrombocytopenia.
- Bone marrow transplantation:
- Chemotherapy of acute leukemias: as severe thrombocytopenia is routinely observed after induction therapy of AML, TPO might be beneficial in this setting.
- Radiation therapy
- Aplastic anemia and other bone marrow failure states
- ITP and thrombocytopenia of HIV
- Harvesting peripheral blood progenitor cells
- Platelet apheresis
Potential side effects of thrombopoietin
Clinical studies shows that TPO is well tolerated. Toxicities such as flu-like symptoms, fatigue or major organ toxicites that occur with other cytokines have not been reported.
- Marrow fibrosis possibly due to abnormal increase in megakaryocytes and with release of PDGF.
- Veno-occlusive disease; TPO may alter endothelial function and lead to an increase in VOD in the bone marrow transplant setting.
- Interaction with other growth factors
Clinical Trials of Thrombopoietin 6,18,24,25
1.Solid tumor chemotherapy:
- In all the clinical studies the administration of either PEG-rhMDGF or rhTPO was found to be safe, and when given before chemotherapy these substances resulted in marked stimulation of platelet production. Results from three clinical trials of PEG-rhMDGF or rhTPO in patients with cancer who were receiving chemotherapy have been reported 18. In all three studies, platelet counts returned to base line significantly faster, and in two of the three studies the nadir platelet counts were higher, in the patients given thrombopoietin than in those given placebo or in the same patients during their first cycle of chemotherapy.
2. Bone marrow transplantation:
- In patients undergoing autologous transplantation using bone marrow stem cells for breast cancer, administration of PEG-rhMGDF accelerated the increase in the platelet count to 20,000/mm3 five to six days and permitted a 48 percent reduction in the use of platelet transfusions as compared with placebo 26. But is not found to be effective in the setting of peripheral blood stem cell transplantation.
3. Delayed recovery or engraftment failure:
- About 10-20% of patients under going bone marrow transplantation do not recover their platelet count and remain transfusion dependent beyond 30 days. Studies with rhTPO in this setting however, doesn't show any difference27 .
- As thrombocytopenia is routinely seen during the induction treatment of acute myeloid leukemia, TPO might be beneficial. However, expression of TPO receptor on leukemic cells was reported to be associated with poor prognosis and a lower response to chemotherapy 28. But recent in vitro studies show that although AML blasts were found to express TPO receptors, there appeared to be no correlation between receptor expression and functional response to MGDF 29. So further studies are needed to settle this issue.
- TPO may have an important use in transfusion medicine. In a randomized controlled trial, MDGF given to normal subjects subcutaneously in a single dose of 3 µg per kilogram of body weight increased the circulating platelet count and yield by two to three fold compared to placebo group 30.
Other thrombopoietic subsances
In addition to thrombopoietin, several other recombinant cytokines (including interleukin-1, interleukin-3, interleukin-6, interleukin-11, granulocyte-macrophage colony-stimulating factor, steel factor (SCF), PIXY-321 and promegapoietin (nterleukin-3-thrombopoietin fusion protein) have both direct and indirect stimulatory effects in vitro and in vivo on cells of the megakaryocytic lineage. However, majority of these cytokines have not had beneficial effects on platelet recovery after myelosuppressive therapy or have had serious side effects. Of all these,only interleukin-11 has passed clinical trials and is approved for use by FDA for secondary prophylaxis against thrombocytopenia after the same type of chemotherapy 31,32. IL-11(oprelvekin) is available as Neumega and dose is 50m g/kg/day for 10-14 days.
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