Thrombopoietin (TPO) is a crucial regulator of platelet production, first introduced in the scientific literature in 1958. Over the years, research has elucidated its essential role in hematopoiesis, the process of blood cell formation. TPO regulates platelet production and maturation through its interaction with the c-Mpl receptor.
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Discovery and Function of Thrombopoietin
Early Discovery
TPO was initially identified as a specific factor for megakaryocytopoiesis and thrombopoiesis. The primary target cells for TPO in the bone marrow are late-stage megakaryocyte progenitors, such as colony-forming unit-megakaryocyte (CFU-MK), which express GpIIb/IIIa (CD41) in rats. Essential for the full maturation of megakaryocytes, TPO does not directly affect platelet shedding from mature megakaryocytes. This specific action underscores its role in ensuring the efficient production of functional platelets necessary for maintaining hemostasis.
Extended Biological Roles
Beyond its pivotal role in megakaryocytopoiesis, TPO has been shown to influence erythroid progenitors in the human bone marrow and erythroid and erythromegakaryocytic progenitors in the mouse embryonic yolk sac. This indicates that TPO's influence extends into the early stages of hematopoiesis. Recombinant human TPO (rHuTPO), particularly in combination with other cytokines, can rescue embryonic erythropoiesis in mice lacking the erythropoietin receptor. Furthermore, TPO supports the production of multiple hematopoietic cell lineages. This broad impact on hematopoietic progenitors is evident in studies using c-Mpl knockout mice and those involving both mouse and human cells. rHuTPO has been shown to enhance recovery from pancytopenia in myelosuppressed mice, and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) has accelerated multilineage hematopoietic recovery in both myelosuppressed mice and nonhuman primates.
Structure and Expression of the TPO Gene
The human TPO gene spans 6.2 kb and contains seven exons. An additional exon was later identified upstream of exon 1. TPO mRNA is constitutively produced in the liver and kidney, with hepatocytes being the primary source. During development from fetus to adult, TPO is predominantly produced in hepatocytes expressing albumin. The blood concentration of TPO in thrombocytopenic individuals with liver diseases does not increase, likely due to impaired production by hepatocytes. This impaired production underscores the critical role of the liver in TPO homeostasis. Furthermore, TPO mRNA is also present in the kidneys and other organs, suggesting additional, albeit lesser, production sites.
In thrombocytopenic states, such as aplastic anemia or immune thrombocytopenia, TPO mRNA levels increase in bone marrow stromal cells, indicating a regulatory mechanism responding to platelet demand. The transcription of the human TPO gene is initiated at multiple sites, with consensus sequences for EVI1, GATA-binding proteins, and ETS family transcription factors present in the 5' flanking region of the gene. These transcription factors are crucial for the activation of TPO gene expression, especially in liver cells. Mutations in the TPO gene have been associated with familial essential thrombocythemia, highlighting the gene's importance in regulating platelet production and its potential role in hematologic disorders.
Structure of the TPO Protein
Human TPO cDNA encodes a polypeptide of 353 amino acids, with the full-length rHuTPO secreted from mammalian cells comprising 332 amino acids after signal peptide cleavage. The NH2-terminal half of the TPO molecule, containing an erythropoietin-like domain, is essential for biological activity and binds to the TPO receptor (c-Mpl). Two disulfide bonds in the NH2-terminal domain are critical for its function. The COOH-terminal domain is highly glycosylated, containing six N-linked and multiple O-linked carbohydrate chains, which are crucial for the protein's stability and secretion.
Fig. 1 Structure of full-length human TPO (Kato T, et al. 1998).
Recent studies using specific antibodies and mutagenesis have provided insights into the structure-function relationships of TPO. The epitope domains of both neutralizing and non-neutralizing antibodies have been mapped, revealing important regions for TPO binding to c-Mpl. Determining the three-dimensional structure of TPO through X-ray crystallography or nuclear magnetic resonance will further elucidate these interactions, enhancing our understanding of TPO's function and facilitating the development of targeted therapies.
Regulation of the Blood Concentration of TPO and Truncation
The blood concentration of TPO, ranging from 0.33 to 1.72 fmol/ml, is maintained by the constitutive low-level production in the liver and the number of c-Mpl receptors on platelets and megakaryocytes. Recent studies have shown that TPO can undergo proteolytic cleavage, particularly by thrombin, which modulates its activity. This cleavage produces truncated TPO species that retain biological activity. Notably, the truncated forms are more active in vitro, suggesting a physiological mechanism for regulating TPO activity in response to platelet demand.
Truncated TPO molecules have been detected in thrombocytopenic animals, indicating the presence of proteolytic enzymes in the blood responsible for generating these forms. Platelets mediate TPO cleavage in the presence of Ca2+, with thrombin generating various TPO polypeptide species. The initial cleavage by thrombin at Arg191 in the COOH-terminal domain increases biological activity, while prolonged digestion at Arg117 in the NH2-terminal domain eventually destroys the activity. These cleavage sites are conserved among species, underscoring their physiological relevance.
Clinical and Biological Implications
Studies on the forms of circulating TPO in individuals with hematopoietic disorders, including aplastic anemia, essential thrombocythemia, and polycythemia vera, have shown that the molecular size distribution of TPO does not significantly differ between these disorders and normal controls. This suggests that the truncated forms of TPO are not markedly related to the pathophysiology of these disorders. However, in thrombocytopenic conditions, an increase in TPO production, rather than proteolytic processing, maybe the primary response to meet the increased demand for platelet production.
The presence of truncated TPO forms in thrombocytopenic states highlights a potential regulatory mechanism for platelet production. Full-length TPO is predominantly present in the circulation, but localized proteolytic events, such as cleavage by thrombin, may enhance TPO activity in response to decreased platelet counts. This local processing could potentiate TPO-stimulated platelet production, ensuring a rapid response to thrombocytopenia.
Conclusion
Thrombopoietin (TPO) is a key regulator of megakaryocytopoiesis and thrombopoiesis, playing a vital role in platelet production and the broader hematopoietic process. Its structure, gene expression, and regulatory mechanisms are complex and finely tuned to maintain hematopoietic homeostasis. The understanding of TPO's role has expanded significantly, revealing its influence on multiple hematopoietic lineages and its potential therapeutic applications in conditions like pancytopenia and thrombocytopenia.
References
- Kaushansky K. Thrombopoietin: a tool for understanding thrombopoiesis. Journal of Thrombosis and Haemostasis. 2003, 1 (7): 1587-92.
- Kaushansky K. Thrombopoietin and its receptor in normal and neoplastic hematopoiesis. Thrombosis Journal. 2016, 14(Suppl 1):40.
- Kato T., et al. Native thrombopoietin: structure and function. Stem Cells. 1998, 16 (5): 322-8.
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