Stem Cells, Vol. 16, No. 5, 322-328,
September 1998
© 1998 AlphaMed Press
Native Thrombopoietin: Structure and Function
Takashi Kato,
Atsushi Matsumoto,
Kinya Ogami,
Tomoyuki Tahara,
Haruhiko Morita,
Hiroshi Miyazaki
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan
Key Words. ELISA • Native thrombopoietin • Posttranslational processing • Proteolysis • Thrombopoietin • Thrombocytopenia • Truncation
Dr. Takashi Kato, Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-1295, Japan.
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Abstract
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Thrombopoietin (TPO), the c-Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c-Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full-length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.
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Introduction
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Thrombopoietin (TPO), also known as c-Mpl ligand, is a primary regulator of the proliferation and maturation of megakaryocytes as well as of platelet production [1, 2]. Since the direct purification of the protein from plasma [3, 4], its affinity purification based on its interaction with c-Mpl [5, 6], and the cloning by various procedures and expression of cDNAs encoding TPO [4, 5, 7-10], more than a hundred biological studies of TPO and c-Mpl have been perfomed with the use of recombinant molecules [11].
TPO was initially identified as a specific factor for megakaryocytopoiesis and thrombopoiesis. The primary target cell population for TPO in bone marrow comprises megakaryocyte progenitors at the late stage of differentiation, such as colony-forming unit-megakaryocyte (CFU-MK) expressing GpIIb/IIIa (CD41) in the rat [12-14], and TPO has been shown to be essential for full maturation of megakaryocytes [15-17]. Interestingly, however, TPO does not exert the direct effect on platelet shedding from mature megakaryocytes [18, 19]. In addition to its role in megakaryocytopoiesis, TPO also affects erythroid progenitors in human bone marrow [20] and erythroid and erythromegakaryocytic progenitors in mouse embryonic yolk sac [21]. Additionally, recombinant human (rHu)TPO in combination with other cytokines rescued in vitro embryonic erythropoiesis in mice lacking erythropoietin receptor [22]. Furthermore, TPO contributes to the production of multiple lineages of hematopoietic cells, as revealed by c-Mpl knockout mice [23, 24], and it supports primitive hematopoietic progenitors in synergy with other cytokines, as demonstrated with both mouse [25-27] and human [28-30] cells. Accordingly, rHuTPO improved the recovery from pancytopenia in myelosuppressed mice [31, 32], and pegylated recombinant human megakaryocyte growth and development factor ([PEG-rHuMGDF]; pegylated recombinant molecule relates to human TPO including the Mpl-binding NH2-terminal domain) accelerated multilineage hematopoietic recovery in myelosuppressed mice [33] and nonhuman primates [34]. Likewise, a single i.v. administration of PEG-rHuMGDF was able to enhance the recovery from thrombocytopenia as well as both primitive and committed hematopoiesis in myelosuppressed mice [35].
It is important for such studies on the extensive biological effects of TPO that we understand the production, activation, structure, transport, and metabolism of both native TPO and c-Mpl. The structure of TPO differs from that of other cytokines. However, whereas recent studies have examined the blood concentration of TPO in individuals with various diseases, information on the structural characteristics of native endogenous TPO has remained limited. In this article, our knowledge of the structure and function of native TPO, based largely on studies with recombinant molecules, will be summarized.
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Structure and Expression of the TPO Gene
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The entire human TPO gene was originally shown to span 6.2 kb and contain six exons and five introns [9]. However, an additional exon was subsequently detected upstream of exon 1 [36]. Southern blot analysis indicates the presence of only a single copy of the TPO gene in the human genome. Although splicing variants of TPO mRNA have been detected [37-39], their specific physiological roles remain unknown.
TPO was shown to be secreted from rat hepatocytes [40]. At all stages of development from fetus to adult, TPO is predominantly produced in hepatocytes expressing albumin but not in other liver cells, such as endothelial cells [41]. Among organs of the murine fetus at 13.5 days postcoitus, TPO mRNA is most abundant in the liver and is apparent by in situ hybridization in hepatocytes at later stages of embryogenesis. Thus, TPO may play a role in hematopoiesis in the embryo. However, the time required for detection of TPO mRNA in hepatocytes by in situ hybridization with digoxigenin-labeled probes (two days at 37°C) indicates that the concentration of TPO in these cells is low. The blood concentration of TPO in thrombocytopenic individuals with liver diseases was shown not to be increased [42], probably because of impaired production of TPO by hepatocytes, although the role of other factors specific to thrombocytopenia, such as consumption and degradation by platelets and spleen, should also be considered. The presence of TPO mRNA in kidney and other organs in adults suggests that they also may produce TPO protein to some extent. The synthesis of TPO mRNA is constitutive in the liver and kidney [38, 43, 44].
In contrast to the constitutive production of TPO mRNA in liver and kidney, the amount of TPO mRNA detected by reverse transcriptase and polymerase chain reaction analysis was increased in the bone marrow and spleen cells of thrombocytopenic mice [45]. In addition, in situ hybridization revealed that the abundance of TPO mRNA in bone marrow stromal cells was increased in thrombocytopenic patients, including those with aplastic anemia or immune thrombocytopenia [46]. According to these observations, TPO expression may be regulated in the bone marrow microenvironment in response to platelet demand.
Transcription of the human TPO gene is initiated at multiple sites. Consensus sequences for EVI1, GATA-binding proteins, and ETS family transcription factors are present in the 5' flanking region of the gene. Analysis of promoter activities in the human liver cell lines HepG2 and PLC [47, 48] revealed that members of the ubiquitously expressed ETS family of transcription factors are essential for activation of TPO gene expression. Given that the human TPO gene was mapped to chromosome 3q26-q28 [9, 36, 37, 49-52], close to the locus of the EVI1 gene at 3q26 that is transcriptionally activated in 3q21q26 syndrome, the possible role of TPO gene expression in the thrombocytosis associated with this syndrome was investigated. However, chromosomal rearrangements of the TPO gene were not detected, and the serum concentration of TPO was not increased in individuals with this condition [50-52]. Recently, however, mutations in the TPO gene have been associated with familial essential thrombocythemia (ET). A deletion of a G nucleotide in the 5' untranslated region of exon 2 [53] and a G
C transversion in the splice donor site of an intron, which results in exon skipping [54], have been identified. In both instances, the translation of the affected transcripts was markedly increased because of the presence of additional initiation codons upstream of the original ATG. Consequently, the blood concentration of TPO in affected individuals is greater than the normal value. However, the increased platelet counts in patients with familial ET are not always explained by TPO or c-Mpl gene mutations as reported [55].
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Structure of the TPO Protein
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Human TPO cDNA encodes a polypeptide of 353 amino acids. The full-length rHuTPO secreted from mammalian cells after cleavage of the signal peptide consists of 332 amino acids ( Fig. 1). As with various truncated TPO proteins purified from animals, the entire human TPO molecule is not directly required for biological activity [56, 57]. These observations indicate that the TPO receptor (c-Mpl) binds the NH2-terminal half of the TPO molecule, which contains an erythropoietin-like domain and retains biological activity. Two disulfide bonds in the NH2-terminal domain are essential for the biological activity of human [56, 57] and mouse [58] TPO. The COOH-terminal domain of the protein is highly glycosylated, containing six N-linked and multiple O-linked carbohydrate chains, so that the molecular mass of the full-length rHuTPO derived from CHO (Chinese hamster ovary) cells is between 80 and 100 kDa as determined by SDS-polyacrylamide gel electrophoresis (PAGE) [59]. This glycosylated COOH-terminal domain is thought to be necessary for survival of TPO in the circulation. In addition, the sugar chains have recently been shown to be important for the secretion of TPO from cells [60].
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Figure 1. Structure of full-length human TPO. Amino acids are shown in single-letter code, and sites of thrombin cleavage are indicated.
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The sequence of a region (A-B loop to B helix) of the NH2-terminal domain of TPO shows similarity to that of the receptor-binding domain of neurotrophins [61], suggesting that this region might participate in the binding of TPO to its receptor. Recently, the structure-function relations of TPO have been examined with the use of specific antibodies [62, 63] and by mutagenesis [62, 64]. Analysis of the epitope domains of both neutralizing and nonneutralizing antibodies as well as of the effects of mutation of specific TPO residues should provide valuable information in this regard. We have thus shown that at least two regions delineated by Asp8 to Gln28 and Ala60 to Arg117 are important for TPO binding to c-Mpl [63]. Further insights into the structure-function relations of TPO should be provided by determination of the three-dimensional structure of TPO by x-ray crystallography or nuclear magnetic resonance.
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Regulation of the Blood Concentration of TPO and Truncation of the TPO Protein
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Given the large size of the liver, the constitutive low-level production of TPO by each hepatocyte may be sufficient to maintain the normal serum concentration of TPO, which ranges from 0.33 to 1.72 fmol/ml that is equivalent to 12 to 61 pg of full-length TPO polypeptide backbone per milliliter (1 fmol corresponds to 35 pg of the full-length TPO polypeptide backbone) [65]. The number of free TPO molecules in the bloodstream is also determined by the receptor (c-Mpl)-ligand (TPO) association; that is, by "c-Mpl mass" on the surface of platelets and megakaryocytes [3, 66]. Recently, the blood concentration of TPO in patients with various diseases has been extensively studied, and the pivotal relation between platelet number and TPO concentration has been confirmed.
However, in addition to its regulation by the number of TPO molecules in the blood, platelet production might also be modulated at the level of TPO activity, specifically, by proteolysis of TPO. All the TPO proteins purified from animals in the thrombocytopenic state were shown to be truncated (Table 1), although they still exhibited biological activity in vitro [3-6, 8, 56]. Such truncation was apparent whether TPO was purified from plasma by multiple conventional steps in the presence of various common protease inhibitors [3, 4, 67] or by affinity chromatography [5, 6], indicating that blood might contain proteolytic enzymes responsible for the generation of these truncated TPO molecules. We demonstrated the cleavage of rHuTPO by platelets in the absence of any exogenous protease and in the presence of Ca2+, and we showed that this proteolysis was mediated by thrombin [59]. Cleavage by thrombin generates various TPO polypeptide species and results in modulation of the activity of TPO in vitro. TPO was initially and rapidly cleaved by thrombin at Arg191 in the COOH-terminal domain, resulting in an increase in biological activity. The activity was eventually destroyed after prolonged digestion by cleavage at Arg117 in the NH2-terminal domain. Importantly, these two thrombin cleavage sites are conserved among species.
Thus, truncation increases the biological activity of TPO, at least in vitro [57, 59]. One possible physiological explanation for the existence of truncated TPO molecules in blood is included in the model shown in Figure 2. According to this model, the modulation of TPO activity by posttranslational processing, such as by specific proteolysis, contributes to the direct regulation of platelet production. When the platelet number decreases as a result of platelet coagulation, therefore, circulating full-length mature TPO molecules are processed by enzymatic cleavage to truncated forms that are biologically more active. Consequently, megakaryocytopoiesis in bone marrow and platelet production are stimulated. It was important to determine whether the full-length form of the mature endogenous protein actually circulates in the bloodstream in humans, and whether truncated forms of TPO appear in the blood of thrombocytopenic patients. To examine these issues directly, we first analyzed the types of TPO circulating in human blood.
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Molecular Forms of Circulating Human TPO
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Although physicochemical analysis of rHuTPO, including its N- and O-linked carbohydrate structures, has been described [68], a precise characterization of native human TPO has not been available because of its extremely low concentration in blood. To examine the forms of endogenous TPO, we applied a highly sensitive enzyme-linked immunosorbent assay (ELISA) [65] in conjunction with chromatography. This ELISA relies on two antibodies: a neutralizing monoclonal IgG (TN1) that recognizes the peptide region between Ala60 and Arg117 in the NH2-terminal domain of TPO [63] serves as the capture antibody, and polyclonal IgG to full-length rHuTPO is used as the detector; thus, TPO peptide fragments that lack the NH2-terminal domain are not detected by the assay. Because the molecular mass of TPO can vary as a result of proteolytic cleavage of the polypeptide backbone or degradation of the attached sugar chains, it is not sufficient to express the amount of TPO in terms of mass. The ELISA detects various TPO species uniformly, so that the molar TPO concentration values obtained directly reflect the number of TPO molecules. TPO was partially purified from plasma with an affinity gel coupled with antibodies to TPO, treated with SDS, and subjected to gel-filtration chromatography under dissociating conditions in the presence of SDS. Although truncated TPO species were detected, the predominant peak was apparent at a molecular size of about 80 to 100 kDa, similar to that of fully glycosylated rHuTPO consisting of 332 amino acid residues. This peak likely corresponds to the full-length mature form of the protein [69].
In addition to gel-filtration chromatography, we also performed SDS-PAGE and immunoblot analysis, which allowed the visualization of full-length TPO in plasma for the first time for any animal species. Although TPO was partially purified from human plasma by a factor of more than 100,000-fold, a large volume of blood proteins still existed in the fraction. However, the band on the immunoblot was clearly visualized; it showed a molecular mass virtually identical to that of the fully glycosylated rHuTPO produced by CHO cells. A subtle difference in the intensity of staining between the plasma protein and rHuTPO was likely due to differences in composition of the carbohydrate chains rather than to a difference in the polypeptide backbone (A. Matsumoto et al., in preparation).
We next examined the molecular size distribution of TPO in individuals with hematopoietic disorders, including aplastic anemia as an example of a myelodeficient disorder, and ET and polycythemia vera as myeloproliferative disorders. The molecular size distributions of TPO and the number of truncated TPO species did not differ markedly between individuals with these various disorders and normal controls. In individuals with aplastic anemia whose plasma concentrations of TPO initially showed a marked increase, most of the additional TPO in the blood did not appear to be truncated. These results indicated that the amount of truncated TPO in the circulation is not related to the pathophysiology of the hematopoietic disorders examined [69].
We had postulated that circulating TPO might undergo proteolytic processing to generate truncated forms, resulting in an increase in TPO activity, in response to an increased demand for platelet production in the thrombocytopenic state. However, the predominant form of circulating TPO does not appear to be cleaved to an extent sufficient to affect TPO activity. It also remains possible that such posttranslational processing may be locally stimulated in response to thrombocytopenia, in addition to the stimulated production of TPO apparent in bone marrow cells. Furthermore, it is possible that truncated forms of TPO are rapidly removed from the circulation as a result of binding to c-Mpl on platelets, making it difficult to detect their generation in the blood.
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The Life History of TPO
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Since its discovery four years ago, several important observations have shed light on the life history of TPO from production to metabolism ( Fig. 3). TPO is produced in hepatocytes, and to a lesser extent, in other organs. The key trigger responsible for stimulation of TPO production in cells remains to be identified. TPO secreted at sites of production enters the bloodstream and is transported to bone marrow, where it acts directly on megakaryocyte progenitors to influence platelet production. Early hematopoiesis in bone marrow is also supported by TPO through its actions on primitive hematopoietic progenitors. TPO molecules also bind to platelets in the bloodstream, an interaction that influences the blood concentration of TPO. The TPO bound to platelets undergoes proteolysis and subsequent further degradation [70]. In addition to the possible modulation of TPO mRNA abundance in bone marrow, local proteolytic events such as the generation of truncated TPO by thrombin also may potentiate TPO-stimulated platelet production. Finally, free TPO and TPO bound to platelets are cleared from the circulation.
Further insight into the molecular regulation of platelet production should be obtained by determination of the structure of native TPO as well as by investigations into the role of soluble c-Mpl.
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Acknowledgments
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We thank all those who contributed to the discovery of TPO and subsequent research and development at Kirin: J.-I. Tanaka, T. Sudo, A. Shimosaka and K. Asano for their continuous support, and T. Kondo at Hokkaido University and S. Kunishima at Nagoya University for valuable discussion of gene mutations in patients with familial ET.
From THROMBOPOIETIN: FROM MOLECULE TO MEDICINE. STEM CELLS 1998;16(suppl 2):11-19. Contact AlphaMed Press for information about this supplement.
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Abstract
Introduction
