First printed in R&D Systems' 1996 Catalog.
Thrombopoietin (Tpo) is a factor that stimulates the development of megakaryocyte precursors of platelets, leading to an increase in the number of circulating platelets in a manner that is analogous to the erythropoietin (Epo) stimulation of erythroid precursors. The existence of such a factor was demonstrated 35 years ago by injecting the serum of rats depleted of platelets into normal rats; the recipient rats responded with an increased number of circulating platelets.1 A long and tedious attempt to obtain thrombopoietin (reviewed in ref. 2) led to the cloning of the Mpl ligand in 1994 by five independent groups.3-7 The Mpl ligand is generally accepted as thrombopoietin (reviewed in ref. 8).
Mpl (the thrombopoietin receptor) was discovered as the product of the gene c-mpl, the normal homolog of the oncogene v-mpl, the transforming gene of a murine myeloproliferative leukemia virus.9,10 Antisense oligonucleotides of c-mpl inhibited colony-forming ability of megakaryocyte progenitors11, and "knockout" mice deficient in c-mpl had an 85% reduction in circulating platelets12 The Mpl receptor proved to be the key to identification and cloning of thrombopoietin.3-7
Mature human Tpo, obtained from the primary translation product by cleavage of a 21-residue signal peptide, is a 332-amino acid (aa) residue polypeptide.3,8,13 It consists of at least two regions. The N-terminal portion (about 154 aa residues) has sequence and general structural homology with Epo. There is 23% sequence identity, with an equal amount of conservative substitution. The two essential cysteines of Epo are conserved in Tpo, and the two factors have a similar distribution of the four alpha helical regions of hematopoietic cytokines. There is a high degree of sequence homology (> 70%) between human, porcine, canine, murine and rat Tpos. The homology is higher in the N-terminal (Epo-like) region than the C-terminal region of the molecule. The C-terminal portion of Tpo has no sequence similarity with other known proteins.
The mass of Tpo predicted from the sequence is about 35 kDa, but masses reported from measurements of material in serum or in culture fluid from recombinant cells vary from 18-70 kDa. This has led to suggestions8,13 that Tpo is highly glycosylated and that it is susceptible to proteolytic processing. There is evidence for the plausibility of each of these suggestions. First, there are six potential N-linked glycosylation sites in the C-terminal half of Tpo. While the effects of glycosylation on thrombopoietic activity or in vivo stability are not known, it is possible that the glycosylated C-terminal half of Tpo confers a longer half-life in plasma, a known effect of glycosylation of Epo. Second, there are two Arg-Arg motifs that are potential sites of proteolytic processing. The first (at residues 153 and 154) is just after the Epo-like N-terminal domain, and the second (at residues 245 and 246) is half way through the C-terminal domain.
As mentioned earlier, the receptor for Tpo is the proto-oncogene c-Mpl, a homolog of an envelope protein of the myeloproliferative leukemia virus.9,10 The human and murine forms of the receptor have been cloned and sequenced and display approximately 81% identity at the amino acid sequence level.9,10,14 Both the human and murine sources show the presence of multiple forms of the Mpl receptor produced as a result of alternative mRNA splicing, including apparently soluble forms lacking transmembrane and cytoplasmic domains.9,10,14 The human and murine Mpl receptors show the conserved sequences and structural organization characteristic of the members of the hematopoietin superfamily of cytokine receptors.15,16 No concensus sequence for kinase activity is found in the cytoplasmic domain of these receptors.9 The intracellular domain of the Mpl receptor has been found to consist of two distinct domains that interact with different signal transduction pathways. One domain is required for induction of Shc phosphorylation and c-fos mRNA synthesis indicating involvement of the Ras signal transduction pathway.17 A separate cytoplasmic domain is involved with activation of the JAK2 kinase and subsequent activation of STAT protein. Apparently conflicting reports suggest that either a STAT-5 like protein18 or STAT1 and STAT317 are the particular STAT proteins involved.
Identification of the types of cells that express the Tpo receptor (Mpl) give a clue to the action of Tpo. Expression of the c-mpl gene has been detected by reverse transcription-polymerase chain reaction (RT-PCR) in pluripotent hematopoietic stem cells (CD34+CD38-), but among lineage-committed cell lines or mature cells its expression is detected only in cells of the megakaryocytic lineage.11 Immunofluorescence studies by flow cytometry give generally confirming results.19 In a population of total hematopoietic cells, Mpl is detected on very few, and most of those are of the megakayocytic lineage, with the brightest staining on polyploid megakaryocytes. Attempts to show that pluripotent stem cells expressed Mpl have been unsuccessful, raising the possibility that RT-PCR experiments detect expression of c-mpl in only a few megakaryocyte progenitors within the population of primitive stem cells. This is an important point because of its relevance to the question of whether Tpo acts to induce differentiation into the megakaryocyte lineage. With murine cells, an AA4+/Sca- population, which is capable of long-term reconstitution of lethally irradiated mice (and thus is pluripotent), is about 50% Mpl+, supporting the idea that Mpl is expressed on primitive stem cells.20
Definition of the thrombopoietic activity of Tpo is difficult because of the multiple cytokines known to have general hematopoietic, including thrombopoietic, activity. Research in this area was recently reviewed by Kaushansky21 The major thrombopoietic cytokines are Stem Cell Factor (SCF), IL-3, IL-6 and IL-11. The question is, where in the sequence from proliferation of stem cells to differentiation into committed cell lines to maturation into mature platelets does Tpo exert its effect and is there synergy with the other thrombopoietic cytokines? The multipotent stem cell differentiates into a committed cell that assumes properties of a megakaryocyte and begins endocytosis with ploidy increasing to 32 to 64, finally fragmenting into platelets. Tpo apparently affects this entire thrombopoietic process8,19-21, with a stronger effect in the latter stages. Both the numbers of megakaryocytes, and their ploidy, are greater in cultures with Tpo than with either SCF or IL-3, IL-6 or IL-11. Indeed, high concentrations of IL-6 or IL-11 in combination with high concentrations of SCF or IL-3 were required to approach the activity of Tpo alone.8,21 Furthermore, complete maturation of megakaryocytes with formation of platelets is observed only in the presence of Tpo.8
The obvious clinical interest is in the possible use of recombinant Tpo to treat thrombocytopenic states produced by pathological conditions or therapeutic treatments such as irradiation.