A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

The complete gene for human thrombopoietin ('I'PO) has been cloned by screening a human genomic library using human TPO cDNA as a probe. This gene is 6.2 kb in length and contains six exons and five introns. It is shown that the human genome contains a single copy of the human TPO gene according to Southern blotting analysis. The transcription initiation site was determined by Sl nuclease mapping. The human TPO gene expressed TFQ activity when transfected into COS-1 cells. The human TPO gene has been mapped to chromosome 3q27 by in situ hybridization using a biotin-labeled probe.

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO 163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO 163 (1) predicted the existence of a potent, lineage-specific soluble factor, which they called thrombopoietin (TPO), that stimulates megakaryocytopoiesis and platelet production. It was not until 1994 that unequivocal evidence for the existence of this elusive molecule was provided by the nearly simultaneous isolation and cloning of TPO by five independent research groups (2-6). This cytokine has proven to be a primary factor in megakaryocytopoiesis from megakaryocyte colony formation to platelet production and the differentiation and proliferation of progenitor cells of multiple hematopoietic lineages (7). As such, TPO is being investigated for its potential to treat thrombocytopenia resulting from AIDS and chemotherapy and radiation treatments for cancer and leukemia and for the in vivo and ex vivo expansion of hematopoietic stem cells for bone marrow transplantation.Human TPO (hTPO) is a heavily glycosylated protein with two distinct regions. The 153-residue N-terminal region is homologous to human erythropoietin (EPO) with which it shares 23% sequence identity and is sufficient for receptor binding and signal transduction (2,3,8). The 179-residue C-terminal region has a large number of proline and glycine residues and six N-linked glycosylation sites. Its function is not known, although recent work indicates a role in secretion and protection from proteolysis (9, 10).The TPO receptor c-Mpl was first identified as an oncogene of the murine myeloproliferative leukemia virus (11, 12) that was able to immortalize hematopoietic progenitor cells and was later cloned from human and mouse (13,14). c-Mpl is expressed in some pluripotent hematopoietic stem cells (15) and in the megakaryocyte lineage from progenitor cells to platelets (16). It is a class I cytokine receptor of the hematopoietic superfamily of receptors and signals by the JAK͞STAT, Ras, and mitogenactivated protein kinase pathways (17-21). Class I hematopoietic receptors bind to their cytokine ligands by Ϸ200-aa Ig-like extracellular domains called cytokine receptor homology (CRH) or hematopoietic receptor domains that contain a distinctive WSXWS sequence motif (13).Cytokines possess two distinct interaction sites that bind with differing affinities [high affinity (nanomolar range) and low affinity (micromolar range)] to the same cytokinerecognition surface of the CRH domain. Crystal structures of human EPO and human growth hormone (hGH) in complex with the extracellular CRH domains of their receptors (22, 23) have shown the cytokine-CRH interaction in detail. However, unlike EPO receptor (EPOR) and hGH receptor (hGHR), which have only one CRH domain, c-Mpl belongs to a subset of hematopoietic ...

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