review

Thrombopoietin from beginning to end Ian S. Hitchcock and Kenneth Kaushansky Department of Medicine, Stony Brook University, Stony Brook, NY, USA

Summary In the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench-to-bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan-haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c-Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non-redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c-Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research. Keywords: thrombopoeitin, megakaryocytes, megakaryocytopoiesis, myeloproliferative disease.

History The term erythropoietin (EPO) was first used in the literature in 1906 to describe the humoral substance responsible for erythropoiesis. At the time, blood platelets were barely distinguishable in the best microscopes, prompting their description with the pejorative phrase ‘the dust of the blood’. However, the work of Carnot and of Wright and others in the early 20th century defined the critical role of blood platelets in coagulation and their origin from the marrow megakaryocyte, ultimately leading Kelemen and Tanos (1958) to coin the term thrombopoietin (TPO, also termed THPO) to

Correspondence: Dr Ian S. Hitchcock, Department of Medicine, Stony Brook University, Stony Brook, NY 11794-8151, USA. E-mail: [email protected]

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

describe the humoral substance responsible for platelet production. In the mid-1960s, several groups began attempting to purify TPO from the plasma of thrombocytopenic animals. These early efforts were severely handicapped by inconvenient and insensitive assays for the hormone, and the attempts failed to produce unequivocal proof of the existence of TPO. With the availability of in vitro megakaryocyte differentiation assays in the 1980s, additional purifications were attempted; however, while some claims were made of its biological activities, attempts to produce a cDNA for TPO, the sine qua non of the existence of a protein, also failed. Occasionally in science, a finding from one field, although by itself important, can have a catalytic effect on a seemingly unrelated area of research. The discovery and characterization of the murine myeloproliferative leukaemia virus (MPLV) had such an influence on the search for TPO. The virus causes an acute myeloproliferative syndrome in infected mice (Wendling et al, 1986). In 1990, the responsible oncogene (v-mpl, now termed Mpl) was cloned, and the protooncogene (c-mpl, also now termed Mpl) obtained 2 years later (Souyri et al, 1990; Vigon et al, 1992). Based on the presence of four spatially conserved cysteine residues and a juxtamembrane pentapeptide sequence (Trp-Ser-NaaTrp-Ser), it was immediately evident that c-mpl encodes a member of the haematopoietic cytokine receptor family (Cosman, 1993), which includes the receptors for EPO, interleukin (IL) 3, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL5, IL7, IL9, IL11 and multiple lymphokines. However, when cloned in 1992 its ligand was unknown; c-mpl encoded an ‘orphan receptor’. Based on the cell from which c-mpl was cloned, the bipotent erythroid/megakaryocytic cell line HEL (Long et al, 1990), we, and others, postulated that the c-Mpl (also termed MPL) ligand might be TPO.

The cloning of thrombopoietin While progress in understanding megakaryocyte biology and in purifying TPO was being made using the c-Mpl receptor, others had developed improved bioassays for TPO and continued to make efforts using conventional purification strategies. Using a modified in vivo assay in which the polyploidy of marrow megakaryocytes was examined, Kuter et al (1994) performed an 11-step purification of ovine thrombopoietin, First published online 6 February 2014 doi:10.1111/bjh.12772

Review although the publication of this work did not report the cloning of the corresponding cDNA. In contrast, using an in vitro megakaryocyte-based assay, scientists at Kirin Pharmaceuticals devised a 12-step conventional purification scheme, obtained sufficient purified TPO from the plasma of thrombocytopenic rats to obtain amino acid sequence, and then cloned cDNA for rat TPO and then multiple species of the protein, including the human hormone (Sohma et al, 1994). Using the c-Mpl proto-oncogene product coupled to affinity matrices, scientists at Genetech and at Amgen obtained sufficient purified porcine and canine TPO, respectively, to allow their amino acid sequencing and cDNA cloning (Bartley et al, 1994; de Sauvage et al, 1994). In contrast to the biochemical purifications utilized by these groups, an expression cloning strategy was used by Lok and Kaushansky to obtain cDNA for murine and then human TPO (Lok et al, 1994). In this approach an IL3 dependent cell line was engineered to express the c-Mpl proto-oncogene. In this way, if the c-Mpl ligand was added to the culture, the cells would proliferate in the absence of IL3. The cells were then chemically mutated and autonomously growing sublines were obtained; although most such sublines produced their own IL3, two sublines were producing the c-Mpl ligand. One of these sublines was used as source for cDNA library construction and an additional functional cloning strategy. Remarkably, the THPO cDNAs obtained from three of these groups were identified nearly simultaneously in February 1994. Initial in vitro experiments using the corresponding recombinant proteins demonstrated the effect of TPO on megakaryocyte maturation, and injections into normal mice resulted in impressive increases in peripheral blood platelet counts and marrow megakaryocytes.

Structure of thrombopoietin The cloned human THPO cDNA encodes thrombopoietin (TPO, THPO), a polypeptide of 353 amino acids including the 21 amino acid secretory leader sequence (Kaushansky, 1998). The mature protein consists of two domains. The amino-terminal 154 residue domain is homologous to EPO, like EPO displays a four helix bundle fold (Feese et al, 2004), and binds to the c-MPL receptor (Bartley et al, 1994). And while the amino-terminal domain of TPO is homologus to EPO, the two proteins do not cross compete for binding to the corresponding receptors (Broudy et al, 1997). The carboxyl-terminal domain of TPO bears no resemblance to any known proteins, and is responsible for two functions. First, as it is modified with multiple sites of both N– and O–linked carbohydrate, the carboxyl-terminal domain greatly prolongs the circulatory half-life of the hormone (Harker et al, 1996). Second, the carboxyl-terminal domain serves as an intramolecular chaperone, aiding in the proper folding of the polypeptide into the mature hormone (Linden & Kaushansky, 2000, 2002; Muto et al, 2000). 260

The sites on TPO that physically interact with c-Mpl are fairly clear. Using site-directed alanine scanning mutagenesis to study the functional activities of 40 solvent-exposed residues of the protein, monoclonal antibody epitope mapping and a phage display binding assay system the residues vital for hormone binding to c-Mpl were localized to the first and fourth predicted a-helices of the molecule and the segment connecting the first and second helices, (Pearce et al, 1997). Subsequently, specific residues responsible for binding in the A helix of the protein (Arg10, Lys14 and Arg17) and helix D (His133, Gln132, Lys138 and Phe141) were identified. Of great interest, an Arg17Cys mutation was described in a family with congenital thrombocytopenia and aplastic anaemia (Dasouki et al, 2013).

The regulation of thrombopoietin expression As would be expected for the physiological regulator of platelet production, plasma concentrations of TPO vary inversely with the platelet count in patients with aplastic anaemia (Nichol et al, 1995). Northern blot analyses of multiple organs reveal that TPO is widely expressed, with mRNA being present at highest levels in the liver of normal animals, but the kidney, smooth muscle and a number of other organs also express the gene (Bartley et al, 1994; Lok et al, 1994; de Sauvage et al, 1994). Based on the capacity of platelets to adsorb thrombopoietin from solution, internalize and destroy it, Kuter and Rosenberg (1995) and Fielder et al (1996) have proposed that the regulation of TPO levels in plasma is dependent entirely on platelet numbers; in patients with thrombocytosis, the steady-state level of hormone production is overwhelmed by platelet-mediated TPO metabolism, and so levels are low; in contrast, in patients with thrombocytopenia, little of the hepatic TPO production is adsorbed by platelets, allowing blood levels to rise. Ancillary data for this assertion comes from the Thpo hemizygous mouse (de Sauvage et al, 1996); loss of one allele of Thpo leads to a 40% reduction in platelet counts. If active regulation of PO exists, then this model of hormone regulation posits that increased expression from the second, normal allele should have corrected the reduction in blood levels and re-established a normal platelet count. These arguments notwithstanding, a growing body of evidence suggests that additional mechanisms operate to affect TPO expression. At baseline, it is very difficult to detect specific mRNA in marrow stromal cells. However, transcript levels are substantially increased in marrow stromal cells in response to thrombocytopenia (McCarty et al, 1995; Guerriero et al, 1997; Sungaran et al, 1997; Hirayama et al, 1998). In addition, a number of inflammatory states are associated with TPO levels above that expected for their platelet count (Cerutti et al, 1997; Socolovsky et al, 1999; Tacke et al, 2002) although this finding has not been universal (Wang et al, 1998). The inflammation-induced increase in TPO expression is mediated by IL6; this well-known inflammatory ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

Review response mediator increases TPO production both in vitro and in vivo (Wolber & Jelkmann, 2000; Kaser et al, 2001; Wolber et al, 2001). The mechanism by which TPO production is regulated in marrow stromal cells is less well understood. It is known that platelet a granule PDGF-BB and FGF2 stimulates, and platelet factor 4, thrombospondin and TGFb inhibits TPO production from cultures of marrow stromal cells (Sungaran et al, 2000); on balance, whole platelet extracts suppresses TPO production. Moreover, all trans retinoic acid (ATRA) increases blood levels of TPO, at both the protein and THPO mRNA levels, a response mediated by a retinoic acid response element (RARE) in the 5′ flanking region of the THPO gene (Kinjo et al, 2004). Thus, accumulating evidence indicates that marrow stromal cells can be induced by exogenous stimuli to produce TPO and help explain the response to inflammatory stimuli. The THPO gene displays an unusual 5′ untranslated region structure. Unlike the majority of genes that initiate translation with the first ATG codon present in the THPO mRNA, THPOtranslation initiates at the eighth ATG codon in the transcript, located in the third exon of a full-length mRNA (Chang et al, 1995). As the eighth ATG is out of frame with but embedded in the short open reading frame of the seventh ATG, translation initiation is inefficient (Morris, 1997). Thus, under normal circumstances little TPO protein is produced for any given amount of THPO mRNA. Although it is not certain whether this molecular arrangement has physiological consequences, it is clear from patients with familial essential thrombocythaemia that mutation of the THPO gene in non-coding sequences can lead to enhanced translation efficiency and thrombocytosis. Four cases of autosomal dominant familial thrombocytosis have been linked to mutations in the region surrounding the initiation codon (Wiestner et al, 1998).

Thrombopoietin and megakaryopoiesis As soon as recombinant TPO was available, it became clear that the hormone was able to drive not only megakaryocyte differentiation as previously thought, but also megakaryocyte progenitor cell proliferation (Kaushansky et al, 1994). Thrombopoietin alone is able to produce near maximal numbers of megakaryocyte colony-forming units (CFU-MK) in vitro and, in combination with a number of other hormones and haematopoietic cytokines, such as IL3, IL11 and stem cell factor (SCF, KITLG), it is able to increase the size of the individual colonies. However without TPO, CFU-MK formation is completely aborted (Kaushansky et al, 1995). In addition to being essential in the early stages of megakaryocyte lineage determination and progenitor proliferation, TPO also plays a number of critical roles in megakaryocyte maturation. In vitro, TPO alone is able to increase megakaryocyte size, ploidy and expression of lineage-specific markers glycoprotein (GP)Ib and GPIIb/IIIa (Kaushansky et al, 1994; ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

Zeigler et al, 1994). Furthermore, studies of megakaryocyte ultrastructure show increased demarcation membrane and the formation of platelet granules following culture with TPO, suggesting that the hormone is critical in priming the megakaryocyte for thrombopoiesis (Kaushansky et al, 1995). However, the final stages of platelet formation and release appear to be TPO-independent, and instead rely on one or more as yet unidentified components of the plasma.

Thrombopoietin and the haematopoietic stem cell With the availability of recombinant TPO and improved methods to purify haematopoietic stem and progenitor cell populations, further study indicated that the biological properties of TPO were not restricted to the megakaryocyte lineage. In vitro studies showed that TPO augmented survival and proliferation of CD34+ haematopoietic progenitor cells, especially when used in combination with IL3 or SCF (Ku et al, 1996; Sitnicka et al, 1996). These data were further supported by the generation of transgenic mice lacking either Thpo or c-Mpl (Mpl). Transplantation of normal haematopoietic stem cell (HSCs) into Thpo / mice resulted in a 15–20-fold decrease in stem cell expansion compared to transplantation into wild-type controls (Fox et al, 2002). Additionally, c–Mpl / mice exhibit a significant reduction in haematopoietic progenitors of all lineages (Alexander et al, 1996a) and have only 10–20% of the normal number of HSCs (Solar et al, 1998). More recent work revealed an intriguing paracrine role for TPO/c–Mpl in maintaining quiescent Tie2+ HSCs at the endosteal niche (Yoshihara et al, 2007). They demonstrated that osteoblasts at the niche release TPO, supporting the quiescence of c-Mpl-expressing HSCs and inhibition of this interaction reduce the number of HSCs at the niche. We now know that TPO also plays a role in HSC maintenance in humans, as congenital forms of amegakaryocytic thrombocytopenia often develop as a result of c-MPL (MPL) mutations, with the majority of such children developing aplastic anaemia in the first 1–5 years of life due to a diminishing pool of HSCs (Ihara et al, 1999; van den Oudenrijn et al, 2000; Tonelli et al, 2000; Ballmaier et al, 2001).

Thrombopoietin and platelets Despite significant advances in our understanding of the cellular mechanisms and function of TPO signalling in HSCs, progenitors and megakaryocytes, the role of TPO and c-Mpl in platelet function remains somewhat elusive. Importantly, platelets express c-Mpl and most of the machinery required for TPO signal transduction, including; JAK2, STAT3, STAT5, AKT and Ras (see below). Superphysiological amounts of TPO (>100 ng/ml) are able to directly activate platelet aggregation in vitro (reviewed in Akkerman, 2006), whilst more physiological concentrations prime platelets for 261

Review stimulation with other agonists, possibly by increasing activity of Ras (van Willigen et al, 2000). TPO also has significant effects on platelet adhesion under flow. Low TPO concentrations (0
01–1 ng/ml) accelerate firm platelet adhesion to von Willebrand factor and prevent de-attachment at higher flow rates, suggesting that TPO may be important in thrombus formation (Van Os et al, 2003), highlighting the need for further in–depth in vivo studies.

Molecular mechanisms of thrombopoietin c-Mpl is a member of the type I cytokine receptor family along with receptors for a number of interleukins, colony stimulating factors and EPO. As with many growth factor receptors, ligand-mediated tyrosine phosphorylation of both the receptor and associated proteins is an integral part of the cellular response to haematopoietic cytokine action (Tojo et al, 1987; Satoh et al, 1992). However, similar to other type I cytokine receptors, c-Mpl lacks intrinsic kinase activity; instead it recruits and directly associates with cytoplasmic kinases to mediate activation of downstream signalling proteins. We now know that the initial stages of type I cytokine receptor phosphorylation and subsequent intracellular signalling rely on Janus kinases (JAKs). JAK2 associates with c-Mpl prior to the receptor being trafficked to the membrane. Indeed, c–Mpl/JAK2 association stabilizes expression of the receptor, increasing the presence of c-Mpl at the membrane (Tong et al, 2006). Extensive research over the last two decades using both c-Mpl-expressing cell lines and primary cells has identified a variety of different proteins activated in the signalling milieu following TPO stimulation. Due to the relatively close homology between c-Mpl and EpoR, initial studies focused on the JAK/STAT pathway and identified JAK2 and TYK2 as the immediate kinases that bind to c-Mpl and become activated following ligand binding (Bacon et al, 1995; Drachman et al, 1995), leading to the activation of both STAT3 and 5 (Drachman & Kaushansky, 1997; Socolovsky et al, 1999). TPO stimulation also causes the phosphorylation and formation of the Shc-Grb2-SOS adaptor protein complex (Sasaki et al, 1995; Hill et al, 1996), activation of phosphatases SHIP and SHPTP-2 and stimulation of both the phosphoinositide3 kinase (PI3K)/AKT (Sattler et al, 1997; Miyakawa et al, 2001) and Raf-1/MAP kinase pathways (Nagata & Todokoro, 1995; Yamada et al, 1995). A number of regions in c-Mpl have been identified as important in receptor function and downstream signalling. In cell lines, truncation of the receptor at 69 residues from the transmembrane domain ablated activation of Shc and STAT3, but the receptor was still able to support cell growth with reduced activation of STAT5, PI3K and ERK1/2 (Alexander et al, 1996b). In vivo studies confirmed these findings when a similarly truncated c-Mpl receptor (at 61 residues from the transmembrane domain) was knocked in to the endogenous c-Mpl locus in mice, and the resting platelet 262

count was shown to be normal (Luoh et al, 2000). However, recovery following myelosupression was significantly attenuated in mice expressing the truncated receptor, suggesting that signalling via the distal regions of c-Mpl is important for maximal megakaryopoiesis or increased platelet production following injury. Given its importance throughout haematopoiesis and the numerous intracellular signalling pathways it activates, stringent negative regulatory mechanisms are required to ensure TPO signalling is tightly controlled. There are two main mechanisms by which TPO regulates its own activity; activation of negative regulators and internalization and degradation of its activated receptor. Of the proteins activated or upregulated in response to TPO, LYN, LNK and suppressors of cytokine signalling (SOCS) have all been identified as mediating important negative feedback. Chemical inhibition of LYN, a member of the Src kinase family, in c–Mplexpressing cell lines, enhanced TPO-mediated ERK1/2 activation and proliferation, and promoted megakaryocyte differentiation in bone marrow cells, suggesting that LYN negatively regulates TPO signalling (Lannutti & Drachman, 2004). This finding was further supported by a Lyn-deficient mouse model, which exhibits increased megakaryopoiesis and a greater signalling response to TPO stimulation (Lannutti et al, 2006). Overexpression of LNK, an adaptor protein, negatively regulates TPO-mediated activation of STAT5 and ERK1/2 and inhibits cell growth in cell lines, as well as attenuating megakaryopoiesis when transiently overexpressed in haematopoietic progenitor cells (Tong & Lodish, 2004). Furthermore, Lnk / mice exhibit greatly increased numbers of bone marrow megakaryocytes and CFU-MKs, and enhanced TPO-mediated activation of ERK1/2, AKT, STAT3 and STAT5 in isolated CD41+ megakaryocytes. Increased expression of SOCS proteins dramatically inhibits TPO signalling by directly binding to and down regulating the c-Mpl receptor and downstream signalling proteins (reviewed in Croker et al, 2008). TPO has been shown to significantly increase SOCS-3 levels in c-Mpl-expressing cell lines, while IFN-a upregulates SOCS-1 to negatively regulate TPO signalling (Wang et al, 2000). In addition to activating negative regulators, TPO-stimulation results in a rapid internalization and degradation of c-Mpl. Adaptor protein-2 (AP2) associates with the c-Mpl intracellular motif Y591RRL, driving clathrin coat formation and endocytosis, while an identical intracellular motif Y521RRL appears to target the internalized receptor to the lysosome (Hitchcock et al, 2008). As well as being subject to lysosomal degradation, activated c-Mpl is also ubiquitinated and targeted for degradation by the proteasome. Following TPO stimulation, polyubiquitination occurs at two intracellular lysine residues, K553 and K573, leading to degradation of the receptor. This is ablated when both residues are mutated to arginine (c-MplK553+573R) (Saur et al, 2010). siRNA knockdown of the E3 ubiquitin ligase CBL reduced TPOmediated c-Mpl ubiquitination, indicating its role in the ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

Review process, although ubiquitination was not completely prevented, suggesting other E3 ubiquitin ligases may also be involved.

Therapeutic potential of thrombopoietin The dramatic increase in our understanding of the structure and function of TPO in the mid-1990’s lead to the generation of thrombopoietic agents for human therapeutic use. The first generation of thrombopoietic drugs included recombinant human (rh) TPO and pegylated megakaryocyte growth and development factor (PEG-rHuMGDF). RhTPO is a glycosylated, full-length peptide produced by CHO cells that is identical the endogenous TPO. When administered intravenously to cancer patients, rhTPO resulted in an increase in platelet numbers after 5 d, which peaked at days 10–14 (Vadhan-Raj et al, 1997, 2000). PEG-rHuMGDF is a pegylated product produced by E. coli containing the 163 amino acid amino- terminus of human TPO. Administered subcutaneously, PEG-rHuMGDF has similar effects to rhTPO (reviewed in Kuter, 2007). The effectivness of rhTPO and PEG-rHuMGDF have been used in clinical trials with varying success. In three studies involving 101 patients that received carboplatinum-based chemotherapeutic regimens, platelet counts returned to baseline significantly faster in the patients that also received rhTPO or PEG-rHuMGDF, compared to those receiving placebo (Fibbe et al, 1995; Molineux et al, 1996; Vadhan-Raj et al, 1997). In a later study, Vadhan-Raj et al (2000) compared the effects of a first cycle of carboplatin-based therapy without the hormone, to a second cycle which was immediately followed by a single dose of rhTPO. Baseline platelet values and the number of days with ≤20 9 109 platelets/l were both significantly improved by the addition of rhTPO. Moreover, the majority of the patients in this study required platelet transfusions after the first cycle of chemotherapy, but only 26% of patients required transfusion following the second cycle with additional TPO treatment. Given the relative success in increasing platelet counts following myeloablation, PEG–rHuMGDF was subsequently used in clinical trials aimed at increasing platelet yields in normal individuals donating platelets by apheresis. Enhancing platelet collections in donors could allow a single donor plateletpheresis to be used for multiple recipients, or alternatively increase the mean platelet increment when the cells were subsequently infused into a single recipient. Although these initial data proved promising (Kuter et al, 1997), all clinical trials were halted after 13 out of 538 healthy volunteers receiving PEGrHuMGDF developed thrombocytopenia as a result of antigenic response to the recombinant drug that cross-reacted with endogenous TPO (Li et al, 2001; Basser et al, 2002; Kuter & Begley, 2002). As a result, all trials involving the first generation of TPO drugs were discontinued. Focus then shifted to the development of novel thrombopoietic agents that could be easily produced and that would ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

not evoke an antigenic response. Cwirla et al (1997) identified a 14 amino acid peptide (Ile-Glu-Gly-Pro-Thr-LeuArg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) which binds to c-Mpl with high affinity, but will not evoke an immune response since it does not share homology with TPO. Four of these linear peptides were then covalently fused to the Fc fragment of the human IgG1 heavy chain, increasing longevity and activity in the circulation (Wang et al, 2004; Bussel et al, 2006). This ‘peptibody’, named romiplostim (also known as AMG-531 and marketed as N-plate; Amgen) bound to the cMpl receptor and successfully stimulated the same downstream signalling pathways as rhTPO (Broudy & Lin, 2004). Romiplostim given subcutaneously increased platelet counts in 80% of patients with idiopathic thrombocytopenic purpura (ITP) and long-term studies have shown that the drug is well tolerated with no signs of antigenic response (reviewed in Kuter, 2007). Romiplostim was approved by the US Food and Drug Administration (FDA) in 2008 for ITP patients that have failed to respond to traditional treatments. Another TPO mimetic, eltrombopag (also known as SB497115 and marketed as Promacta/Revolade; GlaxoSmith Kline), was developed following a large-scale cell-based screening of non-peptide, orally available TPO mimetics (Duffy et al, 2001, 2002a,b; Erickson-Miller et al, 2005; Safonov et al, 2006). Unlike peptide mimetics, eltrombopag does not compete with TPO for binding to c-Mpl, instead interacting with the transmembrane domain of the receptor leading to c-Mpl activation as a result of conformational change (Kim et al, 2007). Platelet counts were increased in ITP patients receiving eltrombopag in clinical trials, although the degree of platelet increase was significantly lower than patients given romiplostim (Kuter, 2008). Eltrombopag was also approved by the FDA in 2008 as a second-line treatment for patients with ITP. Some concerns still exist regarding long-term use of these agents, especially regarding the potential development of bone marrow reticulin formation or outright fibrosis, although these side effects are rare. The development of bone marrow fibrosis has been reported in a small number of patients following romiplostim treatment, but was not severe enough to cause changes in blood counts (Kuter et al, 2007). Additionally, patients with liver failure taking eltrombopag have shown increased portal vein thrombosis following invasive procedures, suggesting that the drug may cause low level platelet activation (Afdhal et al, 2012). The development of the thrombopoietic agents, romiplostim and eltrombopag, and their success in treating patients with ITP, paves the way for the generation of other haematopoietic growth factor mimetics in the future.

The pathobiology of thrombopoietin and its receptor A significant fraction of patients with congenital and acquired states of thrombocytosis and thrombocytopenia are 263

Review due to disorders of TPO or its receptor. Mutations of the THPO gene cause congenital thrombocytopenia and aplastic anaemia and congenital thrombocytosis. Likewise, mutations of c-MPL cause congenital and acquired thrombocytosis and thrombocytopenia. Several distinct mutations of the THPO gene have been identified in patients with congenital thrombocythaemia (Jorgensen et al, 1998; Kondo et al, 1998; Wiestner et al, 1998; Ghilardi & Skoda, 1999). While distinct, each of the mutations thus far described result in greatly enhanced efficiency of translation of THPO mRNA, increasing TPO levels and hence, causing thrombocytosis. To understand the nature of these mutations, one must understand the determinants of mRNA translation efficiency. Over 90% of all proteins are translated from the first ATG codon that appears in the corresponding mRNA. In contrast, TPO is normally translated from the 8th ATG codon. While this arrangement alone does not greatly diminish the efficiency of translation, the fact the 8th ATG resides within an out of frame, 7th open reading frame greatly diminishes translation efficiency. Should a ribosme bind the 7th ATG, it will translate that polypeptide, passing by the 8th ATG and then terminating. Since ribosomes cannot then reinitiate upstream of the (7th cistron) stop codon, only downstream, that ribosome translation cycle does not produce TPO. Only if the 7th ATG is skipped does a ribosome translate TPO from the 8th ATG. All of the described THPO mutants that cause thrombocythaemia do so by improving translation efficiency, either by converting an upstream ATG (ATG 5 or ATG7) into the THPO initiating start codon (by mutation-induced alternate splicing or by a frame shifting deletion), or by creation of a premature stop codon in open reading frame 7, allowing ribosomal re-initiation at ATG8 (Jorgensen et al, 1998; Kondo et al, 1998; Wiestner et al, 1998; Ghilardi et al, 1999; Cazzola & Skoda, 2000). Myeloproliferative neoplasms originate in a pluripotent haematopoietic stem/progenitor cell. Consequently, if growth factors or their receptor play a role in these disorders, then that cytokine receptor must be expressed in haematopoietic stem cells. As noted in the sections above, c-Mpl is present on the repopulating HSC, and genetic elimination of murine Thpo or c-Mpl lead to the conclusion that it plays a critical, non-redundant role in these cells. Following upon the characterization of a constitutively active mutant of the EpoR, Alexander et al (1995) engineered constitutively active receptor mutants of c-Mpl by substituting cysteine residues into the dimer interface domain of the receptor. Factor-dependent haematopoietic cells transduced with the mutant receptor displayed constitutive phosphorylation of both c-Mpl and the signal transduction molecules implicated in c-Mpl function, suggesting a potential for contributing to the tumorigenicity of the mutant cells (Alexander et al, 1995). Subsequent work by Onishi et al (1996) selected for an activating mutation in the transmembrane domain of c-Mpl by using a combination of retrovirus-mediated gene 264

transfer and polymerase chain reaction-driven random mutagenesis. These experimental conclusions were confirmed in man, when activating mutations of c-MPL were identified adjacent to the transmembrane domain of the receptor in patients with congenital thrombocytosis or acquired myeloproliferative neoplasms. An activating mutation of c-MPL at was first identified by Pikman et al (2006) at Trp515 in patients with acquired essential thrombocythaemia or primary myelofibrosis, and a mutation at Asn505 [the same residue, as was identified in the murine studies of Alexander et al, 1995)] was found to be causative in several patients with hereditary thrombocythaemia (Liu et al, 2009). Just as gain of fucntion mutants of THPO or c-MPL cause thrombocytosis or myeloproliferative neoplasms, loss of function mutants of these two genes have been described that cause thrombocytopenia or aplastic anaemia. Homozygous or compound heterozygous missense or nonsense mutation of c-MPL has been found in most patients described with congenital amegakaryocytic thrombocytopenia (CAMT) (Muraoka et al, 1997; Ihara et al, 1999; van den Oudenrijn et al, 2000; Tonelli et al, 2000; Ballmaier et al, 2001). Infants with this disorder usually present with excessive bleeding in the perinatal period, although occasionally the diagnosis is delayed a short time. Bone marrow examination at birth reveals normal cellularity, although only a few megakaryocytes are initially found, and those present are unusually small and hypolobated. In time, however, the marrow becomes aplastic, (Ballmaier et al, 2001) necessitating stem cell transplantation for survival. The majority of such children fail to express c-Mpl on their platelet surfaces, usually due to the presence of non-sense or severe missense codons in the extracellular domain of the receptor, although mutations have been described in nearly every exon of the c-MPL gene. Until recently c-MPL mutations were the only known cause of congenital amegakaryocytic thrombocytopenia. It was rather perplexing why mutations of THPO were not described, as genetic elimination of murine Thpo is a near phenocopy of genetic elimination of murine c-Mpl. Moreover, the development of anti-TPO antibodies can lead to severe thrombocytopenia and occasionally pancytopenia (Basser et al, 2002). Very recently, a family was described who bear two different mutations of the THPO gene (Dasouki et al, 2013). Inheritance of one allele (predicting an unpaired Cys residue within the secretory leader sequence of TPO or a Cys residue that resides at the interface between the two sites on which TPO binds to c-Mpl) is associated with congenital thrombocytopenia, whereas inheritance of one of each mutant allele causes congenital aplastic anaemia. Although the majority of patients with immune thrombocytopenic purpura display enhanced megakaryopoiesis secondary to peripheral immune destruction of platelets, usually due to an autoantibody to platelet GPIIb/IIIa or GPIb, careful platelet turnover studies reveals that not all patients have rapid platelet elimination (Ballem et al, 1987). In such patients it is possible that reduced production of platelets ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

Review might be due to antibodies against thrombopoietin or its receptor. More recent study has borne out this possibility, both as de novo disease, and in response to administration of a genetically modified form of thrombopoietin (Li et al, 2001; Chang et al, 2003; Katsumata et al, 2003). In both of these situations, the clue to the unusual pathophysiology was reduced megakaryopoiesis in the marrow of the patients with presumed immune thrombocytopenic purpura. Soon after the c-Mpl proto-oncogene was identified it became clear that it was expressed on a sizable proportion of blast cells from patients with myeloid malignancies (Vigon et al, 1993). Although there is little evidence that disorders of c-Mpl are involved in the generation of acute myeloid leukaemia or myelodysplastic syndromes, its expression on the surface of these cells appears to adversely affect the response characteristics of the cells and the overall survival of patients (Bouscary et al, 1995; Wetzler et al, 1997; Schroder et al,

References Afdhal, N.H., Giannini, E.G., Tayyab, G., Mohsin, A., Lee, J.W., Andriulli, A., Jeffers, L., McHutchison, J., Chen, P.J., Han, K.H., Campbell, F., Hyde, D., Brainsky, A. & Theodore, D. (2012) Eltrombopag before procedures in patients with cirrhosis and thrombocytopenia. New England Journal of Medicine, 367, 716–724. Akkerman, J.W. (2006) Thrombopoietin and platelet function. Seminars in Thrombosis and Hemostasis, 32, 295–304. Alexander, W.S., Metcalf, D. & Dunn, A.R. (1995) Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity. EMBO Journal, 14, 5569–5578. Alexander, W.S., Roberts, A.W., Nicola, N.A., Li, R. & Metcalf, D. (1996a) Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood, 87, 2162–2170. Alexander, W.S., Maurer, A.B., Novak, U. & Harrison-Smith, M. (1996b) Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation. EMBO Journal, 15, 6531–6540. Bacon, C.M., Tortolani, P.J., Shimosaka, A., Rees, R.C., Longo, D.L. & O’Shea, J.J. (1995) Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3. FEBS Letters, 370, 63–68. Ballem, P.J., Segal, G.M., Stratton, J.R., Gernsheimer, T., Adamson, J.W. & Slichter, S.J. (1987) Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance. The Journal of Clinical Investigation, 80, 33–40. Ballmaier, M., Germeshausen, M., Schulze, H., Cherkaoui, K., Lang, S., Gaudig, A., Krukemeier, S., Eilers, M., Strauss, G. & Welte, K. (2001)

2000). In contrast, however, the adverse prognosis of expression of c-Mpl did not extend to chronic myeloid leukaemia (Kaban et al, 2000). The identification of the c-Mpl receptor and the cloning of TPO nearly 20 years ago provided the tools necessary to develop critical insights into the biology of one of the most enigmatic cells in human biology, the marrow megakaryocyte. Additional studies lead to the conclusion that TPO is more than just a thrombopoietin, with important and nonredundant effects of the HSC. These findings have expanded our understanding of the molecular mechanisms of haematopoiesis, and catalysed the developed of effective TPO-mimics, that are approved for use in certain patients with thrombocytopenia. And while these discoveries have been quite gratifying, many more scientific and clinical questions remain, which will form the basis of many important studies in the future.

c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood, 97, 139–146. Bartley, T.D., Bogenberger, J., Hunt, P., Li, Y.S., Lu, H.S., Martin, F., Chang, M.S., Samal, B., Nichol, J.L., Swift, S., Johnson, M.J., Hsu, R.-Y., Parker, V.P., Suggs, S., Skrine, J.D., Merewether, L.A., Clogston, C., Hsu, E., Hokom, M.M., Hornkohl, A., Choi, E., Pangelinan, M., Sun, Y., Mar, V., McNinch, J., Simonet, L., Jacobsen, F., Xie, C., Shutter, J., Chute, H., Basu, R., Selander, L., Trollinger, D., Sieu, L., Padilla, D., Trail, G., Elliott, G., Izumi, R., Covey, J., Crouse, A., Garcia, W., Xu, J., Del Castillo, J., Biron, S., Cole, M.C.-T., Hu, R., Pacifici, T., Ponting, I., Saris, C., Wen, D., Yung, Y.P., Lin, H. & Rosselman, R.A. (1994) Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell, 77, 1117–1124. Basser, R.L., O’Flaherty, E., Green, M., Edmonds, M., Nichol, J., Menchaca, D.M., Cohen, B. & Begley, C.G. (2002) Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor. Blood, 99, 2599–2602. Bouscary, D., Preudhomme, C., Ribrag, V., Melle, J., Viguie, F., Picard, F., Guesnu, M., Fenaux, P., Gisselbrecht, S. & Dreyfus, F. (1995) Prognostic value of c-mpl expression in myelodysplastic syndromes. Leukemia, 9, 783–788. Broudy, V.C. & Lin, N.L. (2004) AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl. Cytokine, 25, 52–60. Broudy, V.C., Lin, N.L., Sabath, D.F., Papayannopoulou, T. & Kaushansky, K. (1997) Human platelets display high-affinity receptors for thrombopoietin. Blood, 89, 1896–1904. Bussel, J.B., Kuter, D.J., George, J.N., McMillan, R., Aledort, L.M., Conklin, G.T., Lichtin, A.E., Lyons, R.M., Nieva, J., Wasser, J.S., Wiznitzer, I., Kelly, R., Chen, C.F. & Nichol, J.L. (2006)

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP. New England Journal of Medicine, 355, 1672–1681. Cazzola, M. & Skoda, R.C. (2000) Translational pathophysiology: a novel molecular mechanism of human disease. Blood, 95, 3280–3288. Cerutti, A., Custodi, P., Duranti, M., Noris, P. & Balduini, C.L. (1997) Thrombopoietin levels in patients with primary and reactive thrombocytosis. British Journal of Haematology, 99, 281–284. Chang, M.S., McNinch, J., Basu, R., Shutter, J., Hsu, R.Y., Perkins, C., Mar, V., Suggs, S., Welcher, A., Li, L., Lu, H., Bartley, T., Hunt, P., Martin, F., Samal, B. & Bogenberger, J. (1995) Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene. Journal of Biological Chemistry, 270, 511–514. Chang, M., Nakagawa, P.A., Williams, S.A., Schwartz, M.R., Imfeld, K.L., Buzby, J.S. & Nugent, D.J. (2003) Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro. Blood, 102, 887–895. Cosman, D. (1993) The hematopoietin receptor superfamily. Cytokine, 5, 95–106. Croker, B.A., Kiu, H. & Nicholson, S.E. (2008) SOCS regulation of the JAK/STAT signalling pathway. Seminars in Cell & Developmental Biology, 19, 414–422. Cwirla, S.E., Balasubramanian, P., Duffin, D.J., Wagstrom, C.R., Gates, C.M., Singer, S.C., Davis, A.M., Tansik, R.L., Mattheakis, L.C., Boytos, C.M., Schatz, P.J., Baccanari, D.P., Wrighton, N.C., Barrett, R.W. & Dower, W.J. (1997) Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science, 276, 1696–1699. Dasouki, M.J., Rafi, S.K., Olm-Shipman, A.J., Wilson, N.R., Abhyankar, S., Ganter, B., Furness, L.M., Fang, J., Calado, R.T. & Saadi, I. (2013) Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with

265

Review autosomal recessive aplastic anemia. Blood, 122, 3440–3449. Drachman, J.G. & Kaushansky, K. (1997) Dissecting the thrombopoietin receptor: functional elements of the Mpl cytoplasmic domain. Proceedings of the National Academy of Sciences of the United States of America, 94, 2350–2355. Drachman, J.G., Griffin, J.D. & Kaushansky, K. (1995) The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl. Journal of Biological Chemistry, 270, 4979–4982. Duffy, K.J., Darcy, M.G., Delorme, E., Dillon, S.B., Eppley, D.F., Erickson-Miller, C., Giampa, L., Hopson, C.B., Huang, Y., Keenan, R.M., Lamb, P., Leong, L., Liu, N., Miller, S.G., Price, A.T., Rosen, J., Shah, R., Shaw, T.N., Smith, H., Stark, K.C., Tian, S.S., Tyree, C., Wiggall, K.J., Zhang, L. & Luengo, J.I. (2001) Hydrazinonaphthalene and azonaphthalene thrombopoietin mimics are nonpeptidyl promoters of megakaryocytopoiesis. Journal of Medicinal Chemistry, 44, 3730–3745. Duffy, K.J., Price, A.T., Delorme, E., Dillon, S.B., Duquenne, C., Erickson-Miller, C., Giampa, L., Huang, Y., Keenan, R.M., Lamb, P., Liu, N., Miller, S.G., Rosen, J., Shaw, A.N., Smith, H., Wiggall, K.J., Zhang, L. & Luengo, J.I. (2002a) Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 2. Rational design of naphtho[1,2-d]imidazole thrombopoietin mimics. Journal of Medicinal Chemistry, 45, 3576–3578. Duffy, K.J., Shaw, A.N., Delorme, E., Dillon, S.B., Erickson-Miller, C., Giampa, L., Huang, Y., Keenan, R.M., Lamb, P., Liu, N., Miller, S.G., Price, A.T., Rosen, J., Smith, H., Wiggall, K.J., Zhang, L. & Luengo, J.I. (2002b) Identification of a pharmacophore for thrombopoietic activity of small, non-peptidyl molecules. 1. Discovery and optimization of salicylaldehyde thiosemicarbazone thrombopoietin mimics. Journal of Medicinal Chemistry, 45, 3573–3575. Erickson-Miller, C.L., DeLorme, E., Tian, S.S., Hopson, C.B., Stark, K., Giampa, L., Valoret, E.I., Duffy, K.J., Luengo, J.L., Rosen, J., Miller, S.G., Dillon, S.B. & Lamb, P. (2005) Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist. Experimental Hematology, 33, 85–93. Feese, M.D., Tamada, T., Kato, Y., Maeda, Y., Hirose, M., Matsukura, Y., Shigematsu, H., Muto, T., Matsumoto, A., Watarai, H., Ogami, K., Tahara, T., Kato, T., Miyazaki, H. & Kuroki, R. (2004) Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proceedings of the National Academy of Sciences of the United States of America, 101, 1816–1821. Fibbe, W.E., Heemskerk, D.P., Laterveer, L., Pruijt, J.F., Foster, D., Kaushansky, K. & Willemze, R. (1995) Accelerated reconstitution of platelets and erythrocytes after syngeneic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice. Blood, 86, 3308– 3313.

266

Fielder, P.J., Gurney, A.L., Stefanich, E., Marian, M., Moore, M.W., Carver-Moore, K. & de Sauvage, F.J. (1996) Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets. Blood, 87, 2154–2161. Fox, N., Priestley, G., Papayannopoulou, T. & Kaushansky, K. (2002) Thrombopoietin expands hematopoietic stem cells after transplantation. The Journal of Clinical Investigation, 110, 389– 394. Ghilardi, N. & Skoda, R.C. (1999) A single-base deletion in the thrombopoietin (TPO) gene causes familial essential thrombocythemia through a mechanism of more efficient translation of TPO mRNA. Blood, 94, 1480–1482. Ghilardi, N., Wiestner, A., Kikuchi, M., Ohsaka, A. & Skoda, R.C. (1999) Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene. British Journal of Haematology, 107, 310–316. Guerriero, A., Worford, L., Holland, H.K., Guo, G.R., Sheehan, K. & Waller, E.K. (1997) Thrombopoietin is synthesized by bone marrow stromal cells. Blood, 90, 3444–3455. Harker, L.A., Marzec, U.M., Hunt, P., Kelly, A.B., Tomer, A., Cheung, E., Hanson, S.R. & Stead, R.B. (1996) Dose–response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood, 88, 511–521. Hill, R.J., Zozulya, S., Lu, Y.L., Hollenbach, P.W., Joyce-Shaikh, B., Bogenberger, J. & Gishizky, M.L. (1996) Differentiation induced by the cMpl cytokine receptor is blocked by mutant Shc adaptor protein. Cell Growth & Differentiation, 7, 1125–1134. Hirayama, Y., Sakamaki, S., Matsunaga, T., Kuga, T., Kuroda, H., Kusakabe, T., Sasaki, K., Fujikawa, K., Kato, J., Kogawa, K., Koyama, R. & Niitsu, Y. (1998) Concentrations of thrombopoietin in bone marrow in normal subjects and in patients with idiopathic thrombocytopenic purpura, aplastic anemia, and essential thrombocythemia correlate with its mRNA expression of bone marrow stromal cells. Blood, 92, 46–52. Hitchcock, I.S., Chen, M.M., King, J.R. & Kaushansky, K. (2008) YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation. Blood, 112, 2222–2231. Ihara, K., Ishii, E., Eguchi, M., Takada, H., Suminoe, A., Good, R.A. & Hara, T. (1999) Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia. Proceedings of the National Academy of Sciences of the United States of America, 96, 3132–3136. Jorgensen, M.J., Raskind, W.H., Wolff, J.F., Bachrach, H.R. & Kaushansky, K. (1998) Familial thrombocytosis associated with overproduction of thrombopoietin due to a novel splice donor site mutation. Blood, 92, 205a. Kaban, K., Kantarjian, H., Talpaz, M., O’Brien, S., Cortes, J., Giles, F.J., Pierce, S. & Albitar, M. (2000) Expression of thrombopoietin and its receptor (c-mpl) in chronic myelogenous

leukemia: correlation with disease progression and response to therapy. Cancer, 88, 570–576. Kaser, A., Brandacher, G., Steurer, W., Kaser, S., Offner, F.A., Zoller, H., Theurl, I., Widder, W., Molnar, C., Ludwiczek, O., Atkins, M.B., Mier, J.W. & Tilg, H. (2001) Interleukin-6 stimulates thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis. Blood, 98, 2720– 2725. Katsumata, Y., Suzuki, T., Kuwana, M., Hattori, Y., Akizuki, S., Sugiura, H. & Matsuoka, Y. (2003) Anti-c-Mpl (thrombopoietin receptor) autoantibody-induced amegakaryocytic thrombocytopenia in a patient with systemic sclerosis. Arthritis and Rheumatism, 48, 1647–1651. Kaushansky, K. (1998) Thrombopoietin. New England Journal of Medicine, 339, 746–754. Kaushansky, K., Lok, S., Holly, R.D., Broudy, V.C., Lin, N., Bailey, M.C., Forstrom, J.W., Buddle, M.M., Oort, P.J., Hagen, F.S., Roth, G.J., Papayannopoulou, T. & Foster, D.C. (1994) Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature, 369, 568–571. Kaushansky, K., Broudy, V.C., Lin, N., Jorgensen, M.J., McCarty, J., Fox, N., Zucker-Franklin, D. & Lofton-Day, C. (1995) Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development. Proceedings of the National Academy of Sciences of the United States of America, 92, 3234–3238. Kelemen, E., Cserhati, I. & Tanos, B. (1958) Demonstration and some properties of human thrombopoietin in thrombocythemic sera. Acta Haematologica, 20, 350–355. Kim, M.J., Park, S.H., Opella, S.J., Marsilje, T.H., Michellys, P.Y., Seidel, H.M. & Tian, S.S. (2007) NMR structural studies of interactions of a small, nonpeptidyl Tpo mimic with the thrombopoietin receptor extracellular juxtamembrane and transmembrane domains. Journal of Biological Chemistry, 282, 14253–14261. Kinjo, K., Miyakawa, Y., Uchida, H., Kitajima, S., Ikeda, Y. & Kizaki, M. (2004) All-trans retinoic acid directly up-regulates thrombopoietin transcription in human bone marrow stromal cells. Experimental Hematology, 32, 45–51. Kondo, T., Okabe, M., Sanada, M., Kurosawa, M., Suzuki, S., Kobayashi, M., Hosokawa, M. & Asaka, M. (1998) Familial essential thrombocythemia associated with one-base deletion in the 5′-untranslated region of the thrombopoietin gene. Blood, 92, 1091–1096. Ku, H., Yonemura, Y., Kaushansky, K. & Ogawa, M. (1996) Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. Blood, 87, 4544–4551. Kuter, D.J. (2007) New thrombopoietic growth factors. Blood, 109, 4607–4616. Kuter, D.J. (2008) Thrombopoietin and thrombopoietin mimetics in the treatment of thrombocytopenia. Annual Review of Medicine, 60, 33.31–33.14.

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

Review Kuter, D.J. & Begley, C.G. (2002) Recombinant human thrombopoietin: basic biology and evaluation of clinical studies. Blood, 100, 3457– 3469. Kuter, D.J. & Rosenberg, R.D. (1995) The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood, 85, 2720–2730. Kuter, D.J., Beeler, D.L. & Rosenberg, R.D. (1994) The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production. Proceedings of the National Academy of Sciences of the United States of America, 91, 11104–11108. Kuter, D., McCullough, J., Romo, J., DiPersio, J., Peterson, R., Armstrong, S., Menchaca, D., Tomita, D. & Goodnough, L.T. (1997) Treatment of platelet (PLT) donors with pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) increases circulating PLT counts (CTS) and PLT apheresis yields and increases platelet increments in recipients of PLT transfusions. Blood, 90, 2579. Kuter, D.J., Bain, B., Mufti, G., Bagg, A. & Hasserjian, R.P. (2007) Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres. British Journal of Haematology, 139, 351–362. Lannutti, B.J. & Drachman, J.G. (2004) Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of hematopoietic cell lines and primary megakaryocytic progenitors. Blood, 103, 3736–3743. Lannutti, B.J., Minear, J., Blake, N. & Drachman, J.G. (2006) Increased megakaryocytopoiesis in Lyn-deficient mice. Oncogene, 25, 3316–3324. Li, J., Yang, C., Xia, Y., Bertino, A., Glaspy, J., Roberts, M. & Kuter, D.J. (2001) Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood, 98, 3241–3248. Linden, H.M. & Kaushansky, K. (2000) The glycan domain of thrombopoietin enhances its secretion. Biochemistry, 39, 3044–3051. Linden, H.M. & Kaushansky, K. (2002) The glycan domain of thrombopoietin (TPO) acts in trans to enhance secretion of the hormone and other cytokines. Journal of Biological Chemistry, 277, 35240–35247. Liu, K., Martini, M., Rocca, B., Amos, C.I., Teofili, L., Giona, F., Ding, J., Komatsu, H., Larocca, L.M. & Skoda, R.C. (2009) Evidence for a founder effect of the MPL-S505N mutation in eight Italian pedigrees with hereditary thrombocythemia. Haematologica, 94, 1368–1374. Lok, S., Kaushansky, K., Holly, R.D., Kuijper, J.L., Lofton-Day, C.E., Oort, P.J., Grant, F.J., Heipel, M.D., Burkhead, S.K., Kramer, J.M., Bell, L.A., Sprecher, C.A., Blumberg, H., Johnson, R., Prunkard, D., Ching, A.F.T., Mathewes, S.L., Bailey, M.C., Forstrom, J.W., Buddle, M.M., Osborn, S.G., Evans, S.J., Sheppard, P.O., Presnell, S.R., O’Hara, P.J., Hagen, F.S., Roth, G.J. & Foster, D.C. (1994) Cloning and expression of murine thrombopoietin cDNA and

stimulation of platelet production in vivo. Nature, 369, 565–568. Long, M.W., Heffner, C.H., Williams, J.L., Peters, C. & Prochownik, E.V. (1990) Regulation of megakaryocyte phenotype in human erythroleukemia cells. The Journal of Clinical Investigation, 85, 1072–1084. Luoh, S.M., Stefanich, E., Solar, G., Steinmetz, H., Lipari, T., Pestina, T.I., Jackson, C.W. & de Sauvage, F.J. (2000) Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo. Molecular and Cellular Biology, 20, 507–515. McCarty, J.M., Sprugel, K.H., Fox, N.E., Sabath, D.E. & Kaushansky, K. (1995) Murine thrombopoietin mRNA levels are modulated by platelet count. Blood, 86, 3668–3675. Miyakawa, Y., Rojnuckarin, P., Habib, T. & Kaushansky, K. (2001) Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes. Journal of Biological Chemistry, 276, 2494–2502. Molineux, G., Hartley, C.A., McElroy, P., McCrea, C. & McNiece, I.K. (1996) Megakaryocyte growth and development factor stimulates enhanced platelet recovery in mice after bone marrow transplantation. Blood, 88, 1509–1514. Morris, D.R. (1997) Cis-Acting mRNA Structures in Gene-Specific Translational Control. In: PostTranscriptional Gene Regulation (eds. by J.B. Harford & D.R. Morris), pp. 165–180. WileyLiss, Inc, New York. Muraoka, K., Ishii, E., Tsuji, K., Yamamoto, S., Yamaguchi, H., Hara, T., Koga, H., Nakahata, T. & Miyazaki, S. (1997) Defective response to thrombopoietin and impaired expression of c-mpl mRNA of bone marrow cells in congenital amegakaryocytic thrombocytopenia. British Journal of Haematology, 96, 287–292. Muto, T., Feese, M.D., Shimada, Y., Kudou, Y., Okamoto, T., Ozawa, T., Tahara, T., Ohashi, H., Ogami, K., Kato, T., Miyazaki, H. & Kuroki, R. (2000) Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a “shuttle” peptide to help secretion. Journal of Biological Chemistry, 275, 12090–12094. Nagata, Y. & Todokoro, K. (1995) Thrombopoietin induces activation of at least two distinct signaling pathways. FEBS Letters, 377, 497–501. Nichol, J.L., Hokom, M.M., Hornkohl, A., Sheridan, W.P., Ohashi, H., Kato, T., Li, Y.S., Bartley, T.D., Choi, E., Bogenberger, J. (1995) Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapyinduced thrombocytopenia. The Journal of Clinical Investigation, 95, 2973–2978. Onishi, M., Kinoshita, S., Morikawa, Y., Shibuya, A., Phillips, J., Lanier, L.L., Gorman, D.M., Nolan, G.P., Miyajima, A. & Kitamura, T. (1996) Applications of retrovirus-mediated expression cloning. Experimental Hematology, 24, 324–329.

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268

van den Oudenrijn, S., Bruin, M., Folman, C.C., Peters, M., Faulkner, L.B., de Haas, M. & von dem Borne, A.E. (2000) Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia. British Journal of Haematology, 110, 441–448. Pearce, K.H. Jr, Potts, B.J., Presta, L.G., Bald, L.N., Fendly, B.M. & Wells, J.A. (1997) Mutational analysis of thrombopoietin for identification of receptor and neutralizing antibody sites. Journal of Biological Chemistry, 272, 20595–20602. Pikman, Y., Lee, B.H., Mercher, T., McDowell, E., Ebert, B.L., Gozo, M., Cuker, A., Wernig, G., Moore, S., Galinsky, I., DeAngelo, D.J., Clark, J.J., Lee, S.J., Golub, T.R., Wadleigh, M., Gilliland, D.G. & Levine, R.L. (2006) MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Medicine, 3, e270. Safonov, I.G., Heerding, D.A., Keenan, R.M., Price, A.T., Erickson-Miller, C.L., Hopson, C.B., Levin, J.L., Lord, K.A. & Tapley, P.M. (2006) New benzimidazoles as thrombopoietin receptor agonists. Bioorganic & Medicinal Chemistry Letters, 16, 1212–1216. Sasaki, K., Odai, H., Hanazono, Y., Ueno, H., Ogawa, S., Langdon, W.Y., Tanaka, T., Miyagawa, K., Mitani, K., Yazaki, Y. & Hirai, H. (1995) TPO/c-mpl ligand induces tyrosine phosphorylation of multiple cellular proteins including proto-oncogene products, Vav and c-Cbl, and Ras signaling molecules. Biochemical and Biophysical Research Communications, 216, 338–347. Satoh, T., Uehara, Y. & Kaziro, Y. (1992) Inhibition of interleukin 3 and granulocyte-macrophage colony-stimulating factor stimulated increase of active ras.GTP by herbimycin A, a specific inhibitor of tyrosine kinases. Journal of Biological Chemistry, 267, 2537–2541. Sattler, M., Salgia, R., Durstin, M.A., Prasad, K.V. & Griffin, J.D. (1997) Thrombopoietin induces activation of the phosphatidylinositol-3′ kinase pathway and formation of a complex containing p85PI3K and the protooncoprotein p120CBL. Journal of Cellular Physiology, 171, 28–33. Saur, S.J., Sangkhae, V., Geddis, A.E., Kaushansky, K. & Hitchcock, I.S. (2010) Ubiquitination and degradation of the thrombopoietin receptor c-Mpl. Blood, 115, 1254–1263. de Sauvage, F.J., Hass, P.E., Spencer, S.D., Malloy, B.E., Gurney, A.L., Spencer, S.A., Darbonne, W.C., Henzel, W.J., Wong, S.C., Kuang, W.J., Oles, K.J., Hultgren, B., Solberg, L.A. Jr, Goeddel, D.V. & Eaton, D.L. (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature, 369, 533–538. de Sauvage, F.J., Carver-Moore, K., Luoh, S.M., Ryan, A., Dowd, M., Eaton, D.L. & Moore, M.W. (1996) Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. Journal of Experimental Medicine, 183, 651–656. Schroder, J.K., Kolkenbrock, S., Tins, J., KasimirBauer, S., Seeber, S. & Schutte, J. (2000) Analysis of thrombopoietin receptor (c-mpl) mRNA

267

Review expression in de novo acute myeloid leukemia. Leukemia Research, 24, 401–409. Sitnicka, E., Lin, N., Priestley, G.V., Fox, N., Broudy, V.C., Wolf, N.S. & Kaushansky, K. (1996) The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood, 87, 4998–5005. Socolovsky, M., Fallon, A.E., Wang, S., Brugnara, C. & Lodish, H.F. (1999) Fetal anemia and apoptosis of red cell progenitors in Stat5a / 5b / mice: a direct role for Stat5 in Bcl-X(L) induction. Cell, 98, 181–191. Sohma, Y., Akahori, H., Seki, N., Hori, T., Ogami, K., Kato, T., Shimada, Y., Kawamura, K. & Miyazaki, H. (1994) Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Letters, 353, 57–61. Solar, G.P., Kerr, W.G., Zeigler, F.C., Hess, D., Donahue, C., de Sauvage, F.J. & Eaton, D.L. (1998) Role of c-mpl in early hematopoiesis. Blood, 92, 4–10. Souyri, M., Vigon, I., Penciolelli, J.F., Heard, J.M., Tambourin, P. & Wendling, F. (1990) A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell, 63, 1137–1147. Sungaran, R., Markovic, B. & Chong, B.H. (1997) Localization and regulation of thrombopoietin mRNa expression in human kidney, liver, bone marrow, and spleen using in situ hybridization. Blood, 89, 101–107. Sungaran, R., Chisholm, O.T., Markovic, B., Khachigian, L.M., Tanaka, Y. & Chong, B.H. (2000) The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells. Blood, 95, 3094–3101. Tacke, F., Trautwein, C., Zhao, S., Andreeff, M., Manns, M.P., Ganser, A. & Schoffski, P. (2002) Quantification of hepatic thrombopoietin mRNA transcripts in patients with chronic liver diseases shows maintained gene expression in different etiologies of liver cirrhosis. Liver, 22, 205–212. Tojo, A., Kasuga, M., Urabe, A. & Takaku, F. (1987) Vanadate can replace interleukin 3 for transient growth of factor-dependent cells. Experimental Cell Research, 171, 16–23. Tonelli, R., Scardovi, A.L., Pession, A., Strippoli, P., Bonsi, L., Vitale, L., Prete, A., Locatelli, F., Bagnara, G.P. & Paolucci, G. (2000) Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT). Human Genetics, 107, 225–233.

268

Tong, W. & Lodish, H.F. (2004) Lnk inhibits Tpompl signaling and Tpo-mediated megakaryocytopoiesis. Journal of Experimental Medicine, 200, 569–580. Tong, W., Sulahian, R., Gross, A.W., Hendon, N., Lodish, H.F. & Huang, L.J. (2006) The membrane-proximal region of the thrombopoietin receptor confers its high surface expression by JAK2-dependent and -independent mechanisms. Journal of Biological Chemistry, 281, 38930– 38940. Vadhan-Raj, S., Murray, L.J., Bueso-Ramos, C., Patel, S., Reddy, S.P., Hoots, W.K., Johnston, T., Papadopolous, N.E., Hittelman, W.N., Johnston, D.A., Yang, T.A., Paton, V.E., Cohen, R.L., Hellmann, S.D., Benjamin, R.S. & Broxmeyer, H.E. (1997) Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Annals of Internal Medicine, 126, 673–681. Vadhan-Raj, S., Verschraegen, C.F., Bueso-Ramos, C., Broxmeyer, H.E., Kudelka, A.P., Freedman, R.S., Edwards, C.L., Gershenson, D., Jones, D., Ashby, M. & Kavanagh, J.J. (2000) Recombinant human thrombopoietin attenuates carboplatininduced severe thrombocytopenia and the need for platelet transfusions in patients with gynecologic cancer. Annals of Internal Medicine, 132, 364–368. Van Os, E., Wu, Y.P., Pouwels, J.G., Ijsseldijk, M.J., Sixma, J.J., Akkerman, J.W., De Groot, P.G. & Van Willigen, G. (2003) Thrombopoietin increases platelet adhesion under flow and decreases rolling. British Journal of Haematology, 121, 482–490. Vigon, I., Mornon, J.P., Cocault, L., Mitjavila, M.T., Tambourin, P., Gisselbrecht, S. & Souyri, M. (1992) Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor 3superfamily. Proceedings of the National Academy of Sciences of the United States of America, 89, 5640–5644. Vigon, I., Dreyfus, F., Melle, J., Viguie, F., Ribrag, V., Cocault, L., Souyri, M. & Gisselbrecht, S. (1993) Expression of the c-mpl proto-oncogene in human hematologic malignancies. Blood, 82, 877–883. Wang, J.C., Chen, C., Novetsky, A.D., Lichter, S.M., Ahmed, F. & Friedberg, N.M. (1998) Blood thrombopoietin levels in clonal thrombocytosis and reactive thrombocytosis. American Journal of Medicine, 104, 451–455. Wang, Q., Miyakawa, Y., Fox, N. & Kaushansky, K. (2000) Interferon-alpha directly represses megakaryopoiesis by inhibiting thrombopoietin-induced

signaling through induction of SOCS-1. Blood, 96, 2093–2099. Wang, B., Nichol, J.L. & Sullivan, J.T. (2004) Pharmacodynamics and pharmacokinetics of AMG 531, a novel thrombopoietin receptor ligand. Clinical Pharmacology and Therapeutics, 76, 628– 638. Wendling, F., Varlet, P., Charon, M. & Tambourin, P. (1986) MPLV: a retrovirus complex inducing an acute myeloproliferative leukemic disorder in adult mice. Virology, 149, 242–246. Wetzler, M., Baer, M.R., Bernstein, S.H., Blumenson, L., Stewart, C., Barcos, M., Mrozek, K., Block, A.W., Herzig, G.P. & Bloomfield, C.D. (1997) Expression of c-mpl mRNA, the receptor for thrombopoietin, in acute myeloid leukemia blasts identifies a group of patients with poor response to intensive chemotherapy. Journal of Clinical Oncology, 15, 2262–2268. Wiestner, A., Schlemper, R.J., van der Maas, A.P. & Skoda, R.C. (1998) An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nature Genetics, 18, 49–52. van Willigen, G., Gorter, G. & Akkerman, J.W. (2000) Thrombopoietin increases platelet sensitivity to alpha-thrombin via activation of the ERK2-cPLA2 pathway. Thrombosis and Haemostasis, 83, 610–616. Wolber, E.M. & Jelkmann, W. (2000) Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B. Journal of Interferon and Cytokine Research, 20, 499–506. Wolber, E.M., Fandrey, J., Frackowski, U. & Jelkmann, W. (2001) Hepatic thrombopoietin mRNA is increased in acute inflammation. Thrombosis and Haemostasis, 86, 1421–1424. Yamada, M., Komatsu, N., Okada, K., Kato, T., Miyazaki, H. & Miura, Y. (1995) Thrombopoietin induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in a human thrombopoietin-dependent cell line. Biochemical and Biophysical Research Communications, 217, 230–237. Yoshihara, H., Arai, F., Hosokawa, K., Hagiwara, T., Takubo, K., Nakamura, Y., Gomei, Y., Iwasaki, H., Matsuoka, S., Miyamoto, K., Miyazaki, H., Takahashi, T. & Suda, T. (2007) Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell Stem Cell, 1, 685–697. Zeigler, F.C., de Sauvage, F., Widmer, H.R., Keller, G.A., Donahue, C., Schreiber, R.D., Malloy, B., Hass, P., Eaton, D. & Matthews, W. (1994) In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells. Blood, 84, 4045–4052.

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 259–268