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1.  Structure-activity analysis of binding kinetics for NMDA receptor competitive antagonists: the influence of conformational restriction. 
British Journal of Pharmacology  1991;104(1):207-221.
1. The kinetics of action of 17 structurally related NMDA receptor competitive antagonists were measured under voltage clamp in mouse hippocampal neurones. Analysis of the response to rapid changes in antagonist concentration during constant application of agonist was used to estimate microscopic association (kon) and dissociation (koff) rate constants for antagonist binding, assuming a two-equivalent site model for competitive antagonism. Dose-inhibition curves were analysed to estimate antagonist equilibrium dissociation constants. 2. For a series of 11 omega-phosphono, alpha-amino acids kon and koff varied 26 and 107 fold respectively. Rapid association and dissociation rate constants were obtained for flexible antagonist molecules such as D-2-amino-7-phosphonoheptanoic acid (D-AP7): kon 1.4 x 10(7) M-1 s-1; koff 20.3 s-1. For conformationally restrained molecules such as 3S,4aR,6S,8aR-6-phosphonomethyl-decahydroisoquinoline- 3-carboxylic acid (LY 235959), association and dissociation rate constants were much slower: kon 1.1 x 10(6) M-1 s-1; koff 0.2 s-1. For the D- and L-isomers of 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) estimates for kon were similar, but for the L-isomer koff was 10 fold faster than for the D-isomer. 3. For 2-amino-5-phosphonopentanoic acid (AP5) and its piperidine derivative cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755), an increase in chain length of two methylene groups between the omega-phosphono and alpha-carboxylate moieties caused a 1.6 to 1.8 fold decrease in kon with little change in koff. In contrast, for AP5, CPP and its omega-carboxylate analogue, addition of a double bond close to the phosphonate moiety caused a 1.3 to 1.6 fold increase in kon. 4. For antagonists with an omega-tetrazole moiety, kon and koff were 2.8-4.6 times faster than for the parent omega-phosphono compounds. A similar, but smaller increase in kon and koff was observed for antagonists with an omega-carboxylate moiety. 5. The slow kinetics of action of potent NMDA receptor antagonists were not an artefact of buffered diffusion. In neurones equilibrated with 200 microM D-AP7, 2 microM LY 235959 and 10 microM NMDA, a transient agonist response was recorded following a rapid switch to D-AP7-free solution. This can only be explained by differences in the binding kinetics of AP7 and LY 235959, since at equilibrium, with these concentrations, either antagonist essentially eliminates the agonist response to 10 microM NMDA.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC1908275  PMID: 1686203
2.  Human thrombopoietin reduces myocardial infarct size, apoptosis, and stunning following ischaemia/reperfusion in rats 
Cardiovascular research  2007;77(1):44-53.
Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart.
Methods and results
We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 μg/kg iv in vivo. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial KATP channels (5-HD), and sarcolemmal KATP channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 μg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period.
A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and KATP channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.
PMCID: PMC2958050  PMID: 18006466
Ischaemia; thrombopoietin; protein kinases; infarction; K-ATP channel
3.  The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD) 
Nucleic Acids Research  2006;34(8):2355-2363.
Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets.
PMCID: PMC1458284  PMID: 16679454
4.  Extracellular Domain N-Glycosylation Controls Human Thrombopoietin Receptor Cell Surface Levels 
The thrombopoietin receptor (TpoR) is a type I transmembrane protein that mediates the signaling functions of thrombopoietin (Tpo) in regulating megakaryocyte differentiation, platelet formation, and hematopoietic stem cell renewal. We probed the role of each of the four extracellular domain putative N-glycosylation sites for cell surface localization and function of the receptor. Single N-glycosylation mutants at any of the four sites were able to acquire the mature N-glycosylated pattern, but exhibited a decreased Tpo-dependent JAK2–STAT response in stably transduced Ba/F3 or Ba/F3-JAK2 cell lines. The ability of JAK2 to promote cell surface localization and stability of TpoR required the first N-glycosylation site (Asn117). In contrast, the third N-glycosylation site (Asn298) decreased receptor maturation and stability. TpoR mutants lacking three N-glycosylation sites were defective in maturation, but N-glycosylation on the single remaining site could be detected by sensitivity to PNGaseF. The TpoR mutant defective in all four N-glycosylation sites was severely impaired in plasma membrane localization and was degraded by the proteasome. N-glycosylation receptor mutants are not misfolded as, once localized on the cell surface in overexpression conditions, they can bind and respond to Tpo. Our data indicate that extracellular domain N-glycosylation sites regulate in a combinatorial manner cell surface localization of TpoR. We discuss how mutations around TpoR N-glycosylation sites might contribute to inefficient receptor traffic and disease.
PMCID: PMC3355985  PMID: 22649382
cytokine receptor; thrombopoietin; JAK2; N-glycosylation; signal transduction; endoglycosidase H; ER maturation; cell surface traffic
5.  Regulation of polymorphonuclear cell activation by thrombopoietin. 
Journal of Clinical Investigation  1997;99(7):1576-1584.
Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation.
PMCID: PMC507977  PMID: 9120001
6.  Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects 
Annals of Hematology  2010;89(Suppl 1):75-85.
Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety. Hence, a strategy to detect potential antibody formation to the drug and to related endogenous molecules can be useful. The immunogenicity assessment strategy involved both the detection and characterization of binding and neutralizing antibodies. The method for detection was based on a surface plasmon resonance biosensor platform using the Biacore 3000. Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in a neutralization assay. Serum samples from 225 subjects with immune thrombocytopenic purpura (ITP) dosed with romiplostim and 45 ITP subjects dosed with placebo were tested for romiplostim and TPO antibodies. Prior to romiplostim treatment, 17 subjects (7%) tested romiplostim antibody positive and 12 subjects (5%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, 11% of the subjects exhibited binding antibodies against romiplostim and 5% of the subjects with ITP showed binding antibodies against TPO. The antibodies against romiplostim did not cross-react with TPO and vice versa. No cases of anti-TPO neutralizing antibodies were detected in romiplostim-treated subjects. The incidence of anti-romiplostim neutralizing antibodies to romiplostim was 0.4% (one subject); this subject tested negative at the time of follow-up 4 months later. No impact on platelet profiles were apparent in subjects that had antibodies to romiplostim to date. In summary, administration of romiplostim in ITP subjects resulted in the development of a binding antibody response against romiplostim and TPO ligand. One subject developed a neutralizing antibody response to romiplostim that impacted the platelet counts of this subject. No neutralizing antibodies to endogenous TPO were observed.
PMCID: PMC2900600  PMID: 19484238
Immune thrombocytopenic purpura (ITP); Romiplostim; Immunogenicity; TPO; Platelet
7.  Pharmacodynamics-Mediated Drug Disposition (PDMDD) and Precursor Pool Lifespan Model for Single Dose of Romiplostim in Healthy Subjects 
The AAPS Journal  2010;12(4):729-740.
The objective of this study was to characterize the pharmacokinetics and pharmacodynamics (PK-PD) of romiplostim after single-dose administration in healthy subjects. The mean serum romiplostim concentrations (PK data) and mean platelet counts (PD data) collected from 32 subjects receiving a single intravenous (0.3, 1 and 10 μg/kg) or subcutaneous (0.1, 0.3, 1, and 2 μg/kg) dose were fitted simultaneously to a mechanistic PK-PD model based on pharmacodynamics-mediated drug disposition (PDMDD) and a precursor pool lifespan concept. The two-compartment PK model incorporated receptor-mediated endocytosis and linear mechanisms as parallel elimination pathways. The maximal concentration of receptors (assumed to be proportional to the platelet count), the equilibrium dissociation constant, and the first-order internalization rate constant for endocytosis of the drug-receptor complex were 0.022 fg/platelet, 0.131 ng/mL, and 0.173 h−1, respectively. Romiplostim concentration stimulates the production of platelet precursors via the Hill function, where the SC50 was 0.052 ng/mL and Smax was 11.2. The estimated precursor cell and platelet lifespans were 5.9 and 10.5 days, respectively. Model-based simulations revealed that the romiplostim exposure and the platelet response are both dependent on the dose administered and the baseline platelet counts. Also, weekly dosing produced a sustained PD response while dosing intervals ≥2 weeks resulted in fluctuating platelet counts. Thus, the mechanistic PK-PD model was suitable for describing the romiplostim PK-PD interplay (PDMDD), the dose-dependent platelet stimulation, and the lifespans of thrombopoietic cell populations.
PMCID: PMC2976980  PMID: 20963535
lifespan model; pharmacodynamics-mediated drug disposition (PDMDD); platelets; romiplostim; thrombopoiesis receptor agonist
8.  Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling 
Cell Cycle  2011;10(11):1838-1844.
Thrombopoietin (TPO) and its receptor c-Mpl are essential in the regulation of the hematopoietic stem and progenitors cells as well as for the differentiation of megakaryocytes into mature platelets. Once TPO binds to its receptor, an intracellular signaling process is initiated through Janus kinase (JAK-2)-induced phosphorylation of the c-Mpl intracellular domain. Although some protein mediators that transmit the effects of TPO have been identified, many remain undiscovered. Using an unbiased approach with peptide microarrays that contained virtually every Src Homology (SH)2 and Phosphotyrosine Binding (PT B) domains in the human genome, we discovered a previously unreported interaction between c-Mpl at phospho-Tyrosine631 (pY631) and Tensin2, a protein for which limited information is available. Confirming the findings of the microarrays, we discovered that Tensin2 co-precipitates with a pY631 bearing peptide. Furthermore, we found that Tensin2 becomes phosphorylated in a TPO-dependent manner. The functional consequence of Tensin2 was tested via knockdown of Tensin2, which dramatically decreased TPO-dependent cellular proliferation of UT7-TPO cell line as well as their activation of Akt signaling. These studies affirm the use of these arrays as an unbiased screening tool of proteinprotein interactions. We conclude that Tensin2 is an important new mediator in TPO/c-Mpl pathway and has a positive affect on cellular growth, at least in part through its effect on the PI3K/Akt signaling.
PMCID: PMC3233486  PMID: 21527831
tensin2; thrombopoietin; c-Mpl; signal transduction; cellular proliferation
9.  Circulating thrombopoietin levels in normal healthy blood donors and in aplastic anemia patients in relation to disease severity 
Thrombopoietin (TPO) is the key hematopoietic growth factor regulating the production of platelets from bone marrow megakaryocytes and maintaining platelet hemostasis. This study was done to find any relationship between the levels of thrombopoietin and the severity of disease in patients with aplastic anemia.
Materials and Methods:
Serum samples were collected from 52 patients with a confirmed diagnosis of aplastic anemia and 45 normal healthy blood donors of both sexes over a period of 2 years, and TPO was estimated by using commercially available TPO-specific-enzyme-linked immunosorbent assay.
The median TPO level of 1190 pg/ml (range 625-7651 pg/ml) in aplastic anemia patients was significantly higher than the median TPO level of 121.1 pg/ml (81.25-237.7 pg/ml) in normal healthy blood donors (P = 0.000). No significant difference was observed in TPO levels of male and female patients (P = 0.453). The median TPO concentrations observed in very severe aplastic anemia, severe aplastic anemia, and nonsevere aplastic anemia were 2765 pg/ml (range 625-6451 pg/ml), 1190 pg/ml (range 672.1-7651 pg/ml), and 1111.5 pg/ml (range 761.1-2289.2 pg/ml), respectively. TPO in patients of very severe aplastic anemia was significantly higher than patients of nonsevere aplastic anemia (P = 0.043), with no significant relation among rest of the groups.
TPO levels in aplastic anemia patients were significantly higher than in healthy blood donors; however, in aplastic anemia patients TPO levels were significantly higher only in patients with very severe disease.
PMCID: PMC4339937  PMID: 25722577
Aplastic anemia; blood donor; megakaryocytes; thrombopoietin
10.  Biology and Chemistry of Thrombopoietic Agents 
Seminars in hematology  2010;47(3):243-248.
Endogenous thrombopoietin (eTPO) regulates platelet production by increasing the number, ploidy and maturation rate of bone marrow megakaryocytes. Early attempts to treat thrombocytopenia by the administration of recombinant TPO were successful but were complicated by the development of antibodies to one of the recombinant proteins. Two new TPO mimetics have recently been approved by the Food and Drug Administration for the treatment of immune thrombocytopenia (ITP). Romiplostim is a peptide TPO mimetic composed of an IgG Fc fragment to which are attached four 14-amino acid TPO peptides that activate the TPO receptor by binding to the extracytoplasmic domain just like eTPO. Romiplostim is administered as a weekly subcutaneous injection. Eltrombopag is a non-peptide TPO mimetic that is a 442 Da drug that binds to a transmembrane site on the TPO receptor and thereby activates it. It is administered daily as an oral tablet. Administration of both romiplostim and eltrombopag to healthy volunteers produced a dose-dependent rise in platelet count beginning on day 5 and peaking at days 12-15. Both have been highly effective in increasing the platelet count in patients with ITP and are currently being studied in the treatment of other thrombocytopenic conditions (MDS, chemotherapy, liver disease).
PMCID: PMC2904343  PMID: 20620435
11.  Inhibition of GSK-3β promotes survival and proliferation of megakaryocytic cells through a β-catenin-independent pathway 
Cellular signalling  2008;20(12):2317-2323.
The interaction of thrombopoietin (TPO) with its receptor c-Mpl initiates intracellular signals that are critical for megakaryopoiesis. Previously we and others have shown that TPO activates PI3K and Akt and that this pathway is important for megakaryocyte growth. Here, we investigate the importance of the Akt substrate glycogen synthase kinase (GSK)-3β in TPO signaling. GSK-3β is phosphorylated and inhibited by Akt as part of the PI3K pathway. GSK-3β can also be inhibited by Wnt signaling through a distinct mechanism, leading to reduced phosphorylation and accumulation of the transcription factor β-catenin. Therefore, we asked if TPO and Wnt3a can both inhibit GSK-3β in megakaryocytic cells, and if they can act synergistically to promote cell growth. Although both TPO and specific chemical inhibitors of GSK-3β result in increased survival and proliferation in a megakaryocytic cell line model, treatment with Wnt3a failed to increase cell growth either in the absence or presence of TPO, despite inducing high levels of β-catenin. Similarly, expression of a constitutively active version of β-catenin did not increase cell growth either in the absence or presence of TPO, suggesting that the effects of GSK-3β inhibition downstream of TPO signaling are distinct from those induced by Wnt3a and independent of β-catenin. The growth promoting effects of TPO are not mediated by either of the two known GSK-3β targets, cyclin D or HIF-1α. We conclude that GSK-3β is phosphorylated and inhibited by TPO-induced Akt, promoting survival and proliferation in megakaryocytic cells through a pathway that does not involve β-catenin.
PMCID: PMC2677808  PMID: 18804163
GSK-3β; thrombopoietin; β-catenin; megakaryocyte; Wnt3a
12.  Lnk Inhibits Tpo–mpl Signaling and Tpo-mediated Megakaryocytopoiesis 
Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor, c-mpl, activates multiple pathways including signal transducer and activator of transcription (STAT)3, STAT5, phosphoinositide 3-kinase–Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here, we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl, and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)–derived megakaryocyte culture. Consistent with this result, we found that in both BM and spleen, Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition, Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3, STAT5, Akt, and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore, the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast, the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to, but is not essential for, inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus, Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo.
PMCID: PMC2212736  PMID: 15337790
hematopoiesis; megakaryocytes; cytokine receptors; cell proliferation; endomitosis
13.  Romiplostim as a treatment for immune thrombocytopenia: a review 
“Immune thrombocytopenia” (ITP) is an autoimmune disorder that leads to peripheral destruction, as well as a decreased production of platelets. ITP most commonly presents as mild mucocutaneous bleeding. Though it is rare, the leading cause of mortality in persons with ITP is intracranial hemorrhage and those that do not respond to therapy are at increased risk. Our understanding of the pathophysiology of ITP has evolved immensely, especially over the last 60 years. The discovery of the platelet-production stimulator, thrombopoietin (TPO), lent clarity to an earlier hypothesis that inhibition of platelet production at the level of the megakaryocyte, at least in part, accounts for thrombocytopenia in adults with ITP. This facilitated the development of TPO-based therapies to treat ITP. Thrombopoietin receptor agonists are one of the most recent treatments to enter the landscape. Original production of a recombinant human TPO was halted after clinical trials revealed the untoward effect of autoantibodies to the recombinant human TPO with cross-reactivity to endogenous TPO. Next-step development focused on stimulation of the TPO receptor with fewer immunogenic agents. Currently, two such thrombopoietin receptor agonists, romiplostim and eltrombopag, are licensed in the USA to treat thrombocytopenia in adults with persistent or chronic ITP. Ongoing research will assess their efficacy in other immune-mediated and nonimmune-mediated primary and secondary thrombocytopenias.
PMCID: PMC4304598  PMID: 25632241
thrombopoietin; thrombopoietin receptor agonist; megakaryocyte; peptibody
14.  Thrombopoietin as Biomarker and Mediator of Cardiovascular Damage in Critical Diseases 
Mediators of Inflammation  2012;2012:390892.
Thrombopoietin (TPO) is a humoral growth factor originally identified for its ability to stimulate the proliferation and differentiation of megakaryocytes. In addition to its actions on thrombopoiesis, TPO directly modulates the homeostatic potential of mature platelets by influencing their response to several stimuli. In particular, TPO does not induce platelet aggregation per se but is able to enhance platelet aggregation in response to different agonists (“priming effect”). Our research group was actively involved, in the last years, in characterizing the effects of TPO in several human critical diseases. In particular, we found that TPO enhances platelet activation and monocyte-platelet interaction in patients with unstable angina, chronic cigarette smokers, and patients with burn injury and burn injury complicated with sepsis. Moreover, we showed that TPO negatively modulates myocardial contractility by stimulating its receptor c-Mpl on cardiomyocytes and the subsequent production of NO, and it mediates the cardiodepressant activity exerted in vitro by serum of septic shock patients by cooperating with TNF-α and IL-1β. This paper will summarize the most recent results obtained by our research group on the pathogenic role of elevated TPO levels in these diseases and discuss them together with other recently published important studies on this topic.
PMCID: PMC3337636  PMID: 22577249
15.  Thrombopoietin-Mediated Sustained Activation of Extracellular Signal-Regulated Kinase in UT7-Mpl Cells Requires Both Ras–Raf-1- and Rap1–B-Raf-Dependent Pathways 
Molecular and Cellular Biology  2001;21(8):2659-2670.
Thrombopoietin (TPO) regulates growth and differentiation of megakaryocytes. We previously showed that extracellular signal-regulated kinases (ERKs) are required for TPO-mediated full megakaryocytic maturation in both normal progenitors and a megakaryoblastic cell line (UT7) expressing the TPO receptor (Mpl). In these cells, intensity and duration of TPO-induced ERK signal are controlled by several regions of the cytoplasmic domain of Mpl. In this study, we explored the signaling pathways involved in this control. We show that the small GTPases Ras and Rap1 contribute together to TPO-induced ERK activation in UT7-Mpl cells and that they do so by activating different Raf kinases as downstream effectors: a Ras–Raf-1 pathway is required to initiate ERK activation while Rap1 sustains this signal through B-Raf. Indeed, (i) in cells expressing wild-type or mutant Mpl, TPO-induced Ras and Rap1 activation correlates with early and sustained phases of ERK signal, respectively; (ii) interfering mutants of Ras and Rap1 both inhibit ERK kinase activity and ERK-dependent Elk1 transcriptional activation in response to TPO; (iii) the kinetics of activation of Raf-1 and B-Raf by TPO follow those of Ras and Rap1, respectively; (iv) RasV12-mediated Elk1 activation was modulated by the wild type or interfering mutants of Raf-1 but not those of B-Raf; (v) Elk1 activation mediated by a constitutively active mutant of Rap1 (Rap1V12) is potentiated by B-Raf and inhibited by an interfering mutant of this kinase. UT7-Mpl cells represent the second cellular model in which Ras and Rap1 act in concert to modulate the duration of ERK signal in response to a growth factor and thereby the differentiation program. This is also, to our knowledge, the first evidence suggesting that Rap1 may play an active role in megakaryocytic maturation.
PMCID: PMC86897  PMID: 11283246
16.  Thrombopoietin Contributes to Neuronal Damage in Experimental Bacterial Meningitis ▿  
Infection and Immunity  2010;79(2):928-936.
Thrombopoietin (Tpo), which primarily regulates megakaryopoiesis, and its receptor (c-Mpl) are expressed in the brain, where Tpo exhibits proapototic effects on neurons. In the present study, we investigated the implication of Tpo in experimental pneumococcal meningitis. Following intrathecal infection with the encapsulated Streptococcus pneumoniae strain D39, we observed upregulation of Tpo mRNA expression at 12 h and 24 h in brain homogenates of wild-type C57BL/6 mice. c-Mpl mRNA expression was upregulated at 12 h and returned to baseline at 24 h. Compared to wild-type mice, mutants with homozygous Tpo receptor ablation (c-Mpl−/−) displayed reduced microglial activation and neuronal apoptosis in the dentate gyrus. Concentrations of bacteria in blood or cerebrospinal fluid (CSF), as well as CSF pleocytosis, were not significantly different between wild-type and c-Mpl−/− mice. In human postmortem brain, Tpo protein was colocalized to macrophages during encephalitis. In murine primary microglia and RAW264.7 macrophages, upregulation of Tpo mRNA was induced by D39-conditioned medium but not by bacterial lipopeptide or by medium conditioned by pneumococcal mutants defective in hydrogen peroxide formation (ΔspxB) or pneumolysin (Δpln). We conclude that Tpo acts as a mediator of neuronal damage in bacterial meningitis.
PMCID: PMC3028827  PMID: 21149592
17.  Novel Pathways to Erythropoiesis Induced by Dimerization of Intracellular c-Mpl in Human Hematopoietic Progenitors 
Stem cells (Dayton, Ohio)  2012;30(4):697-708.
The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl.
PMCID: PMC3973153  PMID: 22290824
humans; myeloid progenitor cells; erythropoiesis; c-Mpl
18.  Serum Thrombopoietin Levels and Its Relationship With Thrombocytopenia in Patients With Cirrhosis 
Hepatitis Monthly  2014;14(5):e18556.
Patients with cirrhosis usually have thrombocytopenia in discrete levels. The mechanism of thrombocytopenia is thought as splenic sequestration and destruction of platelets, impaired bone marrow generation and diminished hepatic thrombopoietin synthesis.
The aim of this study was to evaluate serum thrombopoietin levels and its relationship with thrombocytopenia at patients with cirrhosis.
Patients and Methods:
Ninety–two cirrhotic patients and 45 healthy controls without history or findings of pathologies that can effect thrombopoietin levels were enrolled by simple random sampling to patient and control groups of this case control study performed at Eskisehir-Turkey. Thrombopoietin was measured in serum samples with a solid phase enzyme-linked immune absorbent assay. Additionally, spleen size and volume index were determined.
Platelet counts were lower in patients with cirrhosis (97000 ± 8000/mm3) than in healthy subjects (240000 ± 51000/mm3, P < 0.001). Significant difference was determined for platelet counts among child A, B and C stages (Child A vs. Child B P < 0.05 Child A vs. Child C P < 0.001–Child B vs. Child C P < 0.05). Serum TPO concentration was higher (69 ± 12 pg/mL) in cirrhotic group than healthy controls (49 ± 9 pg/ml) (P < 0.05). No significant difference in TPO levels were found among the Child A, B and C stages (64 ± 11 pg/mL, 75 ± 13 pg/mL and 68 ± 10 pg/mL, respectively). Spleen size and SVI was significantly higher in the cirrhotic patients than healthy controls (148 ± 14 mm vs. 98 ± 11 mm, P < 0.001-9167 ± 287 cm2 vs. 4118 ± 123 cm2). Significant difference was determined for spleen size and spleen index among child A, B and C stages (Child A vs. Child B P < 0.05 Child A vs. Child C P < 0.001–Child B vs. Child C P < 0.05). TPO levels were significantly different between cirrhotic patients with platelet levels below 50.000/mm3 (n = 16, plt-count: 41000 ± 8300/mm3, TPO levels: 73 ± 7 pg/mL) and above 50.000/mm3 (n = 76, plt-count: 105000 ± 9500/mm3, TPO levels: 65 ± 10 pg/mL) (P < 0.01). In correlation analysis, there was a strong negative correlation between platelet count-spleen size (P < 0.001, r = -0.74) and platelet count–serum TPO levels (P < 0.001, r = -0.71).
Our results suggest that liver cirrhosis does not cause impaired thrombopoietin production even in the late stage of disease. Thrombopoietin has no contribution for the occurrence of thrombocytopenia in cirrhosis; splenic sequestration seems to be the main factor.
PMCID: PMC4071317  PMID: 24976834
Liver Cirrhosis; Thrombocytopenia; Thrombopoietin
19.  Selection between Michaelis–Menten and target-mediated drug disposition pharmacokinetic models 
Target-mediated drug disposition (TMDD) models have been applied to describe the pharmacokinetics of drugs whose distribution and/or clearance are affected by its target due to high binding affinity and limited capacity. The Michaelis–Menten (M–M) model has also been frequently used to describe the pharmacokinetics of such drugs. The purpose of this study is to investigate conditions for equivalence between M–M and TMDD pharmacokinetic models and provide guidelines for selection between these two approaches. Theoretical derivations were used to determine conditions under which M–M and TMDD pharmacokinetic models are equivalent. Computer simulations and model fitting were conducted to demonstrate these conditions. Typical M–M and TMDD profiles were simulated based on literature data for an anti-CD4 monoclonal antibody (TRX1) and phenytoin administered intravenously. Both models were fitted to data and goodness of fit criteria were evaluated for model selection. A case study of recombinant human erythropoietin was conducted to qualify results. A rapid binding TMDD model is equivalent to the M–M model if total target density Rtot is constant, and RtotKD/(KD + C)2 ≪ 1 where KD represents the dissociation constant and C is the free drug concentration. Under these conditions, M–M parameters are defined as: Vmax = kintRtotVc and Km = KD where kint represents an internalization rate constant, and Vc is the volume of the central compartment. Rtot is constant if and only if kint = kdeg, where kdeg is a degradation rate constant. If the TMDD model predictions are not sensitive to kint or kdeg parameters, the condition of RtotKD/(KD + C)2 ≪ 1 alone can preserve the equivalence between rapid binding TMDD and M–M models. The model selection process for drugs that exhibit TMDD should involve a full mechanistic model as well as reduced models. The best model should adequately describe the data and have a minimal set of parameters estimated with acceptable precision.
PMCID: PMC3157300  PMID: 20012173
Michaels–Menten; Target-mediated drug disposition; Nonlinear pharmacokinetics; Erythropoietin
20.  Role of the Distal Half of the c-Mpl Intracellular Domain in Control of Platelet Production by Thrombopoietin In Vivo 
Molecular and Cellular Biology  2000;20(2):507-515.
The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Δ60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Δ60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.
PMCID: PMC85116  PMID: 10611229
21.  Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line 
Molecular and Cellular Biology  1998;18(7):4282-4290.
Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ∼30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.
PMCID: PMC109012  PMID: 9632812
22.  Thrombopoietin protects the brain and improves sensorimotor functions: reduction of stroke-induced MMP-9 upregulation and blood–brain barrier injury 
This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 μg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood–brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 μg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPO's efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.
PMCID: PMC3063625  PMID: 20877384
blood–brain barrier; brain infarction; focal ischemia; matrix metalloproteinase-9; neurologic deficits; stroke; thrombopoietin
23.  Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin 
Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.
PMCID: PMC2192470  PMID: 8627177
24.  Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis 
Stem cell research  2012;9(2):77-86.
Umbilical cord blood (UCB) transplantation has emerged as promising therapy, but is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag’s effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+CD38−, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+CD41−, CD34−CD41+, and CD34−CD41− cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34−C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.
PMCID: PMC3412938  PMID: 22683680
Eltrombopag; thrombopoietin; c-Mpl; CD34+; CD34+CD38−; CD 41+; NOD/SCID xenotransplant; STAT5; STAT3; AKT
25.  Thyroid peroxidase forms thionamide-sensitive homodimers: relevance for immunomodulation of thyroid autoimmunity 
Thyroid peroxidase (TPO) is the key enzyme in thyroid hormone production and a universal autoantigen in Graves’ and other autoimmune thyroid diseases. We wished to explore the expression of TPO and whether it was affected by thionamide antithyroid drugs. We studied recombinant TPO, stably expressed by a Chinese hamster ovary cell line (CHO-TPO) and transiently expressed TPO-enhanced green fluorescent protein (eGFP) and -FLAG fusion proteins. Immunoblotting of CHO-TPO cell extracts showed high-molecular weight (HMW) TPO isoforms that were resistant to reduction, as well as 110 kDa monomeric TPO. Co-immunoprecipitation and enzyme-linked-immunosorbent assay (ELISA) binding studies of FLAG- and eGFP-tagged TPO demonstrated TPO dimerisation. CHO-TPO cells cultured in methimazole (MMI) for 10 days showed a significant reduction in HMW-TPO isoforms at MMI concentrations of 1 µM and above (p < 0.01), whereas monomeric TPO expression was unchanged. We observed a similar reduction in HMW-TPO in CHO-TPO cells cultured in propylthiouracil (10 µM and above). Binding of Graves’ disease patient sera and TPO-Fabs to enzymatically active TPO that was captured onto solid phase was not abrogated by MMI. The cellular localisation of TPO in CHO-TPO cells was unchanged by MMI treatment. Our demonstration of homodimeric TPO and the reduction in HMW-TPO isoforms during thionamide treatment of CHO-TPO cells shows, for the first time, an effect of thionamides on TPO structure. This suggests a structural correlate to the effect of thionamides on TPO enzymatic activity and opens up a novel potential mechanism for thionamide immunomodulation of autoimmune thyroid disease.
PMCID: PMC2757584  PMID: 19669106
Thyroid peroxidase; Homodimerisation; Methimazole; Propylthiouracil; Graves’ disease; Immunomodulation

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