Thrombopoietin Concentrations Research Articles

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

Inhibition of VPAC1 Signaling Raises Platelet Numbers after Myelosuppressive Therapy and in GATA1 Congenital Thrombocytopenia.

Thrombocytopenia is a serious complication in patients undergoing myelosuppressive chemotherapy. Therefore, the development of new strategies to reduce thrombocytopenia or accelerate platelet recovery without adverse effect on the bone marrow (BM) is still warranted. Previously we found that the pituitary adenylate cyclase activating peptide (PACAP) is a physiological inhibitor of platelet activation (JCI, 2004). The receptor for PACAP on megakaryocytes (MKs) and platelets is the Gs coupled receptor VPAC1. We have presently investigated whether the neutralization of the regulatory role of VPAC1 can affect thrombopoiesis, independently of thrombopoietin (TPO). We found that inhibition of VPAC1 signaling stimulates in vitro megakaryopoiesis, since more MKs could be generated from Sca1+ cells, isolated from normal mouse BM by incubation with a neutralizing anti-VPAC1 antibody (Ab) (23A11). 23A11 was also capable of enhancing the later stages of MK proliferation of human cord blood derived CD34+ cells. These in vitro findings were validated in two in vivo animal models of myelosuppressive therapy. The subcutaneous injection of a neutralizing Ab directed against PACAP (PP1A4) or 23A11 (1 mg/kg on days 0,3 and 7) boosted platelet recovery in mice after chemotherapy-induced thrombocytopenia (20 mg/kg busulfan intraperitoneally on days 7 and 10) in a TPO-independent manner. Platelet recovery in mice, injected with PP1A4, was also significantly increased compared to non-treated mice exposed to 8 Gy total body irradiation. Plasma TPO concentrations did not differ between the two groups. We next questioned whether VPAC1 inhibition could rescue the GATA1-defective phenotype due to a developmental arrest in the megakaryoblast stage. FACS analysis of BM Sca1+ cells from GATA1 deficient mice cultured during 12 days in MK differentation medium in vitro showed an increased MK DNA ploidy when 23A11 was added to the culture. A similar observation was made with CD34+ cells from BM of a GATA1 deficient patient with severe thrombocytopenia. GATA1 deficient mice are thrombocytopenic, with a platelet count of 50 +/− 15 x 10(9) plt/L. This platelet number increases to 150 +/− 40 x 10(9) plt/L after subcutaneous injection with 23A11 whereas injection with PBS had no effect. Again, TPO plasma levels in these injected GATA1 deficient mice showed no association between anti-PACAP or anti-VPAC1 treatment. Thus, our data indicate that VPAC1 signaling tempers normal megakaryopoiesis and that elimination of this pathway facilitates platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Essential thrombocythemia in a child with elevated thrombopoietin concentrations and skeletal anomalies

Essential thrombocythemia is a rare myleoproliferative disorder in pediatrics. This myleoproliferative disorder is characterized by excessive proliferation of megakaryocytes and sustained elevation of platelet count. Reactive thrombocytosis is a more common cause of elevated platelet counts among children. We describe a 2-year-old child with essential thrombocythemia, skeletal anomalies, and elevated thrombopoietin concentrations. The child's mother was also subsequently diagnosed with essential thrombocythemia and had elevated thrombopoietin concentrations. Chromosomal studies on the mother, child and other family members were normal.

Hepatocyte growth factor accelerates thrombopoiesis in transgenic mice

Hepatocyte growth factor (HGF) is one of the potent growth factors for liver regeneration and has a strong effect on epithelial and nonepithelial cells. As one of the pleiotropic functions, HGF acts as a hematopoietic regulator in the proliferation and differentiation of hematopoietic progenitors. However, the effect of HGF on the thrombopoietic function remains unclear. The correlation between HGF and thrombopoiesis was investigated in transgenic (TG) mice overexpressing murine HGF controlled by the murine HGF by the metallothionein promoter. Furthermore, the mechanism of thrombocytosis induced by HGF in vitro was analyzed in hepatoma cell line HepG2. Both the platelet count and the serum thrombopoietin (TPO) concentration were significantly higher in TG than in the wild type (WT) control mice. In the liver and spleen, the expression of TPOmRNA in TG was higher than that in WT by real-time polymerase chain reaction. The expressions of transcriptional factor of TPO, GABP-alpha/beta were more increased in TG liver compared to WT. In an in vitro study, HGF induced TPO and GABP-alpha/beta expression and enhanced TPO promoter activity. Therefore, HGF induced thrombopoiesis accompanied with the overexpression of TPO through GABP stimulation.

High Thrombopoietin Concentrations in The Cerebrospinal Fluid of Neonates with Sepsis And Intraventricular Hemorrhage May Contribute to Brain Damage

Thrombopoietin (TPO) and its receptor (TPOR) are expressed in the central nervous system (CNS). Although TPO shares significant homology with various neurotrophins, recent data indicate a proapoptotic function of TPO in the CNS. In this study, TPO concentrations were analyzed in the cerebrospinal fluid (CSF) of neonates. Human neuroblastoma-derived SH-SY5Y cells were established to elucidate the effects of inflammation and hypoxia on neuronal Tpo expression. TPO was detectable in the CSF of 6 of 15 neonates with bacterial infection/sepsis (median 140, range 2-613 pg/mL), 5 of 9 neonates with posthemorrhagic hydrocephalus (median 31, range 1.4-469 pg/mL), 3 of 4 neonates with posthemorrhagic hydrocephalus plus bacterial infection/sepsis or meningitis (median 97, range 6-397 pg/mL), but not in controls ( n = 3). Neither the presence of detectable TPO nor its level in the CSF significantly correlated with any clinical or laboratory parameter. In SH-SY5Y cells, TPO and TPOR expression was detected by RT-PCR and Western blot analysis. In vitro, interleukin-6 (IL-6) did not significantly change Tpo gene expression. In contrast, Tpo mRNA expression significantly decreased under hypoxia, whereas erythropoietin (EPO) mRNA expression increased. In conclusion, our data provide evidence that in neuronal cells, TPO production is regulated by different mechanisms than in hepatocytes.

Familial Essential Thrombocythemia in a Child with Elevated Thrombopoietin Concentrations and Skeletal Anomalies.

We describe a 2-year-old child with an incidental finding of persistent elevated platelet count. The platelet count at the time of referral was 900 × 109/L. His physical examination revealed bony abnormalities of the right upper extremity, and hand. (Photo1) X-Ray of the hand revealed amputation of the fingers of the right hand with rudimentary metacarpal bones, and evidence of shorter radius and ulna of the of the left forearm when compared to the right. The physical examination was otherwise normal with the exception of moderate splenomegaly. Further workup revealed normal iron stores, and no inflammatory process which could explain the thrombocytosis. Chromosomal breakage and rearrangements studies were normal and confirmed a normal male karyotype. A bone marrow aspirate and biopsy revealed megakaryocytic hyperplasia with many binucleated and trinucleated megakaryocytes. (Photo 2) The erythroid and myeloid series were well represented and of normal morphology. Flow cytometry for ALL specific and CMLspecific markers were normal. The platelet count reached 2,800 × 109/L, and serum thrombopoietin level was obtained, which returned 2119.8 pg/ml. The patient was placed on hydroxyurea and aspirin 81mg/day, and reduction in the platelet count occurred. After six months of hydroxyurea therapy the platelet count rose again to pretreatment levels. Hydroxyurea was discontinued and anagrelide started with moderate platelet reduction. Family members were also evaluated. The siblings all had normal platelet counts but the mother had an elevated platelet count 900 × 10 9/L. The bone marrow aspirate of the childs mother was also consistent with ET. The JAK2 kinase mutation was not found in the mother or the child. The childs mother was also found to have an elevated thrombopoietin level of 475.6 pg/ml and was started on hydroxyurea.No connection has been made between our patients congenital anomalies and the thrombocytosis. To our knowledge there have been no other reports of such features in either adult or pediatric patients with ET. Because this childs mother was also subsequently diagnosed with ET, familial ET is a more likely diagnosis. The diagnosis of familial ET is further supported by the elevated TPO concentration in our patient. Sporadic cases of familial ET with elevated thrombopoietin concentrations have been reported. Although ET has been reported in patients as young a 6 weeks, our case is different than other reported pediatric cases of ET because of not only the elevated TPO but the associated skeletal anomalies of our patient. To date neither the child or the mother has suffered any thrombohemorrhagic events, which have been reported to occur at rates varying from 20% to 40%. The mother and child are being followed prospectfully with serial blood counts, serum TPO concentrations. [Display omitted] [Display omitted]

Megakaryocyte development and platelet production

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth-factor for the megakaryocyte (MK) lineage, induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C-Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c-Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.

Suspects in the tale of lupus-associated thrombocytopenia

Immunologically mediated thrombocytopenia is a frequent clinical manifestation in patients with systemic lupus erythematosus (SLE). Autoantibodies targeting platelet membrane glucoproteins have a central role in peripheral platelet destruction. Autoantibodies against thrombopoietin are also present in about one-third of patients, but their pathogenetic role is obscure. Thirty-eight serum samples from SLE patients were tested for anti-platelet antibodies, anti-thrombopoietin antibodies and levels of circulating thrombopoietin. Bone marrow histology was also assessed. Thirty-nine per cent of sera displayed anti-thrombopoietin antibodies and 29% had circulating anti-platelet antibodies. Anti-thrombopoietin antibodies were associated with lower thrombopoietin concentrations, and lower mean platelet values in long-term follow-up. Anti-platelet antibodies were present in about 40% of thrombocytopenic and non-thrombocytopenic individuals but were absent in patients who had recovered from thrombocytopenia, supporting their pathogenetic role. Both autoantibodies were absent in control sera from patients with rheumatoid arthritis and primary Sjögren's syndrome. Decreased bone marrow cellularity, normal or low number of hypolobulated, pyknotic megakaryocytes and stromal alterations were prominent findings in thrombocytopenic SLE patients, suggesting a defect in megakaryopoiesis. These findings were not evident in specimens from patients with idiopathic thrombocytopenic purpura who had increased megakaryocytes, normal cellularity and absence of stromal alterations. In conclusion, peripheral destruction due to platelet autoantibodies, anti-thrombopoetin antibodies, lower effective circulating thrombopoetin and impaired compensatory response due to bone marrow damage interact in SLE and thrombocytopenia ensues.

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD)

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.

Preoperative serum thrombopoietin levels are higher in patients with ovarian cancer than with benign cysts

Objective To assess the diagnostic relevance of serum thrombopoietin (TPO) levels, we compared serum TPO levels between the patients with ovarian cancer and benign ovarian cysts. We also correlated serum TPO concentrations in ovarian cancer patients to know prognostic factors such as disease stage, tumor grade, histological subtype, and residual tumor mass. Study design Preoperative serum TPO levels were measured in women with epithelial ovarian cancer ( n = 51) and women with benign ovarian cysts ( n = 25) using an enzyme-linked immunosorbent assay. Results The serum TPO concentration was significantly elevated in the ovarian cancer patients as compared to the benign patient controls. Platelet counts and TPO in the serum did not correlate in the ovarian cancer patients. However, using a TPO cutoff of 90 pg/ml and a CA125 cutoff of 30 units/ml, when both markers were elevated, the specificity was as high as 92%. Conclusion Serum TPO concentrations may have a role in the diagnostic clinical setting for discerning benign from malignant ovarian tumors.

Platelet count, mean platelet volume and thrombocytopoietic indices in healthy women and men

Gender-dependent differences in platelet count have been demonstrated in few studies. In women platelet count is higher than in men, which seems to reflect different hormonal profiles or a compensatory mechanism associated with menstrual blood loss. The aim of the study was to assess platelet count, mean platelet volume and thrombocytopoietic indices in women and men. The study was conducted on healthy blood donors divided into groups: F — 60 women and M — 65 men. Platelet count and mean platelet volume were determined on a haematological analyser Advia 120, Bayer. The following thrombocytopoietic indices were measured: thrombopoietin concentration (ELISA), percentage of reticulated platelets (flow cytometry, COULTER EPICS XL) and absolute reticulated platelet count. Results Higher platelet count was noted in the group of women 252.35 ± 41.25 × 10 9/l as compared to men 221.87 ± 37.63 × 10 9/l ( p = 0.0002). At the same time women had lower thrombopoietin concentration 156.50 ± 57.18 pg/ml compared to men 180.46 ± 60.98 pg/ml, ( p = 0.03). No statistically significant differences were found in the mean platelet volume, percentage of reticulated platelets or absolute reticulated platelet count between group F and M. Conclusions Platelet count is gender-dependent, being higher in women than in men. Thrombopoietin concentration is gender-dependent and is lower in women than in men. In physiological conditions, there is no correlation between platelet count and thrombopoietin concentration in women ( r = − 0.155) and men ( r = − 0.2586).

Thrombopoietin Production in Wild-Type and Interleukin-6 Knockout Mice with Acute Inflammation

Clinical and laboratory studies indicate that thrombopoietin (TPO) gene expression increases during inflammation. To clarify the role of interleukin 6 (IL-6) in this process, blood cell counts, plasma TPO concentrations, and hepatic and renal TPO mRNA levels were investigated in wild-type and IL-6 knockout mice, with sterile abscesses produced by subcutaneous injection of turpentine oil. Treatment did not cause a change in blood cell counts during the 72 h period of observation. The numbers of thrombocytes and erythrocytes were slightly lower in the IL-6 knockout mice than in the wild-type littermates under all conditions. Plasma IL-6 and TPO concentrations increased on turpentine injection only in the wild-type mice. In addition, turpentine treatment of these caused an increase in hepatic TPO mRNA levels as assessed by competitive polymerase chain reaction (RT-PCR) and real-time PCR, whereas renal TPO mRNA levels were unaltered. TPO mRNA levels did not increase in the livers of IL-6 knockout mice on turpentine treatment. These results support the concept that TPO behaves like an acute-phase protein in that its synthesis is induced by IL-6 in the liver.

Thrombopoietin in the Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

Despite the recent evidence of the localization of thrombopoietin (TPO) and its receptor in the central nervous system (CNS), TPO protein concentrations in the cerebrospinal fluid (CSF) remained to be clarified. We previously reported that serum TPO is increased in children with meningitis. To determine changes in TPO concentrations in the CSF by meningitis and to explore the relationship between serum and CSF TPO concentrations, we measured TPO concentrations in 110 CSF samples and 33 serum/CSF pairs from 11 bacterial meningitis, 49 aseptic meningitis, and 50 nonmeningitis children. In only 12% (13 of 110) of CSF samples (0 bacterial meningitis, 8 aseptic meningitis, and 5 controls), TPO concentrations could be determined (24.1 +/- 29.0 pg/ml). CSF TPO concentrations did not significantly differ among the three groups and did not correlate with age. TPO concentrations in all serum samples were detectable, and mean concentrations in bacterial meningitis (510.6 +/- 237.0 pg/ml) were significantly higher than those in aseptic meningitis (136.6 +/- 71.6, p < 0.01) and controls (181.3 +/- 88.3, p < 0.01). These findings suggest that TPO is not produced in the CNS of patients with meningitis and that TPO did not cross the blood-brain barrier even during meningeal infection.

Thrombopoietin Level Is Increased in the Serum of Asphyxiated Neonates: A Prospective Controlled Study

Thrombopoietin (TPO) is a growth factor that controls platelet production. Despite the known association of chronic hypoxia and acute asphyxia with hematologic changes, TPO had not been studied in neonatal asphyxia. To assess TPO concentrations in the serum of asphyxiated and nonasphyxiated neonates, and examine any correlation with the severity of asphyxia. This prospective study was carried out on 32 asphyxiated neonates and 30 control subjects admitted at Cairo University Medical Center. Asphyxia was defined if two of the following were found: (1) Apgar score </=3 at 1 minute or </=6 at 5 minutes, (2) umbilical cord arterial pH </=7.2 combined with base deficit >/=-10 and (3) clinical evidence of perinatal asphyxia. Encephalopathy was classified clinically according to Sarnat's stages during the first day of life. Platelet count and TPO level (pg/ml) were measured at 1st, 3rd and 7th day of life. : TPO measured on the first day of life did not differ between cases and controls (900.2+/-526.4 vs 726.6+/-441.9 pg/ml, p=0.2). It increased on the 3rd day of life and was significantly higher in asphyxiated infants compared to controls (1291.4+/-627.9 vs 885.5+/-400.3 pg/ml, respectively; p=0.004). This difference remained significant in a logistic regression model controlling for birth weight, sex and mode of delivery (regression coefficient=476.9+/-146.8; p=0.002). In asphyxiated infants (n=32), encephalopathy was classified as mild (n=17), moderate (n=10) and severe (n=5). TPO correlated with the degree of clinical severity on the 7th day of life (r=0.59, p=0.003). TPO did not differ between survivors (n=24) and nonsurvivors (n=8) within the asphyxia group (1197.1+/-596.8 vs 1613.1+/-605.9 pg/ml; p=0.09). Platelet counts correlated negatively with TPO measured on day 1 (r=-0.415; p=0.02), day 3 (r=-0.64; p=0.001) and day 7 (r=-0.562; p=0.007). TPO increased and correlated with severity of asphyxia at 3 and 7 days of life. It correlated negatively with the platelet count at all times.

Thrombopoietin and Interleukin-6 in Children with Pneumonia-Associated Thrombocytosis

The aim of this study was to investigate why some, but not all, children develop thrombocytosis during the course of pneumonia. The retrospective study included 40 healthy children and 75 children with pneumonia: 17 patients with platelet count within the reference values, i.e., platelet count </=450 x 10(9)/L, and 58 with thrombocytosis >450 x 10(9)/L. Erythrocyte sedimentation rate, leukocyte and platelet counts, and concentrations of hemoglobin, C-reactive protein, interleukin-6 and thrombopoietin were determined in the blood of patients and control groups of children. Patients with thrombocytosis were slightly younger (3.0 +/- 1.8 years and median 2.5 years, respectively) than patients with normal platelet count (3.8 +/- 2.4 years and median 4 years, respectively). Additionally, according to clinical and radiological findings, pneumonia in children with thrombocytosis had a more severe and protracted course. Serum thrombopoietin concentrations were found to be 91.2 +/- 41.7 ng/L (range: 14.3-166.7 ng/L) in patients with normal platelet count (313 +/- 70 x 10(9)/L, range: 206-428 x 10(9)/L). In patients with thrombocytosis (581 +/- 131 x 10(9)/L, range: 450-830 x 10(9)/L) serum thrombopoietin ranged from 63.6 to 1115.9 ng/L (526.6 +/- 268.4 ng/L). In these patients both concentration of hemoglobin (114 +/- 12 g/L) and iron (4.3 +/- 1.3 micromol/L) significantly decreased as compared with the control group. Study results suggested the possible development of reactive thrombocytosis in children with pneumonia. As platelets are involved in inflammatory reaction, reactive thrombocytosis might be part of the mechanism of defense. Reactive thrombocytosis may develop as a sequel of either anemia or inflammatory reaction (or both).

Endogenous thrombopoietin levels during the clinical management of acute myeloid leukaemia

Thrombocytopenia represents a major problem in the management of acute myeloid leukaemia (AML). The data regarding the alterations of endogenous thrombopoietin (TPO) regulation during the clinical course of AML are limited. The aim of this study was to investigate endogenous TPO dynamics in association with platelets during the clinical course of AML. We serially measured both TPO and platelets concurrently over the entire treatment period of newly diagnosed patients receiving both remission induction and consolidation chemotherapies. The median concentration of TPO in AML patients at the initial diagnosis was 469.71 pg/ml and increased significantly during the aplastic period due to remission induction chemotherapy (median: 1085.33 pg/ml) but then decreased to a level (median: 45.26 pg/ml) encountered in the healthy control subjects (median: 56.90 pg/ml). In the cytopenic period due to consolidation treatment, TPO level again increased significantly to a high level (median: 891.38 pg/ml) during the platelet nadir, but decreased toward normal (median: 100.75 pg/ml) after the thrombocytopenic period had elapsed. In conclusion, endogenous TPO levels exhibit an inverse fluctuation in relation to platelet counts during the clinical course of AML. Pharmacological stimulation of thrombopoiesis in AML with novel molecules, including the recombinant thrombopoietins and the small peptide agonists, should be based on a critical administration strategy that must consider the endogenous levels of TPO. TPO levels in distinct AML disease states may explain the unsuccessful recombinant TPO trials and could help to design better strategies for ‘pharmacological stimulation of thrombopoiesis’ in AML.

Pruning the Brain

Thrombopoietin (TPO) and erythropoietin (EPO) both act as hematopoietic growth factors: TPO stimulates platelet formation, whereas EPO stimulates erythrocyte formation. Noting that EPO acts as a survival factor in the developing and stressed adult brain, Ehrenreich et al. investigated the possible role of brain TPO. Whereas the amount of mouse brain EPO mRNA decreased between embryonic day 11 (E11) and E15, that of TPO continued to rise into adulthood. Similarly, brain EPO protein expression was decreased in the adult compared with that at E11 or E13, whereas TPO protein expression was greater in the adult. In primary cultures of hippocampal neurons, hypoxia stimulated an increase in EPO mRNA and protein, whereas it caused a decrease in TPO mRNA and protein. Under normoxic conditions, concentrations of TPO that stimulated hematopoietic stem cell proliferation unexpectedly promoted the death of cultured hippocampal neurons, an effect that was blocked by EPO as well as by granulocyte colony-stimulating factor, neurotrophin-3, or brain-derived neurotrophic factor (BDNF). Pharmacological inhibition of the protein kinase JAK2, extracellular signal-regulated kinase (ERK1/2), or caspase-3 blocked TPO-induced neuronal death, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) blocked the ability of EPO to rescue neurons from TPO-mediated death. TPO enhanced apoptosis of young neurons (neurons that expressed β-tubulin III) in juvenile rats in a model of moderate hypoxic-ischemic brain injury. Thus, whereas other growth factors enhance neuronal survival, the authors propose that TPO may contribute to the pruning of extraneous neurons that takes place during late stages of brain development. H. Ehrenreich, M. Hasselblatt, F. Knerlich, N. von Ahsen, S. Jacob, S. Sperling, H. Wolge, K. Vehmeyer, K.-A. Nave, A.-L. Sirén, A hematopoietic growth factor, thrombopoietin, has a proapoptotic role in the brain. Proc. Natl. Acad. Sci. U.S.A. 102 , 862-867 (2005). [Abstract] [Full Text]

Factors Affecting Thrombopoietin Production from Bone Marrow Stromal Cells.

Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis and therefore also the most important determinant of the number of platelets in circulation. The regulation of TPO blood concentration is complex, with at least a significant component mediated by removal and degradation of the hormone by the mature circulating platelet mass. In this way, if the production of TPO were fixed, when the number of platelets rises, an increased amount of TPO is removed from the plasma, resulting in an inverse relationship between platelet counts and TPO concentration. However, several studies hint at the existence of additional mechanisms in which TPO production is altered in response to physiological and pathologic conditions. We, and others, have previously reported that TPO mRNA is increased in the bone marrow of mice or humans after either immune- or radiation-mediated thrombocytopenia, although hepatic levels remain unchanged by this manipulation. To further explore the mechanism(s) of this effect, we utilized in vitro marrow stromal cell models to study the effects of blood proteins on TPO production. As an initial hypothesis we determined if platelet-derived proteins in serum might suppress TPO production from both a marrow stromal cell line, OP9, or primary murine marrow stromal cells derived from long-term hematopoietic cultures. As assessed by quantitative RT-PCR we found that TPO mRNA levels increase a mean of 2.8-fold±0.7 (n = 3) twelve hours following the removal of serum from the culture. Similar results were obtained from primary murine marrow stromal cells; TPO mRNA levels rose a mean of 2.9-fold±0.9 (n = 4) within sixteen hours of serum deprivation. In contrast, TPO transcript levels were unaffected by the same manipulation of serum in the hepatocyte cell line HEPA1c1c7. As the removal of serum might have induced a stress response in the cells, we tested whether other cell stressors might mimic this response; we found that neither UV irradiation nor treatment with toxic metals such as nickel, cobalt, or cadmium produced any rise in TPO mRNA in marrow stromal cells. Furthermore, to test whether a cell cycle arrest triggered by serum deprivation might mediate these effects, we treated stromal cells with cell cycle inhibitors, but failed to find any affect on TPO transcript levels in stromal cells. Further biochemical fractionation of serum suggested that one or more distinct proteins is responsible for this effect, demonstrated by the ability of both ammonium sulfate precipitation and ion exchange chromatography to partition the suppressive effects of serum. However, the active agent in serum is not TPO itself, as the addition of 150 ng/ml of the pure hormone did not suppress stromal cell TPO transcript levels. Knowledge of this novel regulatory mechanism should be useful in treating platelet disorders and perhaps also during stem cell transplantation, a setting in which TPO is known to play a vital role.

Multiple Cycles of Recombinant Human Thrombopoietin Therapy in a Patient with Chronic Refractory Idiopathic Thrombocytopenic Purpura.

Thrombopoietin (TPO) is the key regulator of megakaryocytepoiesis and platelet production. TPO binds to its specific receptor, c-Mpl, on the surfaces of megakaryocytes, and may promote the proliferation, differentiation and maturation of megakaryocytes, and finally increase the circulating platelet count. The role of TPO in the pathogenesis of idiopathic thrombocytopenic purpura (ITP) is not certain. Plasma concentrations of TPO in ITP patients were similar to or little lower than that in healthy subjects. Therefore it is possible that supplemental TPO could significantly promote platelet production and increase platelet counts in ITP patients. Here, we report the result of multiple cycles of recombinant human thrombopoietin (rhTPO) therapy in a patient with refractory ITP. The patient, a 42-year-old woman, was admitted to our department on December 30, 2003. She had suffered from chronic ITP for more than 4 years. The patient had been treated with glucocorticosteroids, immunosuppressive agents and splenectomy. No sustained response could be achieved. The diagnosis of chronic refractory ITP was made. There were petechiae and gingival bleeding on admission. Liver and spleen were not palpable. Hemoglobin was 142g/L, white blood cell count 7.6×10 9/L, platelet count 15×10 9/L. Bone marrow aspiration revealed that erythroid and myeloid development were normal, megakaryocytes were increased in number and no dysplastic features. After an informed consent was obtained from the patient, rhTPO (Sunshine Pharmaceutical Corporation, China) was administrated subcutaneously at dosage of 1.0 μg/kg, daily for 14 days or until platelet count sustained more than 50×109/L. Anti-rhTPO antibodies were determined weekly by ELISA. Three cycles of rhTPO therapy was given with 6, 13 and 8 dosing for each cycle. The platelet counts before each cycle were all less than10×109/L and increased above 50×109/L on day 5, 11 and 8 of rhTPO administration, respectively. The peak platelet counts of 456, 130 and 82×109/L were reached on day 9, 15 and 13 for each cycle. Then platelet count decreased gradually. The durations of platelet count more than 50×109/L in 3 cycles were 13, 7 and 10 days respectively. No increase of WBC count and Hb level occurred. No liver and kidney function damage, abnormal coagulation functions or thrombosis developed during the treatment. rhTPO antibodies were not detectable. The result indicated that rhTPO could transiently increase peripheral platelet counts of the patient with chronic refractory ITP. It was uncertain why peak platelet counts declined and durations of platelet count more than 50×109/L shortened when multiple cycles of rhTPO were given.

Endothelial Cells Mediate the Repair of Human Bone Marrow Stem and Progenitor Cells Following Radiation Injury.

The purposeful misuse of ionizing radiation has been recognized as a major bioterrorism threat in the United States. Although the myeloablative effects of ionizing radiation exposure have been well established, definitive treatments for potential victims of radiation injury are lacking. We have recently demonstrated that BM stem cells can be harvested from mice following exposure to lethal dose total body irradiation and can subsequently recover multilineage repopulating potential via ex vivo culture with microvascular endothelial cells (EC). However, it remains to be determined whether human hematopoietic stem cells (HSC) can be recovered in a similar manner following high dose radiation injury. In this study, we examined the capacity for ex vivo culture with and without primary human endothelial cells to support the cellular repair and recovery of primary human BM CD34+ cells following exposure to high dose irradiation. Exposure of primary BM CD34+ cells to 400 cGy resulted in a 40% decline in total viable cells and an 89% decline in CD34+CD38− cells compared to input despite 10 day culture with optimal concentrations of thrombopoietin, stem cell factor, and flt-3 ligand alone. Conversely, co-culture of 400 cGy irradiated BM CD34+ cells with primary human brain endothelial cells (LS-01) resulted in a 6-fold and 22-fold increase in total cells and CD34+CD38− cells compared to input, respectively. Non-contact cultures with LS-01 resulted in comparable recovery of total cells and the CD34+CD38− subset by 10 days. Concordantly, TSF-cultured progeny contained 5-fold greater numbers of early apoptotic and necrotic cells within the total and CD34+CD38− fractions as compared to the progeny of contact and non-contact endothelial cultures. Colony forming cell (CFC) assays demonstrated that 400 cGy exposure caused an 8-fold reduction in CFU-total compared to normal BM CD34+ cells and no recovery of CFU-GM, BFU-E, or CFU-Mix was observed following TSF culture. Co-culture with LS-01 resulted in the recovery of 50% of CFU-total compared to normal BM CD34+ cells. In order to assess HSC content post-irradiation, NOD/SCID mice were transplanted with normal BM CD34+ cells, 400 cGy irradiated BM CD34+ cells, and the progeny of 400 cGy irradiated cells following ex vivo culture. 100% of mice transplanted with normal BM CD34+ cells (7.5 x 105) demonstrated human hematopoietic engraftment at 6 weeks, whereas 0% of mice transplanted with 400 cGy irradiated BM CD34+ cells showed detectable human repopulation. Twenty-five percent of mice transplanted with 400 cGy irradiated/LS-01 cultured cells demonstrated human repopulation, whereas 0% of mice transplanted with 400 cGy irradiated/TSF-cultured cells showed human engraftment. These data demonstrate that ionizing radiation has a profoundly toxic effect on both human HSC and committed progenitor cells. Endothelial cells and endothelial cell-derived soluble factors appear to provide anti-apoptotic signals to BM progenitor cells following radiation damage, allowing cellular repair of committed progenitors as well as cells with in vivo repopulating capacity. Cytokine combinations alone, such as TSF, appear ineffective toward rescuing human BM stem/progenitor cells from radiation damage. Studies are ongoing to optimize the application of endothelial cells and endothelial cell-derived growth factors to stimulate HSC repair following radiation injury.