Blockade of Thrombopoietin Reduces Organ Damage in Experimental Endotoxemia and Polymicrobial Sepsis.

BACKGROUND AND PURPOSE: Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions. METHODS: We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model. RESULTS: In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 µm to 70.74±3.60 µm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 µm to 57.50±1.89 µm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration. CONCLUSION: Our results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis.

Evaluation of oral-motor movements and facial mimic in patients with head and neck burns by a public service in Brazil

OBJECTIVES: The purpose of this study was to analyze the characteristics of oral-motor movements and facial mimic in patients with head and neck burns. METHODS: An observational descriptive cross-sectional study was conducted with patients who suffered burns to the head and neck and who were referred to the Division of Orofacial Myology of a public hospital for assessment and rehabilitation. Only patients presenting deep partial-thickness and full-thickness burns to areas of the face and neck were included in the study. Patients underwent clinical assessment that involved an oral-motor evaluation, mandibular range of movement assessment, and facial mimic assessment. Patients were divided into two groups: G1 - patients with deep partial-thickness burns; G2 - patients with full-thickness burns. RESULTS: Our final study sample comprised 40 patients: G1 with 19 individuals and G2 with 21 individuals. The overall scores obtained in the clinical assessment of oral-motor organs indicated that patients with both second- and third-degree burns presented deficits related to posture, position and mobility of the oral-motor organs. Considering facial mimic, groups significantly differed when performing voluntary facial movements. Patients also presented limited maximal incisor opening. Deficits were greater for individuals in G2 in all assessments. CONCLUSION: Patients with head and neck burns present significant deficits related to posture, position and mobility of the oral myofunctional structures, including facial movements. .

Use of romiplostim in patients with chronic idiopathic thrombocytopenic purpura during perioperative period.

INTRODUCTION: In patients with chronic idiopathic thrombocytopenic purpura (cITP), the platelet count tends to be quite variable and, in the majority of cases, specific therapy is not warranted on a regular basis. However, patients with low platelet count (<30 nL) or with bleeding complications would require therapy, such as prednisolone, intravenous immunoglobulin infusions, splenectomy and/or immunosuppression. Romiplostim, a thrombopoietin agonist, has also proven to be useful in improving platelet counts. cITP can be associated with bleeding complications perioperatively. As such, a higher platelet count is warranted (approximately 80 nL), particularly for invasive surgeries, such as orthopaedic surgery, cardio-thoracic surgery, head and neck surgery and abdominal surgery, where risk of bleeding is quite high already. AIM: The aim of this study is to evaluate the safety and efficacy of short-term use of romiplostim, perioperatively. METHODS: Patients with chronic ITP requiring major surgical interventions were enrolled in the study. Patients with malignancies or myelodysplastic syndromes, major bleeding disorders, under 18 years of age or pregnancy were excluded. CONCLUSION: This study has shown that the use of romiplostim is safe and effective in improving platelet counts preoperatively and that this could achieve excellent haemostasis, with no associated bleeding complications or rebound thrombocytopenia. A larger study involving multiple centres is required to verify these findings.

Paraneoplastic thrombocytosis in ovarian cancer.

BACKGROUND: The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS: We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS: Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS: These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Anti-erythropoietin and anti-thrombopoietin antibodies induced after administration of recombinant human erythropoietin.

Recombinant human erythropoietin (rhEPO) has been successfully used for correcting renal anemia. However, recent studies have raised some concerns about the safety of rhEPO treatment due to its immunogenic side effect - pure red cell aplasia (PRCA). We now report a case of development of anti-EPO neutralizing antibodies (Abs) implicated in thrombocytopenia as well as erythrocytopenia. A 35-year-old man had a history of administering rhEPO (epoetin alfa, epoetin beta and darbepoetin alfa) for 2years to treat renal anemia. The hematological parameters were collected. Anti-EPO, anti-platelet, and anti-thrombopoietin (TPO) Ab assays were performed to test the presence of autoreactive Abs. After performing antibody assays due to severe resistance to rhEPO treatment, a high titer of anti-EPO neutralizing Abs was detected. However, unexpectedly, this patient also showed thrombocytopenia rather than PRCA. We investigated the cause of the marked thrombocytopenia and found anti-TPO Abs in patient serum. To our best knowledge, this is the first report of the development of anti-TPO Abs during rhEPO treatment for anemia.

Safety and antithrombotic efficacy of moderate platelet count reduction by thrombopoietin inhibition in primates.

Most heart attacks and strokes are caused by blood clots (thrombi) that block the vasculature. Because disease-causing arterial thrombosis depends on blood platelets, platelet inhibitors such as aspirin and clopidogrel effectively decrease the risk of thrombosis; however, they also impair platelet-dependent hemostasis that staunches bleeding from wounds and can therefore produce excessive bleeding. Experimental studies show that a reduction in the number of platelets also inhibits thrombosis, but these treatments also interfere with platelet function. Because normal hemostasis requires that the platelet concentration remain within a physiological range in the circulation, we evaluated whether lowering the number of circulating platelets--but only to a value still within the normal range--by inhibiting platelet formation in the bone marrow inhibits acute thrombogenesis in baboons. We reduced the platelet count with an inhibitor against the megakaryocyte-promoting hormone thrombopoietin and then showed that experimental occlusive thrombogenesis on collagen-coated vascular grafts was reduced, without impairment of primary hemostasis. These results suggest that suppressing platelet production without interfering with the hemostatic function of platelets may offer a safe alternative to current therapies for prevention of stroke and heart attack triggered by blood clotting.

Novel thrombopoietic agents: a review of their use in idiopathic thrombocytopenic purpura.

The underlying problem in idiopathic thrombocytopenic purpura (ITP) has traditionally been recognized as accelerated platelet destruction. However, recent studies have provided evidence that the pathophysiology of ITP is more complex, and impaired platelet production has emerged as one of the mechanisms contributing to the thrombocytopenia. On these grounds, second-generation thrombopoietic agents have been used in clinical trials to stimulate platelet production in ITP patients who are not responsive to standard treatments. These new molecules bear no structural resemblance to thrombopoietin (TPO) but still bind and activate the TPO receptor. Studies have been completed for two TPO receptor agonists: romiplostim (formerly AMG 531) and eltrombopag (formerly SB497115). Romiplostim is a recombinant protein defined as a peptibody. Results of phase I-II trials published recently demonstrated that romiplostim given as a weekly subcutaneous injection for 1-6 weeks results in doubling of platelet counts and an increase to >50 x 10(9)/L in most treated patients with minimal adverse effects. Eltrombopag is an orally available, small organic compound. In a randomized, double-blind, placebo-controlled phase III trial, ITP patients were given daily oral treatment with placebo or eltrombopag 50 mg. Platelet responses were observed in 59% of eltrombopag-treated patients and in 16% of patients in the placebo arm. No significant adverse events were seen. Other thrombopoietic agents in development, such as AKR-501 (formerly YM 477), appear promising in healthy volunteers. Ongoing phase III clinical trials will reveal the potential of these agents in the management of ITP prior to splenectomy and for long-term maintenance therapy, as well as their relative benefit compared with standard of care treatment.

New drugs for familiar therapeutic targets: thrombopoietin receptor agonists and immune thrombocytopenic purpura.

Various agents to treat immune thrombocytopenic purpura (ITP) have been developed on the principle that stimulating the thrombopoietin (TPO) receptor would increase platelet production. First-generation agents--recombinant human thrombopoietin (rHuTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF)--showed promise, but observations of antibody formation to PEG rHuMGDF led to the discontinuation of development of both agents. Second-generation agents--the TPO peptide mimetics, TPO non-peptide mimetics, and TPO agonist antibodies--have been developed to reduce or eliminate the problem of antigenicity. Clinical studies for some of these agents, such as AMG 531 (romiplosim, Nplate) and eltrombopag (Promacta), are demonstrating their relative safety and efficacy in increasing platelet counts in patients with ITP; AMG 531 and eltrombopag are in late-stage clinical development and are able to stimulate platelet production in patients with ITP. Some differences in safety profiles have been described and are undergoing further study. There are currently seven second-generation TPO receptor agonists that have been reported in the literature, representing the potential advantages--and continuing challenges--with this novel class of platelet-stimulating therapies for ITP and possibly thrombocytopenia in other disease states as well.

Grafting of thrombopoietin-mimetic peptides into cystine knot miniproteins yields high-affinity thrombopoietin antagonists and agonists.

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high-affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin-mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c-Mpl receptor agonists with EC(50) values in the low nanomolar or picomolar range. One selected miniprotein-derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand-induced dimerization.

Increased megakaryocytopoiesis in Lyn-deficient mice.

Previous studies in cell lines have shown Lyn kinase to be a negative regulator of thrombopoietin (TPO)-induced proliferation. To further investigate the role of Lyn during megakaryocytopoiesis, Lyn-deficient mice (lyn(-/-)) were analyzed. We observed that lyn(-/-) mice have more bone marrow-derived GPIIB (CD41) and Mpl(+) cells when compared to their wild-type littermates. In addition, colony-forming unit-megakaryocytes (CFU-MK) are increased and TPO-induced expansion of primary marrow cells yielded a greater number of mature megakaryocytes (MKs) with increased nuclear ploidy. Histopathology of bone marrow and spleens from lyn(-/-) mice showed an increase in the number of MKs. Mechanistic studies revealed that TPO stimulation of MKs from lyn(-/-) mice did not affect phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 3, STAT5, or MAP kinase kinase (MEK). Lyn-deficient MKs supported greater TPO-mediated phosphorylation and kinase activity of both Erk1/2 (mitogen-activated protein kinase, MAPK) and Akt. In contrast, there was a reduction of tyrosine phosphorylation of the inositol phosphatase, SHIP. This is the first direct evidence using primary MKs from Lyn-deficient mice that confirms our prior data from cell lines that Lyn kinase is a negative regulator of TPO signaling.

Role of beta(1)-integrins and their associated tetraspanin molecules in fibronectin-enhanced megakaryopoiesis.

BACKGROUND: We have shown previously that fibronectin (FN) together with megakaryocyte (Mk) growth and development factor (MGDF) enhanced generation of Mk progenitors determined by colony-forming unit (CFU)-Mk assay. MGDF can activate beta(1)-integrins on progenitor cells and increase binding to FN. We studied the role of beta(1)-integrin-tetraspanin complexes by which FN may enhance megakaryopoiesis. METHODS: Cord blood CD34(+) cells were cultured for up to 8 days in serum-free medium containing IL-3, IL-6 and SCF with or without MGDF in the presence or absence of FN. Immunofluorescence flow cytometry was used to monitor changes in beta(1)-integrin-tetraspanin complexes. CFU-Mk assay was used to assess Mk commitment. RESULTS: The cocktail of cytokines irrespective of the presence of MGDF altered the percentage expression of beta(1)-integrins CD49d and CD49e on CD34(+) cells. CD49d expression fell initially (98% to 15%) and then rose to 75%, whereas CD49e progressively increased over the 8 days of culture, from 5.4% to 22%. However, with the addition of FN, similar changes in the expression of beta(1)-integrins were observed but the expression was maintained at a higher level. MGDF and FN increased expression of tetraspanin molecules, CD63 and CD151, as well as their co-expression with the beta(1)-integrins on both the CD34(+) and CD34(-) cells (e.g. and increase from 0% to 80% co-expression of CD49d and CD151 on CD34(+)). Blocking of beta(1)-integrins or the tetraspanin CD151 with the respective MAb reduced Mk progenitor generation in a stromal cell model. DISCUSSION: FN enhanced Mk progenitor generation through modulation of beta(1)-integrin-tetraspanin complexes, such as CD151/CD49d.

Ticlopidine inhibits the prothrombotic effects of thrombopoietin and ameliorates survival after supralethal total body irradiation.

Thrombopoietin modulates the response of platelets to several agonists and, on the other hand, those agonists can be released following irradiation. Thus, we have determined the effects of thrombopoietin, on its own and in combination with ticlopidine, an anti-platelet drug, on platelet activation, thrombosis formation and survival of irradiated C57BL6/J mice. Administration of thrombopoietin 2 h after 9 Gy total body irradiation increased the 125I-fibrin deposition in mouse tissues and accelerated platelet consumption as revealed by an enhanced drop in platelet counts. Additionally, the number of activated platelets, i.e. those expressing P-selectin on their membrane, was higher in thrombopoietin-treated mice as compared to the placebo group, regardless ex vivo stimulation with agonists. These effects of thrombopoietin on platelet activation and consumption were reduced when mice were pretreated with ticlopidine. The combination of ticlopidine with thrombopoietin almost fully promoted 180-day survival, reaching the same efficacy as bone marrow transplantation, while only 30% of the mice treated with thrombopoietin alone survived. In summary, thrombopoietin induces long term-mortality of irradiated mice probably through platelet-mediated thrombosis and thus, ticlopidine efficiently counteracts these adverse effects of thrombopoietin.

Transforming growth factor-beta1 interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells.

OBJECTIVE: Thrombopoietin (TPO) and transforming growth factor-beta(1) (TGF-beta(1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta(1) interferes directly with TPO-induced signal transduction in hematopoietic cells, we compared the regulatory effects in the TPO-responsive cell lines Mo-7e and HEL. MATERIALS AND METHODS: The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation (3H thymidine incorporation), viability (trypan blue exclusion), and protein expression and phosphorylation (Western blot). RESULTS: TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation, whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO. TPO-induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an inhibitor of Janus kinase-2, which completely blocked TPO-induced proliferation. As demonstrated by Western blotting, TGF-beta1 reduced the TPO-stimulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor (Na3VO4), which suggests that TGF-beta1 activated a phosphatase. Although STAT3 also was activated by TPO, STAT3 activation remained unaltered by TGF-beta1. CONCLUSION: Taken together, these data suggest that TGF-beta1 modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TPO-induced signal transduction, particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5.

Characterization of the truncated thrombopoietin variants.

Thrombopoietin (Tpo) is a specific cytokine which regulates megakaryocyte differentiation and maturation. We isolated a truncated mouse Tpo cDNA, the product of which turned out to function neither as an active Tpo variant nor as an antagonist. To define the functional domains of the Tpo molecule further, various truncated and point-mutated Tpo molecules were prepared and their biological activity was assayed. It was found that deletion of the amino terminal side of a potential proteolytic cleavage site, Arg-Arg motif, caused complete loss of Tpo's activity, and that point-mutants lacking one of four conserved cysteine residues lost Tpo activity. We also noticed that Tpo activity was inhibited by the reducing agent. Thus, it was concluded that the amino terminal half of the Tpo is sufficient for Tpo activity, and that the cysteine residues, especially the last cysteine residue located two amino acids away from the Arg-Arg motif, are critical for this activity.