Las trombopoyetinas en el tratamiento de la púrpura trombocitopénica inmunitaria y otras trombocitopenias / Thrombopoietines in the treatment of the thrombocytopenic purpura and other thrombocytopenics

La purpura trombocitopénica inmunitaria y las trombocitopenias secundarias representan condiciones patológicas graves cuyo tratamiento plantea diversos grados de dificultad. La aproximación terapéutica convencional ha sido la administración de esteroides, la esplenectomía y el uso de inmunoglobulina intravenosa u otros tipos de anticuerpos (e.g., anti-D). La mejor comprensión de la fisiología y fisiopatología de la trombopoyesis aunado a los avances en biología molecular ha permitido el desarrollo de una nueva aproximación terapéutica, la aplicación de las trombopoyetinas sintéticas o no inmunogénicas. Dentro de este grupo resaltan dos compuestos: el romiplostin (una proteína de fusión) y el eltrombopag (un compuesto sintético de bajo peso molecular). Ambas se encuentran disponibles comercialmente. Los estudios clínicos indican que estos medicamentos tienen un efecto satisfactorio en el tratamiento de las trombocitopenias, particularmente en los casos refractarios a los tratamientos convencionales.

Immune thrombocytopenic purpura and the secondary thrombocytopenias are conditions potentially severe with diverse degrees of treatment difficulties. Steroids administration, splenectomy and the use of intravenous immunoglobulin and other antibodies (e.g., anti-D) had been the conventional therapy. The better understanding of the thrombopoiesis physiology and physiopathology togetter with the biology advances have permitted the development of a new terapheutic approach: the use of synthetic or nonimmunogenic thrombopoietines. Among this group highlights composites: romiplostim (a fusion protein) and eltrombopag (a synthetic composite with low molecular wheigt). Both are already available and produce a satisfactory effect particularly in nonrespondent cases to the conventional treatment.

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets.

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.

[Signaling pathways involved in megakaryopoiesis]. / Vías de señalización implicadas en la megacariopoyesis.

Hematotoxicology has been studied with less interest than other fields associated with atmospheric pollution. There is limited knowledge about on the effects that certain atmospheric pollutants may provoke in the blood and bone marrow. Suspended particle pollution has become an area of scientific inquiry due to the contaminants adhering to its surface. We have identified the association of inhaled vanadium and variations in megakaryopoyesis and thrombopoyesis. Platelets are the smallest elements in the blood, but they play a strategic role in hemostasis. They are derived from the largest cell of the bone marrow, the megakaryocite. This cell size is about 150 microm, with apolyploid nucleus and unknown origin until few years ago. When TPO was cloned in 1994 the knowledge about megakaryocyte began to growth exponentially, elucidating the mechanisms of proliferation, differentiation and release of platelets. More information is still needed in order to translate knowledge into clinical application for diseases such as thrombocytopenia or thrombocytosis. A review of the current concepts of megakaryopoiesis and its regulation, with emphasis on signaling pathways are presented in this paper; a classification in TPO-dependent and TPO-independent is also detailed. In addition, we review some diseases associated with changes in the signaling pathway of megakaryopoyesis, as well as possible perspectives in this field, including toxicology.

Junín virus infection of human hematopoietic progenitors impairs in vitro proplatelet formation and platelet release via a bystander effect involving type I IFN signaling.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN alpha/beta) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2(-/-) mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels.

Vías de señalización implicadas en la megacariopoyesis / Signaling pathways involved in megakaryopoiesis

La hematotoxicología es un área poco estudiada en nuestro país y es limitado el conocimiento sobre el efecto que ciertos contaminantes atmosféricos inducen en la sangre y en la médula ósea. La contaminación por partículas suspendidas ha cobrado más interés, por los contaminantes que se adhieren a su superficie. Un ejemplo es el benceno, relacionado con aplasia medular y leucemia. Algunos metales que también están en las partículas inhaladas son hematotóxicos. Uno de ellos es el vanadio, que nuestro grupo ha identificado como un agente inductor de alteraciones en la megacariopoyesis, lo que motivó esta revisión. Las plaquetas desempeñan un papel muy importante en la hemostasia y derivan de la célula más grande de la médula ósea: el megacariocito. Hasta hace algunos años desconocíamos casi todo del megacariocito, pero con la clonación de la trombopoyetina, en 1994, la principal hormona reguladora de la producción plaquetaria, ha existido un desarrollo acelerado en el conocimiento de la megacariopoyesis. Este artículo hace una revisión de la megacariopoyesis y su regulación, con énfasis en las vías de señalización implicadas. Además, se mencionan algunas enfermedades relacionadas y se discuten las perspectivas de investigación de este proceso, con énfasis en la toxicología.

Hematotoxicology has been studied with less interest than other fields associated with atmospheric pollution. There is limited knowledge about on the effects that certain atmospheric pollutants may provoke in the blood and bone marrow. Suspended particle pollution has become an area of scientific inquiry due to the contaminants adhering to its surface. We have identified the association of inhaled vanadium and variations in megakaryopoyesis and thrombopoyesis. Platelets are the smallest elements in the blood, but they play a strategic role in hemostasis. They are derived from the largest cell of the bone marrow, the megakaryocite. This cell size is about 150 microm, with apolyploid nucleus and unknown origin until few years ago. When TPO was cloned in 1994 the knowledge about megakaryocyte began to growth exponentially, elucidating the mechanisms of proliferation, differentiation and release of platelets. More information is still needed in order to translate knowledge into clinical application for diseases such as thrombocytopenia or thrombocytosis. A review of the current concepts of megakaryopoiesis and its regulation, with emphasis on signaling pathways are presented in this paper; a classification in TPO-dependent and TPO-independent is also detailed. In addition, we review some diseases associated with changes in the signaling pathway of megakaryopoyesis, as well as possible perspectives in this field, including toxicology.

[Megakaryopoietic cytokine levels in patients with essential thrombocythemia and their relationship with clinical and biochemical features]. / Niveles de citoquinas megacariocitopoyeticas en pacientes con trombocitemia esencial y su relacion con caracteristicas clinicas y bioquimicas.

Megakaryopoiesis and platelet production are driven by transcription factors and cytokines present in bone marrow environment. Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by high platelet count and megakaryocytic hyperplasia. In the present work we evaluated plasmatic levels of cytokines involved in megakaryocytic development in a group of patients with ET that were not on treatment, as well as thrombopoietin (TPO) levels before and during anagrelide treatment. The assays were carried out using ELISA techniques. Among the cytokines mainly involved in proliferation of megakaryocytic progenitors, interleukin 3 (IL-3) levels were found increased in patients compared to normal controls (p = 0.0383). Granulocyte-macrophage colony stimulating factor and stem cell factor levels were normal. Interleukin 6, as well as interleukin 11 and erythropoietin (EPO), cytokines mainly related to megakaryocytic maturation, were normal. Plasma TPO levels before treatment were within the normal range and increased during treatment but the difference was not statistically significant. Patients who displayed spontaneous platelet aggregation had higher plasma TPO levels compared to those who did not (p = 0.049). We did not find any relationship between cytokine levels and clinical or laboratory parameters. The high IL-3 levels seen in some patients with ET could contribute to megakaryocytic proliferation. The simultaneous occurrence of higher TPO levels and elevated platelet count could be a predisposing factor for the development of spontaneous platelet aggregation in ET patients.

Macrophage derived signalling regulates negatively the megakaryocyte compartment.

Megakaryocytopoiesis is the process by which stem cells go through a process of commitment, proliferation and differentiation leading to the production of platelets. In the mouse, this process is accomplished within the bone marrow (BM) and spleen microenvironment and is carried out by regulatory molecules and accessory cells including macrophages, fibroblasts and endothelial-like cells. Previously, we have reported that macrophage depletion following administration of liposomal clodronate (LIP-CLOD) provokes enhancement of both, megakaryocytopoiesis and thrombocytopoiesis. In this report, we investigated the changes in the compartment of megakaryocyte progenitor cells (MK-CFU), their correlation with plasmatic thrombopoietin (TPO) and TPO transcription levels after macrophage depletion. LIP-CLOD-treated mice showed an increase of the MK-CFU in BM and spleen. Concerning TPO plasma levels, kinetic studies revealed a 1.5- and 1.3-fold increase in the TPO concentration at 12 and 24 hr of treatment. We also show evidence of regulation of TPO transcription in the liver and spleen. Although empty liposomes also enhanced TPO gene regulation in these organs, transcriptional TPO up regulation correlated with an increase of protein synthesis only in those animals where macrophages were effectively removed. Taken together, these results suggest that BM and spleen macrophages derived signalling regulates negatively the megakaryocyte compartment.