Rosana F. Marta

JAK2V617F mutation in platelets from essential thrombocythemia patients: correlation with clinical features and analysis of STAT5 phosphorylation status.

Objective:  JAK2V617F mutation rate in granulocytes from essential thrombocythemia (ET) patients ranges from 12% to 57%. Our aim was to evaluate the frequency of this mutation in the megakaryocyte/platelet lineage, and to analyze its clinical associations in ET. In addition, we determined whether this mutation leads to constitutive phosphorylation of STAT5 in platelets.

State-of-the-Art Review : Effectiveness of Anagrelide in the Treatment of Symptomatic Patients with Essential Thrombocythemia

We prospectively evaluated the effect of anagrel ide on platelet counts and the clinical manifestations of micro vascular circulation disturbances in 17 newly diagnosed pa tients with essential thrombocythemia. Ten patients had symp toms related to thrombocythemia, eight at the time of starting anagrelide treatment. The platelet counts before anagrelide treatment and during maintained remission of essential throm bocythemia by anagrelide were 980 (range, 610-2030) and 378 (range, 212-546) x 109/L, respectively. Spontaneous platelet aggregation was found in 6 patients (35%), which disappeared on remission of essential thrombocythemia in five cases (P = 0.02). Essential thrombocythemia-related microvascular thrombotic and hemorrhagic symptoms disappeared with the normalization of platelet count in all cases during maintained remission of essential thrombocythemia by long term continu ous anagrelide treatment with a follow-up period of between 2 and 6 years. However, ET-related symptoms reappeared in three patients, coinciding with increased platelet count up to 600 x 109/L caused by anagrelide dose reduction. We conclude that reduction of increased platelet to normal (< 400 x 109/L) in symptomatic patients with essential thrombocythemia through use of maintained anagrelide treatment is associated with the disappearance of spontaneous platelet aggregation and the complete relief of thrombotic and hemorrhagic manifestations.

Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood‐derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose‐dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma‐induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet‐adsorbed plasma induced its reversion, suggesting the involvement of auto‐antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti‐GPIIb‐IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC‐1 binding and β3 integrin phosphorylation while the presence of anti‐glycoprotein Ia‐IIa auto‐antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.

Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets

Familial platelet disorder with a predisposition to acute myelogenous leukemia (FPD/AML) is an inherited platelet disorder caused by a germline RUNX1 mutation and characterized by thrombocytopenia, a platelet function defect, and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1‐regulated genes involved in these defects.

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Summary.  Background:  Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource‐limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina.

Dysregulation of stromal derived factor 1/CXCR4 axis in the megakaryocytic lineage in essential thrombocythemia.

This study investigated the involvement of chemokines including stromal derived factor 1 (SDF‐1), interleukin 8 (IL‐8), growth‐related oncogene alpha (GRO‐α) and their receptors, CXCR4, CXCR2 and CXCR1 in essential thrombocythemia (ET), a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. Fifty‐three ET patients were studied. Plasma levels of SDF‐1, IL‐8 and GRO‐α, evaluated by enzyme‐linked immunosorbent assay, and flow cytometric analysis of CXCR1 and CXCR2 on the platelet membrane, were found to be normal in ET patients. CXCR4 expression on platelet surface as well as platelet CXCR4 mRNA detected by real‐time reverse transcription polymerase chain reaction, were decreased. Platelet CXCR4 internalization rate was normal while SDF‐1‐induced platelet aggregation was delayed, decreased or absent. Immunohistochemical staining revealed that megakaryocytes were also affected. CXCR4 decrease was not observed either in peripheral white blood cells or in circulating CD34+ precursors. These results show that CXCR4 is decreased in the megakaryocytic lineage in ET, mainly due to a reduced CXCR4 production, and an abnormal platelet response to SDF‐1. This report is the first to describe platelet and megakaryocytic CXCR4 deficiency in a human disease and the presence of this abnormality in a megakaryocytic‐related illness highlights the important role of SDF‐1/CXCR4 axis in platelet development.

Production of functional platelet-like particles by the megakaryoblastic DAMI cell line provides a model for platelet biogenesis

The aim of this study was to evaluate cell maturation and the platelet production capacity of the megakaryoblastic DAMI cell line, to characterize platelet-like particles produced and to investigate the mechanisms involved in their production. DAMI cell maturation was induced by phorbol myristate acetate (PMA) and thrombopoietin (TPO). Expression levels of GATA-1, Fli-1 and NF-E2 were evaluated using real-time PCR and western blot. Platelet-like particles were characterized by the presence of GPIb and GPIIb by flow cytometry, while the soluble fragment of GPIb, glycocalicin, was detected by enzyme immunoassay. Dense and alpha granules were evaluated by mepacrine staining and thrombospondin-1 detection, respectively, and by electron microscopy. Functional capacity of platelet-like particles was studied by measuring P-selectin membrane after thrombin stimulation by flow cytometry and actin polymerization using phalloidin-FITC by immunofluorescence. We found that stimulation of DAMI cells with high concentration of PMA and TPO induced the expression of transcription factors GATA-1 and Fli-1 followed by an increase in the isoform a of NF-E2. Mature DAMI cells give rise to extensions resembling proplatelets and later, produce platelet-like particles expressing GPIIb and GPIb on their surface and containing dense and alpha granules, which were confirmed by electron microscopy. Platelet functionality was demonstrated by the increase in P-selectin membrane expression after thrombin stimulation and by their ability to spread on fibrinogen matrices. DAMI cell line induced to differentiate into mature megakaryocytes is able to produce functional platelets providing a suitable model to study the mechanisms involved in platelet generation.

Platelets possess functional TGF-β receptors and Smad2 protein

TGF-β1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-β receptors on platelets, the effect of TGF-β1 on platelet aggregation and the underlying intracellular mechanisms. TGF-β receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-β receptor type II and mRNA codifying for TβRI and TβRII were found in platelets. We demonstrated that TGF-β1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-β1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-β1. Besides, TGF-β1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-β1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-β1 on platelet aggregation could play a role during pathological states in which circulating TGF-β1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

Systemic disorders in Argentine haemorrhagic fever

Patients with Argentine haemorrhagic fever have mainly central nervous system involvement and haemorrhagic manifestations, with leucopenia and thrombocytopenia. Activation of blood coagulation, fibrinolysis, the complement system and cytokines was observed in the acute phase of the disease.

Anagrelide platelet-lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms

Anagrelide represents a treatment option for essential thrombocythemia patients. It lowers platelet counts through inhibition of megakaryocyte maturation and polyploidization, although the basis for this effect remains unclear. Based on its rapid onset of action, we assessed whether, besides blocking megakaryopoiesis, anagrelide represses proplatelet formation (PPF) and aimed to clarify the underlying mechanisms.