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

BACKGROUND AND OBJECTIVES: 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. METHODS AND RESULTS: Exposure of cord blood-derived megakaryocytes to anagrelide during late stages of culture led to a dose- and time-dependent inhibition of PPF and reduced proplatelet complexity, which were independent of the anagrelide-induced effect on megakaryocyte maturation. Whereas anagrelide was shown to phosphorylate cAMP-substrate VASP, two pharmacologic inhibitors of the cAMP pathway were completely unable to revert anagrelide-induced repression in megakaryopoiesis and PPF, suggesting these effects are unrelated to its ability to inhibit phosphodiesterase (PDE) 3. The reduction in thrombopoiesis was not the result of down-regulation of transcription factors which coordinate PPF, while the myosin pathway was identified as a candidate target, as anagrelide was shown to phosphorylate the myosin light chain and the PPF phenotype was partially rescued after inhibition of myosin activity with blebbistatin. CONCLUSIONS: The platelet-lowering effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregulated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide's cardiovascular side-effects and antiplatelet activity. Further work in this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with an improved pharmacologic profile.

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.

BACKGROUND: 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. METHODS: Lumiaggregometry, α-granule and dense granule content and release, platelet ultrastructure, αIIb ß3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assays and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1. RESULTS: A severe decrease in platelet aggregation, defective αIIb ß3 integrin activation and combined αδ storage pool deficiency were found. However, whereas the number of dense granules was markedly reduced, α-granule content was heterogeneous. A trend towards decreased platelet spreading was found, and ß3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in the level of transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind to the NF-E2 promoter in primary megakaryocytes, and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression. CONCLUSIONS: The FPD/AML platelet function defect represents a complex trait, and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights the RUNX1 target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function.

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

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. METHODS: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. RESULTS: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. CONCLUSIONS: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.

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.

Monocyte IL-2Ralpha expression is associated with thrombosis and the JAK2V617F mutation in myeloproliferative neoplasms.

The development of bone marrow fibrosis and thrombosis are main causes of morbidity in essential thrombocythemia (ET). Monocyte activation has been associated to the production of fibrosis-related cytokines and pro-thrombotic factors. The aim of this study was to identify new markers of monocyte activation in Phi-negative myeloproliferative neoplasms and to search for their relationship with clinical features. Forty-five patients comprising 30 ET, eight myelofibrosis and seven polycythemia vera were included. We evaluated the alpha subunit of IL-2 receptor (CD25) on monocytes, basal and LPS-induced IL-1beta release from mononuclear cells, and monocyte TGF-beta mRNA content. Patients who had thrombotic events displayed higher monocyte CD25 levels (6.2%) than those without symptoms (1.3%) and controls (2.6%), p=0.0006. JAK2V617F-positive patients had higher monocyte CD25 expression levels (4.7%), than JAK2V617F-negative (2.6%), p=0.0213. Patients with myeloproliferative neoplasms had similar monocyte CD25 expression than controls, both, in basal conditions and after cell adhesion. IL-1beta release and TGF-beta mRNA levels were normal. In conclusion, increased monocyte CD25 expression is associated with history of thrombosis and is also up-regulated in patients harboring JAK2V617F mutation. The finding of increased CD25 levels together with normal IL-1beta and TGF-beta production reveals a selective monocyte activation profile in myeloproliferative neoplasms.

Platelets possess functional TGF-beta receptors and Smad2 protein.

TGF-beta1 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-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta 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-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 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-beta1 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-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 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.

Variation of PDGF, TGFbeta, and bFGF levels in essential thrombocythemia patients treated with anagrelide.

We studied 15 patients with essential thrombocythemia (ET) before treatment and after normalization of platelet count by anagrelide. Significantly increased plasma levels of PDGF, TGFbeta, and bFGF were found. Patients with mild reticulin fibrosis in bone marrow had higher PDGF levels. During treatment, plasma TGFbeta and bFGF levels remained elevated in most patients (P < 0.0001 and P < 0.01, respectively). Intraplatelet PDGF levels were low before treatment (P < 0.006) and normal on hematological remission, without relation with the presence or absence of reticulin fibrosis in bone marrow. Intraplatelet TGFbeta levels were normal regardless of the platelet count. Intraplatelet bFGF levels were raised before (P < 0.001) and during treatment (P < 0.01). By immunostaining, TGFbeta and bFGF were seen in megakaryocytes and lymphocytes with a similar pattern of intensity in patients and controls, suggesting that other cells might also contribute to the raised plasma values. We believe that the plasma increment of these cytokines suggests that they play a role in the pathogenesis of ET. The normal PDGF plasma level found during treatment may be in relation with the platelet count. However, the persistent increase of TGF-beta in plasma and bFGF both in plasma and platelets may indicate dysregulation of cytokine synthesis in TE.

Effectiveness of anagrelide in the treatment of symptomatic patients with essential thrombocythemia.

We prospectively evaluated the effect of anagrelide on platelet counts and the clinical manifestations of microvascular circulation disturbances in 17 newly diagnosed patients with essential thrombocythemia. Ten patients had symptoms related to thrombocythemia, eight at the time of starting anagrelide treatment. The platelet counts before anagrelide treatment and during maintained remission of essential thrombocythemia by anagrelide were 980 (range, 610-2030) and 378 (range, 212-546) x 10(9)/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 continuous 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 10(9)/L caused by anagrelide dose reduction. We conclude that reduction of increased platelet to normal (< 400 x 10(9)/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.

Relationship between hematopoietic growth factors levels and hematological parameters in Argentine hemorrhagic fever.

Argentine hemorrhagic fever (AHF) is a viral disease caused by Junin virus and characterized by hematologic and neurological involvement. The main hematologic features are leukopenia, thrombocytopenia, and bone marrow hypoplasia. Hematopoietic growth factors serum levels were measured by ELISA technique in forty-eight patients with confirmed diagnosis of AHF. Patients were classified according to the clinical picture in 15 severe (SCF), 17 moderate (MoCF), and 16 mild (MiCF) cases. Erythropoietin levels were decreased in 28 of 45 patients and raised in 4 SCF patients. Twenty-four of 38 patients had high G-CSF levels at admittance in accordance with clinical picture severity, while IL-3, GM-CSF, and TGF-beta were normal in most cases. A direct correlation was found between G-CSF and TNF-alpha levels. Thrombopoietin levels were found to be raised in 19 of 21 patients. In conclusion, the low levels of Epo may contribute to the severe bone marrow erythroblastopenia described in AHF patients, while G-CSF seems to be a marker of illness severity.

[Thromboxane B2 and platelet derived growth factor in essential thrombocythemia treated with anagrelide]. / Tromboxano B2 y factor de crecimiento derivado de las plaquetas en la trombocitemia esencial tratada con anagrelide.

We present herein studies carried out in 17 patients with essential thrombocythemia before treatment with anagrelide and on remission. Ten patients had symptoms related to thrombocythemia, 8 of them at the time of starting treatment. The plasmatic levels of TXB2 and PDGF were measured by ELISA technique. Before treatment, PDGF values corrected for the platelet count were lower than controls, 0.48 ng/10(5) platelets (0.13-1.93) and 0.92 ng/10(5) platelets (0.33-1.16) respectively (p = 0.02), and they were not different from the results obtained during remission. Count-corrected TXB2 levels before treatment were higher than the control group, 1.0 ng/10(5) platelets (0.04-14.4) and 0.25 ng/10(5) platelets (0.13-0.39) respectively (p = 0.04); these values decreased during remission 0.86 ng/10(5) platelets (0.07-9.8) (p = 0.04), although they were still above normal values (p = 0.008). Symptoms disappeared with the normalization of platelet counts in all cases. These results show that patients with essential thrombocythemia during remission have a tendency to normalize the count-corrected TXB2 values.

Tromboxano B2 y factor de crecimiento derivado de las plaquetas en la trombocitemia esencial tratada con anagrelide. / [Thromboxane B2 and platelet derived growth factor in essential thrombocythemia treated with anagrelide]

We present herein studies carried out in 17 patients with essential thrombocythemia before treatment with anagrelide and on remission. Ten patients had symptoms related to thrombocythemia, 8 of them at the time of starting treatment. The plasmatic levels of TXB2 and PDGF were measured by ELISA technique. Before treatment, PDGF values corrected for the platelet count were lower than controls, 0.48 ng/10(5) platelets (0.13-1.93) and 0.92 ng/10(5) platelets (0.33-1.16) respectively (p = 0.02), and they were not different from the results obtained during remission. Count-corrected TXB2 levels before treatment were higher than the control group, 1.0 ng/10(5) platelets (0.04-14.4) and 0.25 ng/10(5) platelets (0.13-0.39) respectively (p = 0.04); these values decreased during remission 0.86 ng/10(5) platelets (0.07-9.8) (p = 0.04), although they were still above normal values (p = 0.008). Symptoms disappeared with the normalization of platelet counts in all cases. These results show that patients with essential thrombocythemia during remission have a tendency to normalize the count-corrected TXB2 values.

Proinflammatory cytokines and elastase-alpha-1-antitrypsin in Argentine hemorrhagic fever.

Argentine hemorrhagic fever (AHF) is a disease caused by Junin virus. In the acute phase, patients present hematologic and neurologic involvement with high levels of interferon-alpha and tumor necrosis factor-alpha (TNF-alpha. Nineteen patients with a confirmed diagnosis of AHF were studied: six severe, four moderate and nine mild cases. Serum levels of interleukin-6 (IL-6), IL-6 soluble receptor (IL-6sR), IL-8, IL-10, and elastase-alpha1-antitrypsin complex (E-alpha 1AT) were assayed by ELISAs. Levels of IL-6, IL-8, and IL-10 were high in nine, 12, and 13 patients, respectively, while levels of IL-6sR were high in two patients and low in one patient. Seven patients had increased levels of E-alpha1AT. Significant correlations were found between levels of both IL-8 and IL-10 with those of TNF-alpha as well as between IL-8 and E-alpha 1AT. These data demonstrate activation of pro-inflammatory and anti-inflammatory cytokine pathways, and statistical analysis showed differences among the clinical forms of illness. This study shows that IL-8 plays an essential role in neutrophil activation in AHF patients as demonstrated in other infectious diseases.

[Catastrophic antiphospholipid syndrome. Communication of 2 forms of presentation]. / Síndrome antifosfolipídico catastrófico. Comunicación de dos formas de presentación.

Antiphospholipid syndrome is characterized by recurrent fetal loss, arterial and venous thromboses, thrombocytopenia and circulating antiphospholipid antibodies. Few patients have a rapidly progressive, fatal outcome. We report two young patients with systemic lupus erythematosus and antiphospholipid antibodies who died after a short course of disease. Although clinical and laboratory findings differed in both patients--small vessel thromboses and microangiopathic hemolytic anemia mimicking thrombotic thrombocytopenic purpura predominated in one of the patients while small and medium size vessel thromboses without hemolysis were present in the other case--autopsy revealed widespread visceral thromboses in both of them, features consistent with a diagnosis of catastrophic antiphospholipid syndrome. This syndrome has not been reported to occur in association with Pneumocistis carinii pneumonia as we describe in one of our patients.

Síndrome antifosfolipídico catastrófico. Comunicación de dos formas de presentación. / [Catastrophic antiphospholipid syndrome. Communication of 2 forms of presentation]

Antiphospholipid syndrome is characterized by recurrent fetal loss, arterial and venous thromboses, thrombocytopenia and circulating antiphospholipid antibodies. Few patients have a rapidly progressive, fatal outcome. We report two young patients with systemic lupus erythematosus and antiphospholipid antibodies who died after a short course of disease. Although clinical and laboratory findings differed in both patients--small vessel thromboses and microangiopathic hemolytic anemia mimicking thrombotic thrombocytopenic purpura predominated in one of the patients while small and medium size vessel thromboses without hemolysis were present in the other case--autopsy revealed widespread visceral thromboses in both of them, features consistent with a diagnosis of catastrophic antiphospholipid syndrome. This syndrome has not been reported to occur in association with Pneumocistis carinii pneumonia as we describe in one of our patients.

[Markers of thrombin generation in kidney failure of children with epidemic hemolytic uremic syndrome]. / Marcadores de generación de trombina en insuficiencia renal de niños con sindrome urémico hemolítico epidémico.

We present herein the levels of the early markers of blood coagulation activation and TNF-alpha in 12 children with the epidemic form of the hemolytic-uremic syndrome, median age 16 months, range 12-18. All patients recovered from the disease within 2 to 4 weeks. Four blood samples were collected: at admission, 1 week and 2 weeks later and on remission. Prothrombin fragments 1 + 2 (F1 + 2), thrombin-antithrombin complex (TAT) and tumor necrosis factor alpha (TN-alpha) were assayed by commercial ELISA techniques, while von Willebrand factor (vWf) was measured by Laurell's method. At admission, F1 + 2 and TAT levels were 7.8 nM (3.7-12.3) and 22.7 ng/ml (8-76), respectively. Besides, significant correlations were obtained for F1 + 2 levels vs blood creatinine, r: 0.57 p < 0.001; F1 + 2 vs urea, r: 0.66 p < 0.001; TAT vs blood creatinine, r: 0.77 p < 0.001; TAT vs blood urea, r: 0.59 p < 0.001. Median vWf value at admission in 11/12 children was 260% (170-420), correlating with F1 + 2, r: 0.77 p < 0.001 and with TAT, r: 0.41 p < 0.01. Such values tended to normalize with the improvement of the disease. A negative correlation was found for platelet count vs F1 + 2, r: -0.64 p < 0.001. TNF-alpha levels were increased in 5/12 children, 22.2 pg/ml (17.2-53.7). These results may be attributable to similar stimuli on endothelial cells.

Early markers of blood coagulation and fibrinolysis activation in Argentine hemorrhagic fever.

Junin virus, an arenaviridae, is the etiological agent of Argentine hemorrhagic fever. In addition to thrombocytopenia, patients present several alterations in both the blood coagulation and the fibrinolytic system, but diffuse intravascular coagulation could not be demonstrated. To investigate further the activation status of the two systems, levels of thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2, protein C, total and free protein S, C4bBP, antithrombin III, t-PA, PAI-1 and D-dimer were measured. Fourteen patients with a confirmed diagnosis of Argentine hemorrhagic fever were included in the study, 2 were severe, 3 moderate and 9 mild clinical cases, but hemorrhages were slight throughout. Blood samples were collected for 6 consecutive days on admission and on remission. At admission TAT and F1 + 2 levels were increased in 13/14 patients, reaching 0.33 nM (0.06-0.87) and 2.16 nM (0.96-6.5), respectively. PC was low in 4 cases, fPS in 6 and tPS in 2, whereas C4bBP and ATIII values were within normal range. t-PA and D-dimer levels were high in 11/14 patients, reaching 20 ng/ml (2.7-106) and 1660 ng/ml (877-3780) respectively, while PAI-1 was considerably increased in the 2 severe cases and normal in the remainder. These results suggest low level though persistent process of blood coagulation and fibrinolysis activation in this viral hemorrhagic disease. We believe these abnormalities may lead to the well described bleeding manifestations in these patients.

[Immunoperoxidase in the studies of immune thrombocytopenias]. / La inmunoperoxidasa en el estudio de las trombocitopenias inmunológicas.

The usefulness of different techniques to measure platelet bound IgG has been reviewed by George. We present here the results obtained with a technique designed to measure membrane bound IgG employing an anti-human IgG labeled with peroxidase and using O-dianisidine-H2O2 to reveal the enzymatic activity. We studied 152 patients with chronic autoimmune thrombocytopenic (ATP) including 120 adults and 32 children (age below 15 years old), diagnosed by exclusion of diseases that may be associated with thrombocytopenic purpura of either immune or nonimmune mechanisms. Besides, 79 patients with thrombocytopenia related to other diseases were also evaluated. The normal values in 215 controls were 188 +/- 4 IgG molec/platelet (mean +/- SE), while in the whole population of chronic ATP the results were 4714 +/- 344, p < 0.001. In pediatric cases the results had a tendency to values higher than in adults. A negative correlation was found between the number of platelets and the amount of bound IgG, r = 0.41 p < 0.001. IgG bound platelets were also increased in treated patients at relapse. The percent of normal IgG bound platelet was 4.5% in patients with a platelet count below 50,000/microliters and 39% in those with normal platelet number. Patients with secondary thrombocytopenia had elevated IgG/platelet while the values were normal in patients with thrombocytopenia of unknown etiology. We conclude that the immunoperoxidase technique is useful to establish the immunologic nature of thrombocytopenia.