Glucocorticoids regulate thrombopoiesis by remodeling the megakaryocyte transcriptome.

BACKGROUND: Glucocorticoids are widely known for their immunomodulatory action. Their synthetic analogs are used to treat several autoimmune diseases, including immune thrombocytopenia. However, their efficacy and mechanisms of action in immune thrombocytopenia are not fully understood. OBJECTIVES: To investigate the mechanism of glucocorticoid actions on platelet production. METHODS: The actions of glucocorticoids on platelet production were studied combining in vivo, ex vivo and in vitro approaches. RESULTS: Dexamethasone reduced bleeding in mice and rapidly increased circulating young platelet counts. In vitro glucocorticoid treatment stimulated proplatelet formation by megakaryocytes and platelet-like particle release. This effect was blocked by glucocorticoid receptor antagonist RU486, indicating a glucocorticoid receptor-dependent mechanism. Genome-wide analysis revealed that dexamethasone regulates the expression of >1000 genes related to numerous cellular functions, including predominant cytoplasm and cytoskeleton reorganization. Dexamethasone and other glucocorticoids induced the expression of Gda (the gene encoding guanine deaminase), which has been reported to have a role in dendrite development. Inhibition of guanine deaminase enzymatic activity blocked dexamethasone stimulation of proplatelet formation, implicating a critical role for this enzyme in glucocorticoid-mediated platelet production. CONCLUSION: Our findings identify glucocorticoids as new regulators of thrombopoiesis.

Pathogenic mechanisms contributing to thrombocytopenia in patients with systemic lupus erythematosus.

SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].

Publisher Correction: Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia.

Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.

Platelet Apoptosis in Adult Immune Thrombocytopenia: Insights into the Mechanism of Damage Triggered by Auto-Antibodies.

Mechanisms leading to decreased platelet count in immune thrombocytopenia (ITP) are heterogeneous. This study describes increased platelet apoptosis involving loss of mitochondrial membrane potential (ΔΨm), caspase 3 activation (aCasp3) and phosphatidylserine (PS) externalization in a cohort of adult ITP patients. Apoptosis was not related to platelet activation, as PAC-1 binding, P-selectin exposure and GPIb-IX internalization were not increased. Besides, ITP platelets were more sensitive to apoptotic stimulus in terms of aCasp3. Incubation of normal platelets with ITP plasma induced loss of ΔΨm, while PS exposure and aCasp3 remained unaltered. The increase in PS exposure observed in ITP platelets could be reproduced in normal platelets incubated with ITP plasma by adding normal CD3+ lymphocytes to the system as effector cells. Addition of leupeptin -a cathepsin B inhibitor- to this system protected platelets from apoptosis. Increased PS exposure was also observed when normal platelets and CD3+ lymphocytes were incubated with purified IgG from ITP patients and was absent when ITP plasma was depleted of auto-antibodies, pointing to the latter as responsible for platelet damage. Apoptosis was present in platelets from all patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies but was absent in the patient with anti-GPIa-IIa auto-antibodies. Platelet damage inversely correlated with platelet count and decreased during treatment with a thrombopoietin receptor agonist. These results point to a key role for auto-antibodies in platelet apoptosis and suggest that antibody-dependent cell cytotoxicity is the mechanism underlying this phenomenon.

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 ß3 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.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle.

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.