Publications by authors named "Daria Fedorova"

7 Publications

  • Page 1 of 1

Platelet function and bleeding at different phases of childhood immune thrombocytopenia.

Sci Rep 2021 Apr 30;11(1):9401. Epub 2021 Apr 30.

National Medical Research Center of Pediatric Hematology, Oncology and Immunology Named After Dmitry Rogachev, Russian Ministry of Healthcare, 1 Samory Mashela Str, Moscow, Russia, 117997.

Immune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.
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http://dx.doi.org/10.1038/s41598-021-88900-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087794PMC
April 2021

Heterogeneity of Integrin αβ Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis.

Int J Mol Sci 2020 Apr 25;21(9). Epub 2020 Apr 25.

National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, 1 Samory Mashela St, Moscow 117198, Russia.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin αβ functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.
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http://dx.doi.org/10.3390/ijms21093035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246588PMC
April 2020

Platelet function and blood coagulation system status in childhood essential thrombocythemia.

Platelets 2020 Nov 19;31(8):1001-1011. Epub 2019 Dec 19.

Oncology and Immunology, Federal Research and Clinical Centre of Pediatric Hematology , Moscow, Russian Federation, Russia.

Childhood essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder. The quality of life of ET patients may decrease as a result of ischemic and hemorrhagic complications of unclear origin. Our goal was to characterize the hemostatic system in children with ET. We genotyped and investigated blood samples from 20 children with ET in a prospective case series study using platelet aggregation, functional flow cytometry (FC) assay and standard clotting assays. Three children had a mutation, 4 had mutations in and 13 were triple-negative. Myelofibrosis in stage 1-2 was detected in 3 children. Three patients had bleeding episodes and seven had ischemic events. Aggregation in response to collagen, adenosine diphosphate, and ristomycin was decreased in all patients. In FC, significant changes in the whole patient group compared to the healthy children control group were decrease in the resting forward scatter and PAC1 binding (activated GPIIb/IIIa) level. For the activated platelets, dense granules release (by mepacrine), PAC1, and GPIIb/IIIa levels were significantly decreased. GPIb/V/IX, -selectin, and phosphatidylserine levels manifested only moderate differences. Forward and side scatter changes in response to stimulation (representing shape change) and dense granules release were significantly lower in the 3 patients with bleeding than in the 17 patients without hemorrhage. Activated partial thromboplastin time was slightly prolonged, prothrombin index was slightly shortened and thrombin time was normal, while fibrinogen was mildly decreased in the ET patients. It could be concluded that the observed platelet function defects could be related to bleeding in ET, and be potentially used as a marker.
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http://dx.doi.org/10.1080/09537104.2019.1704710DOI Listing
November 2020

Flow cytometry for pediatric platelets.

Platelets 2019 4;30(4):428-437. Epub 2018 Oct 4.

a Cellular Hemostasis and Thrombosis Lab , National Medical Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Russian Ministry of Healthcare , Moscow , Russian Federation.

The ability of platelets to carry out their hemostatic function can be impaired in a wide range of inherited and acquired conditions: trauma, surgery, inflammation, pre-term birth, sepsis, hematological malignancies, solid tumors, chemotherapy, autoimmune disorders, and many others. Evaluation of this impairment is vitally important for research and clinical purposes. This problem is particularly pronounced in pediatric patients, where these conditions occur frequently, while blood volume and the choice of blood collection methods could be limited. Here we describe a simple flow cytometry-based screening method of comprehensive whole blood platelet function testing that was validated for a range of pediatric and adult samples (n = 31) in the hematology hospital setting including but not limited to: classic inherited platelet function disorders (Glanzmann's thrombasthenia; Bernard-Soulier, Wiscott-Aldrich, and Hermasky-Pudlak syndromes, MYH9-dependent thrombocytopenia), healthy and pre-term newborns, acute and chronic immune thrombocytopenia, chronic lympholeukemia, effects of therapy on platelet function, etc. The method output includes levels of forward and side scatter, levels of major adhesion and aggregation glycoproteins Ib and IIb-IIIa, active integrins' level based on PAC-1 binding, major alpha-granule component P-selectin, dense granule function based on mepacrine uptake and release, and procoagulant activity quantified as a percentage of annexin V-positive platelets. This analysis is performed for both resting and dual-agonist-stimulated platelets. Preanalytical and analytical variables are provided and discussed. Parameter distribution within the healthy donor population for adults (n = 72) and children (n = 17) is analyzed.
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http://dx.doi.org/10.1080/09537104.2018.1513473DOI Listing
May 2019

Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.

PLoS One 2015 24;10(4):e0125522. Epub 2015 Apr 24.

VelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.

In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125522PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409293PMC
April 2016

Hsp90 Co-localizes with Rab-GDI-1 and regulates agonist-induced amylase release in AR42J cells.

Cell Physiol Biochem 2009 4;24(5-6):369-78. Epub 2009 Nov 4.

Hunter College, New York, NY 10010, USA.

Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI:Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.
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http://dx.doi.org/10.1159/000257429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711583PMC
February 2010
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