Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Novel SLFN14 mutation associated with macrothrombocytopenia in a patient with severe haemorrhagic syndrome.

BACKGROUND: Platelet-type bleeding disorder 20 (BDPLT20), as known as SLFN14-related thrombocytopenia, is a rare inherited thrombocytopenia (IT). Previously, only 5 heterozygous missense mutations in the SLFN14 gene have been reported. METHODS: A comprehensive clinical and laboratory examination of a 17-year-old female patient with macrothrombocytopenia and severe mucocutaneous bleeding was performed. Examination was carried out using standardized questionnaires to assess bleeding, high-throughput sequencing (Next Generation Sequencing), optical and fluorescence microscopy, flow cytometry with activation and analysis of intracellular calcium signaling of platelets, light transmission aggregometry and thrombus growth in the flow chamber. RESULTS: Analysis of the patient's genotype revealed a previously undescribed c.655 A > G (p.K219E) variant in the hotspot of the SLFN14 gene. Immunofluorescence and brightfield examination of platelets in the smear showed heterogeneity in cells size, including giant forms over 10 µm (normal size 1-5) in diameter, with vacuolization and diffuse distribution of ß1-tubulin and CD63. Activated platelets showed impaired contraction and shedding/internalization of GPIb. GP IIb/IIIa clustering was increased at rest and attenuated upon activation. Intracellular signalling study revealed impaired calcium mobilization upon TRAP 35.97 nM (reference range 180 ± 44) and CRP-XL 10.08 nM (56 ± 30) stimulation. Aggregation with ADP, collagen, TRAP, arachidonic acid and epinephrine was impaired in light transmission aggregometry; agglutination with ristocetin persisted. In the flow chamber with a shear rate of 400 s-1 platelet adhesion to collagen and clot growth were impaired. CONCLUSION: The revealed disorders of phenotype, cytoskeleton and intracellular signaling explain the nature of SLFN14 platelet dysfunction and the patient's severe hemorrhagic syndrome.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

De novo TP53 germline activating mutations in two patients with the phenotype mimicking Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, associated with mutations in ribosomal protein (RP) genes. Growing data on mutations in non-RP genes in patients with DBA-like phenotype became available over recent years. We describe two patients with the phenotype of DBA (onset of macrocytic anemia within the first year of life, paucity of erythroid precursors in bone marrow) and germline de novo variants in the TP53 gene. Both patients became transfusion independent, probably due to L-leucine therapy. The possible role of TP53 variants should be considered in patients with DBA-like phenotype and no mutations in RP genes.

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

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.

Heterogeneity of Integrin αIIbß3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis.

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 αIIbß3 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.

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

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 JAK2V617F mutation, 4 had mutations in CALR 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, P-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.

Flow cytometry for pediatric platelets.

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.

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

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.

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

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.