scholarly journals Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

2019 ◽  
Author(s):  
Steven J. Grzegorski ◽  
Zhilian Hu ◽  
Yang Liu ◽  
Xinge Yu ◽  
Allison C. Ferguson ◽  
...  
Keyword(s):  
Late Onset ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Early Adulthood ◽  
List Type ◽  
Key Points ◽  
Severe Impairment ◽  
Biosynthesis Activation ◽  
Pleiotropic Functions

AbstractThe ability to prevent blood loss in response to injury is a critical, evolutionarily conserved function of all vertebrates. Prothrombin (F2) contributes to both primary and secondary hemostasis through the activation of platelets and the conversion of soluble fibrinogen to insoluble fibrin, respectively. Complete prothrombin deficiency has never been observed in humans and is incompatible with life in mice, limiting the ability to understand the entirety of prothrombin’sin vivofunctions. We have previously demonstrated the ability of zebrafish to tolerate loss of both pro- and anticoagulant factors that are embryonic lethal in mammals, making them an ideal model for the study of prothrombin deficiency. Using genome editing with TALENs, we have generated a null allele in zebrafishf2. Homozygous mutant embryos develop normally into early adulthood, but demonstrate eventual complete mortality with the majority of fish succumbing to internal hemorrhage by 2 months of age. We show that despite the extended survival, the mutants are unable to form occlusive thrombi in both the venous and arterial systems as early as 3-5 days of life, and we were able to phenocopy this early hemostatic defect using direct oral anticoagulants. When the equivalent mutation was engineered into the homologous residues of human prothrombin, there were severe reductions in secretion and activation, suggesting a possible role for kringle 1 in thrombin maturation, and the possibility that the F1.2 fragment has a functional role in exerting the procoagulant effects of thrombin. Together, our data demonstrate the conserved function of thrombin in zebrafish, as well as the requirement for kringle 1 for biosynthesis and activation by prothrombinase. Understanding how zebrafish are able to develop normally and survive into early adulthood without prothrombin will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.Key PointsDisruption of the kringle 1 domain of prothrombin leads to severe impairment of biosynthesis, activation, and activityProthrombin deficiency is compatible with normal development in zebrafish but leads to inability to form clots followed by early mortality

scholarly journals Improved computational analysis of ribosome dynamics from 5’P degradome data using fivepeseq

2020 ◽  
Author(s):  
Lilit Nersisyan ◽  
Maria Ropat ◽  
Vicent Pelechano
Keyword(s):  
Computational Analysis ◽  
Developmental Stages ◽  
Biological Information ◽  
List Type ◽  
Computational Tools ◽  
Ribosome Stalling ◽  
Specific Analysis ◽  
Key Points ◽  
Reproducible Analysis

ABSTRACTIn eukaryotes, 5’-3’ co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus 5’P degradome sequencing, in addition to informing about RNA decay, also provides valuable information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome, however computational tools for their reproducible analysis are lacking. Here we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5’P degradome data. This tool performs both metagene and gene specific analysis, and allows to easily investigate codon specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene specific ribosome pauses in S. cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific frameshifts. To show its wide applicability, we investigate more complex 5’P degradome from A. thaliana and discover both motif-specific ribosome protection associated with particular developmental stages, as well as generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5’P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis.KEY POINTSAnalysis of 5’P degradome data with fivepseq informs about global and gene-specific translational features.Frameshifts in translation-related genes in S. cerevisiae may be linked to ribosome stalling.Ribosome protection at termination and codon motifs are linked to development in A. Thaliana.

scholarly journals Direct oral anticoagulants compared with vitamin K antagonists for acute venous thromboembolism: evidence from phase 3 trials

Blood ◽  
2014 ◽  
Vol 124 (12) ◽  
pp. 1968-1975 ◽  
Author(s):  
Nick van Es ◽  
Michiel Coppens ◽  
Sam Schulman ◽  
Saskia Middeldorp ◽  
Harry R. Büller
Keyword(s):  
Venous Thromboembolism ◽  
Major Bleeding ◽  
Vitamin K Antagonists ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Similar Efficacy ◽  
Efficacy And Safety ◽  
Phase 3 ◽  
Key Points ◽  
Acute Venous Thromboembolism

Key Points DOACs have similar efficacy as VKAs in the treatment of acute symptomatic VTE, but significantly reduce the risk of major bleeding. The efficacy and safety of DOACs in the treatment of acute VTE are consistent in clinically important subgroups.

scholarly journals Rivaroxaban dose adjustment using thrombin generation in severe congenital protein C deficiency and warfarin-induced skin necrosis

2018 ◽  
Vol 2 (2) ◽  
pp. 142-145 ◽  
Author(s):  
Neethu Menon ◽  
Ravi Sarode ◽  
Ayesha Zia
Keyword(s):  
Skin Necrosis ◽  
Protein C ◽  
Dose Adjustment ◽  
Thrombin Generation ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Protein C Deficiency ◽  
Thrombin Generation Assay ◽  
Key Points ◽  
Congenital Protein C Deficiency

Key Points Rivaroxaban was efficacious and safe in a child with protein C deficiency to prevent the recurrence of skin necrosis or venous thrombosis. The dosage of direct oral anticoagulants in children with thrombophilia is unclear; a thrombin generation assay may be useful to adjust it.

scholarly journals FXa-α2-Macroglobulin Complex Neutralizes Direct Oral Anticoagulants Targeting FXa In Vitro and In Vivo

2018 ◽  
Vol 118 (09) ◽  
pp. 1535-1544 ◽  
Author(s):  
Georges Jourdi ◽  
Isabelle Gouin-Thibault ◽  
Virginie Siguret ◽  
Sophie Gandrille ◽  
Pascale Gaussem ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Rotational Thromboelastometry ◽  
Α2 Macroglobulin ◽  
Number Of Patients ◽  
Attractive Candidate

Increasing number of patients are treated with direct oral anticoagulants (DOAC). An antidote for dabigatran inhibiting thrombin (idarucizumab) is available but no antidote is yet approved for the factor Xa (FXa) inhibitors (xabans). We hypothesized that a complex between Gla-domainless FXa and α2-macroglobulin (GDFXa-α2M) may neutralize the xabans without interfering with normal blood coagulation.Purified α2M was incubated with GDFXa to form GDFXa-α2M. Affinity of apixaban and rivaroxaban for GDFXa-α2M was only slightly decreased compared to FXa. Efficacy and harmlessness of GDFXa-α2M were tested in vitro and in vivo. Stoichiometric excess of GDFXa-α2M neutralized rivaroxaban and apixaban as attested by clot waveform assay and rotational thromboelastometry, whereas GDFXa-α2M alone had no effect on these assays. Efficacy and pro-thrombotic potential of GDFXa-α2M were also assessed in vivo. Half-life of GDFXa-α2M in C57BL6 mice was 4.9 ± 1.1 minutes, but a 0.5 mg/mouse dose resulted in uptake saturation such that 50% persistence was still observed after 170 minutes. Single administration of GDFXa-α2M significantly decreased the rivaroxaban-induced bleeding time (p < 0.001) and blood loss (p < 0.01). GDFXa-α2M did not increase D-dimer or thrombin–antithrombin complex formation, suggesting a lack of pro-thrombotic potential.GDFXa-α2M is therefore an attractive candidate for xaban neutralization neither pro- nor anticoagulant in vitro as well as in vivo.

scholarly journals Comparative effectiveness of direct oral anticoagulants and warfarin in patients with cancer and atrial fibrillation

2018 ◽  
Vol 2 (3) ◽  
pp. 200-209 ◽  
Author(s):  
Surbhi Shah ◽  
Faye L. Norby ◽  
Yvonne H. Datta ◽  
Pamela L. Lutsey ◽  
Richard F. MacLehose ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ischemic Stroke ◽  
Cancer Patients ◽  
Comparative Effectiveness ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Patients With Cancer ◽  
Key Points

Key Points In AF and cancer patients, rate of bleeding is lower with apixaban, similar in dabigatran and rivaroxaban users, compared to warfarin users. Ischemic stroke rates did not differ among anticoagulant users. Incident VTE risk was lower in all DOAC compared with warfarin users.

scholarly journals Direct oral anticoagulants for treatment of HIT: update of Hamilton experience and literature review

Blood ◽  
2017 ◽  
Vol 130 (9) ◽  
pp. 1104-1113 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Menaka Pai ◽  
Lori-Ann Linkins
Keyword(s):  
Literature Review ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Key Points

Key Points New data plus a literature review documented new thrombosis in only 1 (2.2%) of 46 patients with acute HIT who were treated with rivaroxaban. The literature review found similarly favorable results, albeit with fewer patients, when apixaban and dabigatran were used to treat acute HIT.

scholarly journals Longitudinal single cell fate of hematopoiesis in vivo using cellular barcoding and DiSNE movie visualization

2018 ◽  
Author(s):  
Jerry Gao ◽  
Dawn S. Lin ◽  
Edmund Crampin ◽  
Shalin H. Naik
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Cell Fate ◽  
Data Interpretation ◽  
List Type ◽  
Hematopoietic Progenitors ◽  
Developmental Patterns ◽  
Hematopoietic Reconstitution ◽  
Key Points

AbstractIdentifying the progeny of many single progenitor cells simultaneously can be achieved by tagging progenitors with unique heritable DNA barcodes, and allows inferences of lineage relationships, including longitudinally. While this approach has shed new light on single cell fate heterogeneity, data interpretation remains a major challenge. In this study, we applied our developmental interpolated t-Distributed Stochastic Neighbor Embedding (DiSNE) movie approach to visualize the clonal dynamics of hematopoietic reconstitution in primates and identify novel developmental patterns, namely a potential cluster of hematopoietic progenitors with early T cell and later granulocyte production.Key pointsComplex single cell haematopoietic fate heterogeneity can be visualized and assessed with tSNE pie mapsDiSNE movie visualization of in vivo haematopoiesis allows “play back” of the waves of haematopoiesisIdentification of novel hematopoietic progenitors with early T cell and later granulocyte production

scholarly journals Interaction of RSC chromatin remodelling complex with nucleosomes is modulated by H3 K14 acetylation and H2B SUMOylation in vivo

2020 ◽  
Author(s):  
Neha Jain ◽  
Davide Tamborrini ◽  
Brian Evans ◽  
Shereen Chaudhry ◽  
Bryan J. Wilkins ◽  
...  
Keyword(s):  
Histone Variants ◽  
Chromatin Remodelling ◽  
List Type ◽  
Atpase Subunit ◽  
Chromatin Remodelling Complex ◽  
Key Points ◽  
Target Sites ◽  
Nucleosome Binding

AbstractChromatin remodelling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair and transcription. A key question is how these complexes find their target sites on chromatin. Here, we use genetically encoded photo-crosslinker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC (remodels the structure of chromatin) complex, on the nucleosome in living yeast. We find that the interaction of the Sth1 bromodomain with the H3 tail depends on K14 acetylation by Gcn5. This modification does not recruit RSC to chromatin but mediates its interaction with neighbouring nucleosomes. We observe a preference of RSC for H2B SUMOylated nucleosomes in vivo and show that this modification moderately enhances RSC binding to nucleosomes in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but its mode of nucleosome binding differs between interphase and mitosis. In sum, our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications most likely in combination with other components such as histone variants and transcription factors.Key PointsIn vivo photo-crosslinking reveals the footprint of the ATPase subunit of RSC on the nucleosome.RSC binds to H3 K14ac nucleosomes via the C-terminal bromodomain of its ATPase-subunit Sth1.RSC preferentially localizes to H2B-SUMOylated nucleosomes.

scholarly journals A cutting-edge approach unravels a novel role for CDK6 in leukemic progenitor cells

2020 ◽  
Author(s):  
Eszter Doma ◽  
Isabella Maria Mayer ◽  
Tania Brandstoetter ◽  
Barbara Maurer ◽  
Reinhard Grausenburger ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Molecular Mechanisms ◽  
List Type ◽  
Robust Method ◽  
Cell Homing ◽  
Key Points

AbstractStudies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (lin-, Sca-1+, c-Kit+) cells which closely resemble MPP1 cells. HPCLSK reconstitute hematopoiesis in lethally irradiated recipient mice over more than eight months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD or MLL-AF9 their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSK can be applied to transgenic mice and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, Cdk6-/- HPCLSKs induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.Key pointsWe describe the generation of murine cell lines (HPCLSK) which reliably mimic hematopoietic/leukemic progenitor cells.Cdk6-/- BCR/ABLp210 HPCLSKs uncover a novel role for CDK6 in homing.

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