ResearchPad - platelet-biology-its-disorders https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Dynamin 2 is required for GPVI signaling and platelet hemostatic function in mice]]> https://www.researchpad.co/article/elastic_article_11012 Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2fl/fl Pf4-Cre (Dnm2Plt/) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2Plt/ platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2Plt−/− platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2Plt−/− platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

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<![CDATA[Complications of whole-exome sequencing for causal gene discovery in primary platelet secretion defects]]> https://www.researchpad.co/article/N480f3f5e-1966-477b-a136-d1c9dd6e8efd

Primary platelet secretion defects constitute a heterogeneous group of functional defects characterized by reduced platelet granule secretion upon stimulation by different agonists. The clinical and laboratory heterogeneity of primary platelet secretion defects warrants a tailored approach. We performed a pilot study in order to develop DNA sequence analysis pipelines for gene discovery and to create a list of candidate causal genes for platelet secretion defects. Whole-exome sequencing analysis of 14 unrelated Italian patients with primary secretion defects and 16 controls was performed on Illumina HiSeq. Variant prioritization was carried out using two filtering approaches: identification of rare, potentially damaging variants in platelet candidate genes or by selecting singletons. To corroborate the results, exome sequencing was applied in a family in which platelet secretion defects and a bleeding diathesis were present. Platelet candidate gene analysis revealed gene defects in 10/14 patients, which included ADRA2A, ARHGAP1, DIAPH1, EXOC1, FCGR2A, ITPR1, LTBP1, PTPN7, PTPN12, PRKACG, PRKCD, RAP1GAP, STXBP5L, and VWF. The analysis of singletons identified additional gene defects in PLG and PHACTR2 in two other patients. The family analysis confirmed a missense variant p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene as potentially causal. In summary, exome sequencing revealed potential causal variants in 12 of 14 patients with primary platelet secretion defects, highlighting the limitations of the genomic approaches for causal gene identification in this heterogeneous clinical and laboratory phenotype.

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<![CDATA[Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI]]> https://www.researchpad.co/article/5c1c2d93d5eed0c4844646cd

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.

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<![CDATA[Comparison of up-front treatments for newly diagnosed immune thrombocytopenia -a systematic review and network meta-analysis]]> https://www.researchpad.co/article/5bf50e63d5eed0c484862467

Corticosteroids such as prednisolone and dexamethasone have been established as up-front therapy for the treatment of newly diagnosed immune thrombocytopenia. Recent studies have indicated that other treatments such as rituximab or thrombopoietin receptor agonist can also be effective choices. We performed a systematic review and network meta-analysis to establish a clinically meaningful hierarchy of efficacy and safety of treatments for newly diagnosed primary immune thrombocytopenia in adults. Randomized controlled trials evaluating medical treatments for newly diagnosed immune thrombocytopenia were included. Reviewers independently extracted data and assessed the risk of bias. The main outcome was the sustained response (platelet count >30×109/L for 3–6 months after completion of treatments), while overall response (platelet count >30×109/L for 2–4 weeks after initiation of the up-front treatment) and therapy-related adverse events were the secondary endpoints. A total of 21 randomized controlled trials (1898 patients) were included in this study. Our main findings were a significantly better sustained response in the recombinant human thrombopoietin+dexamethasone and rituximab+dexamethasone arms compared to those of conventional therapies (prednisolone and dexamethasone monotherapy). Moreover, recombinant human thrombopoietin+dexamethasone and +prednisolone improved early overall response compared to prednisolone, dexamethasone, and rituximab-containing regimens. Therapy-related adverse events showed similar profiles and were tolerable in all treatment arms. Regimens containing recombinant human thrombopoietin agonist may be beneficial up-front therapies in addition to the conventional corticosteroid monotherapies. Future head-to-head trials including these regimens and rituximab-containing treatments are necessary in order to overcome the limitations of the small number in our study and determine the most suitable initial therapies for newly diagnosed immune thrombocytopenia.

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