What’s new in using platelet research? To unravel thrombopathies and other human disorders

This review on platelet research focuses on defects of adhesion, cytoskeletal organisation, signal transduction and secretion. Platelet defects can be studied by different laboratory platelet functional assays and morphological studies. Easy bruising or a suspected platelet-based bleeding disorder is of course the most obvious reason to test the platelet function in a patient. However, nowadays platelet research also contributes to our understanding of human pathology in other disciplines such as neurology, nephrology, endocrinology and metabolic diseases. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet research and disorders with a broader clinical phenotype. Classical thrombopathies involve disorders of platelet adhesion such as Glanzmann thrombastenia and Bernard-Soulier syndrome, defective G protein signalling diseases with impaired phospholipase C activation, and abnormal platelet granule secretion disorders such as gray platelet disorder and delta-storage pool disease. Other clinical symptoms besides a bleeding tendency have been described in MYH9-related disorders and Duchenne muscular dystrophy due to adhesion defects, and also in disorders of impaired Gs signalling, in Hermansky Pudlack disease and Chediak Higashi disease with abnormal secretion. Finally, platelet research can also be used to unravel novel mechanisms involved in many neurological disorders such as depression and autism with only a subclinical platelet defect

Transform-domain analysis of packet delay in network nodes with QoS-aware scheduling

In order to differentiate the perceived QoS between traffic classes in heterogeneous packet networks, equipment discriminates incoming packets based on their class, particularly in the way queued packets are scheduled for further transmission. We review a common stochastic modelling framework in which scheduling mechanisms can be evaluated, especially with regard to the resulting per-class delay distribution. For this, a discrete-time single-server queue is considered with two classes of packet arrivals, either delay-sensitive (1) or delay-tolerant (2). The steady-state analysis relies on the use of well-chosen supplementary variables and is mainly done in the transform domain. Secondly, we propose and analyse a new type of scheduling mechanism that allows precise control over the amount of delay differentiation between the classes. The idea is to introduce N reserved places in the queue, intended for future arrivals of class 1

A dominant gain-of-function mutation in universal tyrosine kinase <i>SRC </i>causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies

The Src family kinase (SFK)member SRC is amajor target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, whichwe confirmedwith in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patientswith myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of a-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC formMKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC- positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets andMKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors. © 2016 by the American Association for the Advancement of Science; all rights reserved

Methylation Defect in Imprinted Genes Detected in Patients with an Albright's Hereditary Osteodystrophy Like Phenotype and Platelet Gs Hypofunction

Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations.We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls (36 ± 3 vs. 29 ± 3%; p<0.001); a pattern that is reversed to XL hypomethylation found in PHPIb. Interestingly, XL hypermethylation was associated with reduced XLalphaS protein levels in the patients' platelets. Methylation for NESP and ExonA/B was significantly different for some but not all patients, though most patients have site-specific CpG methylation abnormalities in these amplicons. Since some AHO features are present in other imprinting disorders, the methylation of IGF2, H19, SNURF and GRB10 was quantified. Surprisingly, significant IGF2 hypermethylation (20 ± 10 vs. 14 ± 7%; p<0.05) and SNURF hypomethylation (23 ± 6 vs. 32 6%; p<0.001) was found in patients vs. controls, while H19 and GRB10 methylation was normal.In conclusion, this is the first report of methylation defects including GNAS in patients with an AHO-like phenotype without endocrinological abnormalities. Additional studies are still needed to correlate the methylation defect with the clinical phenotype

A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder

Background: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. Results: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. Conclusions: This study presents the first patient with a proven genetic GPVI defect.status: publishe

Guanine nucleotide-dependent inhibition of phospholipase C in human endothelial cells

Treatment of intact human umbilical vein endothelial cells with NaF results in a dose-dependent biphasic response in both prostacyclin and inositol phosphate production: the stimulation observed with 10-20 mM NaF decreases with higher concentrations. High concentrations of NaF furthermore reduce thrombin- or A23187-stimulated prostacyclin production. Direct assay of phospholipase C activity in cell homogenates shows a similar biphasic response to NaF, also after chelation of Ca2+; addition of AlCl3 shifts the inhibition toward lower NaF concentrations. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) also causes a dose-dependent biphasic response in inositol phosphate formation in permeabilized cells and homogenates; a higher inhibitory concentration of GTP gamma S abolishes the stimulation of inositol phosphate production by low NaF concentrations. A high concentration of NaF furthermore inhibits the non-G-protein-dependent activation of phospholipase C by deoxycholate. NaF also induces a dose-dependent biphasic response in cyclic AMP formation in intact cells, indicating that the inhibition of phospholipase C at higher NaF concentrations does not result from a rise in cyclic AMP. The data are compatible with the existence of a guanine nucleotide-dependent, cyclic AMP-independent, phospholipase C-inhibitory pathway in endothelial cells.status: publishe

Models for Capacity Demand Estimation in a TV Broadcast Network with Variable Bit Rate TV Channels

Mobile TV is growing beyond the stage of experimentation and evaluation and is (about) to become part of our daily lives. Additionally, it is being delivered through heterogeneous networks and to a variety of receiving devices, which implies different versions of one and the same video content must be transported. We propose two (approximate) analytic methods for capacity demand estimation in a (mobile) TV broadcast system. In particular, the methods estimate the required transport capacity for a bouquet of channels offered on request and in different versions (video formats or in different quality) over a multicast-enabled network, encoded in non-constant bit rate targeting constant quality. we compare a transport strategy where the different versions (of one channel) are simulcast; to a scalable video encoding (SVC) transport. strategy, where all resolutions (of one channel) are embedded in one flow. In addition, we validate the proposed analytic methods with simulations. A realistic mobile TV example is considered with two transported resolutions of the channels: QVGA and VGA. We demonstrate that not always capacity gain is achieved with SVC as compared to simulcast since the former comes with some penalty rate and the gain depends on the system parameters