Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (∼15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets

Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions

At sites of vessel wall damage, the primary hemostatic reac- tion involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Ad- hered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)- bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (Û200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interac- tion of PMNs with a fibrin network

What is important in evaluating health care quality? An international comparison of user views

BACKGROUND: Quality of care from the perspective of users is increasingly used in evaluating health care performance. Going beyond satisfaction studies, quality of care from the users' perspective is conceptualised in two dimensions: the importance users attach to aspects of care and their actual experience with these aspects. It is well established that health care systems differ in performance. The question in this article is whether there are also differences in what people in different health care systems view as important aspects of health care quality. The aim is to describe and explain international differences in the importance that health care users attach to different aspects of health care. METHODS: Data were used from different studies that all used a version of the QUOTE-questionnaire that measures user views of health care quality in two dimensions: the importance that users attach to aspects of care and their actual experience. Data from 12 European countries and 5133 individuals were used. They were analysed using multi-level analysis. RESULTS: Although most of the variations in importance people attach to aspects of health care is located at the individual level, there are also differences between countries. The ranking of aspects shows similarities. 'My GP should always take me seriously' was in nearly all countries ranked first, while an item about waiting time in the GP's office was always ranked lowest. CONCLUSION: Differences between countries in how health care users value different aspects of care are difficult to explain. Further theorising should take into account that importance and performance ratings are positively related, that people compare their experiences with those of others, and that general and instrumental values might be related through the institutions of the health care system

Neutrophil Adhesion to Fibrinogen and Fibrin Under Flow Conditions Is Diminished by Activation and L-Selectin Shedding

The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by b2-integrins. However, the influence of physiologic flow con- ditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil acti- vation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of) neutro- phil adhesion, real-time video-assisted microscopy and custom- made software were used. Under flow conditions, ad- herent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the ad- herence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10Ï7 mol/L) or 4b-phorbol, 12-myristate, 13-ace- tate (PMA; 100 ng/mL) resulted in approximately 50% inhibi- tion of adhesion to fibrin and a more random distribution (cluster index Ú0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respec- tively. Under these conditions, anti-L-selectin antibodies or neuraminidase did not affect adhesion. These results indi- cate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin-mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interac- tion. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation

DDA1, a novel factor in transcription-coupled repair, modulates CRL4CSA dynamics at DNA damage-stalled RNA polymerase II

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.<br/

Transition of care: experiences and preferences of patients across the primary/secondary interface – a qualitative study

BACKGROUND: Coordination between care providers of different disciplines is essential to improve the quality of care, in particular for patients with chronic diseases. The way in which general practitioners (GP's) and medical specialists interact has important implications for any healthcare system in which the GP plays the role of gatekeeper to specialist care. Patient experiences and preferences have proven to be increasingly important in discussing healthcare policy. The Dutch government initiated the development of a special website with information for patients on performance indicators of hospitals as well as information on illness or treatment.In the present study we focus on the transition of care at the primary - secondary interface with reference to the impact of patients' ability to make choices about their secondary care providers. The purpose of this study is to (a) explore experiences and preferences of patients regarding the transition between primary and secondary care, (b) study informational resources on illness/treatment desired by patients and (c) determine how information supplied could make it easier for the patient to choose between different options for care (hospital or specialist). METHODS: We conducted a qualitative study using semi-structured focus group interviews among 71 patients referred for various indications in the north and west of The Netherlands. RESULTS: Patients find it important that they do not have to wait, that they are taken seriously, and receive adequate and individually relevant information. A lack of continuity from secondary to primary care was experienced. The patient's desire for free choice of type of care did not arise in any of the focus groups. CONCLUSION: Hospital discharge information needs to be improved. The interval between discharge from specialist care and the report of the specialist to the GP might be a suitable performance indicator in healthcare. Patients want to receive information, tailored to their own situation. The need for information, however, is quite variable. Patients do not feel strongly about self-chosen healthcare, contrary to what administrators presently believe

Identification of host cell factors required for intoxication through use of modified cholera toxin

We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.Fundação para a Ciência e a Tecnologia (Fellowship

Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding

The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface

Human RAD18 Interacts with Ubiquitylated Chromatin Components and Facilitates RAD9 Recruitment to DNA Double Strand Breaks

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2