An in vitro study of the adhesion of blood platelets onto vascular catheters. Part I

The adhesion of human blood platelets onto vascular catheters was studied using a specially designed perfusion chamber. Polyurethane catheters were exposed to citrated human blood for different periods (up to 20 min) and at different wall shear rates (190, 260, 330 sec-1). The rate of platelet adhesion was determined using 111In-labeled platelets, while the morphology of adhering platelets was investigated using scanning electron microscopy. A linear increase in platelet adhesion was found within the first 10 min of perfusion, after which a plateau value was reached. The number of adhering platelets did not vary significantly with the shear rates applied, which may indicate that within the range of shear rates studied, the adhesion of platelets onto the catheter surface is mainly determined by the rate of the reaction between the platelets and the material surface. Catheters coated with a conjugate of heparin and albumin showed a four- to five-fold reduction in platelet adhesion as compared to uncoated catheters. This reduction in platelet adhesion was not only due to the presence of albumin moieties at the surface but also to the presence of heparin residues in the adsorbed albumin-heparin conjugate

A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme

AbstractRegulation of deubiquitinating enzyme (DUB) activity is an essential step for proper function of cellular ubiquitin signals. UAF1 is a WD40 repeat protein, which binds and activates three important DUBs, USP1, USP12 and USP46. Here, we report the crystal structure of the USP12-Ub/UAF1 complex at a resolution of 2.8Å and of UAF1 at 2.3Å. In the complex we find two potential sites for UAF1 binding, analogous to what was seen in a USP46/UAF1 complex. In line with these observed dual binding states, we show here that USP12/UAF1 complex has 1:2 stoichiometry in solution, with a two-step binding at 4nM and 325nM respectively. Mutagenesis studies show that the fingers sub-domain of USP12 interacts with UAF1 to form the high affinity interface. Our activation studies confirm that the high affinity binding is important for activation while the second UAF1 binding does not affect activation. Nevertheless, we show that this two step binding is conserved in the well-studied USP12 paralog, USP1. Our results highlight the interfaces essential for regulation of USP12 activity and show a conserved second binding of UAF1 which could be important for regulatory functions independent of USP12 activity

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

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

RNF168 Ubiquitinates K13-15 on H2A/H2AX to Drive DNA Damage Signaling

SummaryUbiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR

A review of hyperfibrinolysis in cats and dogs

The fibrinolytic system is activated concurrently with coagulation; it regulates haemostasis and prevents thrombosis by restricting clot formation to the area of vascular injury and dismantling the clot as healing occurs. Dysregulation of the fibrinolytic system, which results in hyperfibrinolysis, may manifest as clinically important haemorrhage. Hyperfibrinolysis occurs in cats and dogs secondary to a variety of congenital and acquired disorders. Acquired disorders associated with hyperfibrinolysis, such as trauma, cavitary effusions, liver disease and Angiostrongylus vasorum infection, are commonly encountered in primary care practice. In addition, delayed haemorrhage reported in greyhounds following trauma and routine surgical procedures has been attributed to a hyperfibrinolytic disorder, although this has yet to be characterised. The diagnosis of hyperfibrinolysis is challenging and, until recently, has relied on techniques that are not readily available outside referral hospitals. With the recent development of point‐of‐care viscoelastic techniques, assessment of fibrinolysis is now possible in referral practice. This will provide the opportunity to target haemorrhage due to hyperfibrinolysis with antifibrinolytic drugs and thereby reduce associated morbidity and mortality. The fibrinolytic system and the conditions associated with increased fibrinolytic activity in cats and dogs are the focus of this review article. In addition, laboratory and point‐of‐care techniques for assessing hyperfibrinolysis and antifibrinolytic treatment for patients with haemorrhage are reviewed

The Consumer Quality Index Hip Knee Questionnaire measuring patients' experiences with quality of care after a total hip or knee arthroplasty

<p>Abstract</p> <p>Background</p> <p>The Dutch Consumer Quality Index Hip Knee Questionnaire (CQI Hip Knee) was used to assess patients' experiences with and evaluations of quality of care after a total hip (THA) or total knee arthroplasty (TKA). The aim of this study is to evaluate the construct validity and internal consistency reliability of this new instrument and to assess its ability to measure differences in quality of care between hospitals.</p> <p>Methods</p> <p>Survey data of 1,675 subjects who underwent a THA or TKA were used to evaluate the psychometric properties. Exploratory factor analyses were performed and item-total correlations and inter-factor correlations were calculated to assess the construct validity of the instrument. Reliability analyses included tests of internal consistency (Cronbach's alpha coefficients). Finally, multilevel analyses were performed to assess the ability of the instrument to discriminate between hospitals in quality of care.</p> <p>Results</p> <p>Exploratory factor analyses indicated that the survey consisted of 21 items measuring five aspects of care (i.e. communication with nurses, communication with doctors, communication with general practitioner, communication about new medication, and pain control). Cronbach's alpha coefficients ranged from 0.76 to 0.90 indicating good internal consistency. The survey's ability to discriminate between hospitals was partly supported by multilevel analysis. Two scales (i.e. communication with nurses and communication with doctors) were able to measure differences between hospitals with respect to patients' experiences with quality of care. Logistic multilevel analyses indicated that hospitals explained part of the variation between patients in receiving information.</p> <p>Conclusion</p> <p>These findings suggest that the CQI Hip Knee is reliable and valid for use in Dutch health care. Health care providers or health plans can use this survey to measure patients' experiences with hospital care and to identify variations in care between hospitals.</p

Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS

The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates