Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size

We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.     

Prophylaxis in severe forms of von Willebrand's disease: results from the von Willebrand Disease Prophylaxis Network (VWD PN)

The bleeding patterns of severe von Willebrand's disease (VWD) adversely affect quality of life, and may be life threatening. There is a presumed role for prophylaxis with VWF‐containing concentrates, but data are scarce. The von Willebrand Disease Prophylaxis Network (VWD PN) was formed to investigate the role of prophylaxis in clinically severe VWD that is not responsive to other treatment(s).Using a retrospective design, the effect of prophylaxis was studied. Availability of records to document, or reliably assess, the type and frequency of bleeding episodes prior to, and after, the initiation of prophylaxis was required. Annualized bleeding rates were calculated for the period prior to prophylaxis, during prophylaxis and by primary bleeding indication defined as the site accounting for more than half of all bleeding symptoms. The Wilcoxon signed‐rank test of differences in the medians was used. Sixty‐one subjects from 20 centres in 10 countries were enrolled. Data for 59 were used in the analysis. The median age at onset of prophylaxis was 22.4 years. Type 3 VWD accounted for the largest number (N = 34, 57.6%). Differences in bleeding rates within individuals during compared with before prophylaxis were significant for the total group (P < 0.0001), and for those with primary bleeding indications of epistaxis (P = 0.0005), joint bleeding (P = 0.002) and GI bleeding (P = 0.001). The effect of prophylaxis was similar among those age < 18 years and those ≥18. One person developed an inhibitor during treatment. We conclude that prophylactic treatment of VWD is efficacious.     

Serum 25-hydroxyvitamin D levels are inversely associated with systemic inflammation in severe obese subjects

Obesity is frequently characterized by a reduced vitamin D bioavailability, as well as insulin-resistance and a chronic inflammatory response. We tested the hypothesis of an independent relationship between serum concentrations of 25-hydroxyvitamin D (25[OH]D) and several circulating inflammatory markers in a cohort of severely obese individuals. Cross-sectional study was carried out among obese patients undergoing a clinical evaluation before bariatric surgery in our University Hospital. Serum 25(OH)D, fasting and post load glucose and insulin, high-sensitive C-reactive protein (hs CRP), fibrinogen, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin, adiponectin and lipid profile were collected. Insulin-resistance was assessed by insulin sensitivity index (ISI). Total body fat (FAT kg), total percent body fat (FAT%) and truncal fat mass (TrFAT) were assessed with dual-energy X-ray absorptiometry. A total of 147 obese subjects (89 women, 37.8 ± 7.1 years) with mean body mass index (BMI) of 43.6 ± 4.3 kg/m² were enrolled. Patients in the lowest tertile of 25(OH)D were significantly more obese with a higher amount of TrFAT, more insulin-resistant, and had higher levels of fasting and post-challenge glucose (p < 0.05 for all). In a multivariate regression analysis, serum 25(OH)D was inversely related to significant levels of hs CRP, IL-6 and TNF-α after accounting for age, gender, season of recruitment, BMI, FAT kg and TrFAT (p < 0.01 for all). In extremely obese subjects, 25(OH)D serum concentrations are inversely associated with several biomarkers of systemic inflammation, regardless of the total quantity of fat mass.     

Analysis of Hemochromatosis Gene Mutations in 52 Consecutive Patients with Polycythemia Vera

A literature review reports increased erythrocyte indices [hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration] in subjects with hereditary hemochromatosis (HH). We, therefore, screened 52 consecutive patients with polycythemia vera for 12 HH gene mutations, comparing iron status and red cell parameters between patients positive or negative for HH gene mutations. Our results support the evidence that there is no association between these two conditions.     

Pioglitazone improves glucose metabolism and modulates skeletal muscle TIMP-3–TACE dyad in type 2 diabetes mellitus: a randomised, double-blind, placebo-controlled, mechanistic study

Aims/hypothesis

Pioglitazone (PIO) is a peroxisome proliferator-activated receptor (PPAR)γ agonist insulin-sensitiser with anti-inflammatory and anti-atherosclerotic effects. Our objective was to evaluate the effect of low-dose PIO (15 mg/day) on glucose metabolism and inflammatory state in obese individuals with type 2 diabetes.

Methods

A randomised, double-blind, placebo-controlled, mechanistic trial was conducted on 29 patients with type 2 diabetes treated with metformin and/or sulfonylurea. They were randomised to receive PIO or placebo (PLC) for 6 months, in a 1:1 ratio. Participants were allocated to interventions by central office. All study participants, investigators and personnel performing measurements were blinded to group assignment. At baseline and after 6 months patients underwent: (1) OGTT; (2) muscle biopsy to evaluate expression of TNF-α, tissue inhibitor of metalloproteases 3 (TIMP-3) levels, TNF-α converting enzyme (TACE) expression and enzymatic activity; (3) euglycaemic–hyperinsulinaemic clamp; (4) measurement of plasma high-sensitivity C-reactive protein (hsCRP), plasminogen activator inhibitor type-1 (PAI-1), TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), adiponectin and fractalkine (FRK). The interventions were PIO 15 mg/day vs placebo and the main outcomes measured were absolute changes in whole-body insulin sensitivity, insulin secretion and inflammatory state.

Results

Fifteen participants were randomized to receive PIO and 14 participants were randomized to receive PLC. Eleven participants completed the study in the PIO group and nine participants completed the study in the PLC group and were analysed. Fasting plasma glucose and HbA_1c decreased modestly (p?<?0.05) after PIO and did not change after PLC. M/I (insulin-stimulated whole-body glucose disposal), adipose tissue insulin resistance (IR) index, insulin secretion/IR (disposition) index and insulinogenic index improved significantly after PIO, but not after PLC. Circulating MCP-1, IL-6, FRK, hsCRP and PAI-1 levels decreased in PIO- as compared with PLC-treated patients, while TNF-α did not change. TNF-α protein expression and TACE enzymatic activity in muscle were significantly reduced by PIO but not PLC. Adiponectin levels increased significantly after PIO as compared with PLC treatment. Given that the mean TACE enzymatic activity level at baseline in the PIO group was 0.29?±?0.07 (fluorescence units [FU]), and at end of study decreased to 0.05 vs 0.14 in the PLC group, the power to reject the null hypothesis that the population means of the PIO and PLC groups are equal after 6 months is greater than 0.80. Given that M/I was 2.41?±?0.35 μmol kg^?1 min^?1 (pmol/l)^?1 at baseline and increased by 0.55 in the PIO and 0.17 in the PLC groups, the power to reject the null hypothesis that the population means of the PIO and PLC groups are equal after 6 months is greater than 0.85. The type I error probability associated with this test of this null hypothesis is 0.05. No serious adverse events occurred in either group.

Conclusions/interpretation

Low-dose PIO (15 mg/day) improves glycaemic control, beta cell function and inflammatory state in obese patients with type 2 diabetes.

Trial registration

Clinical.Trial.gov NCT01223196

Funding

This study was funded by TAKEDA.     

Checking an integrated model of web accessibility and usability evaluation for disabled people

A combined objective-oriented and subjective-oriented method for evaluating accessibility and usability of web pages for students with disability was tested. The objective-oriented approach is devoted to verifying the conformity of interfaces to standard rules stated by national and international organizations responsible for web technology standardization, such as W3C. Conversely, the subjective-oriented approach allows assessing how the final users interact with the artificial system, accessing levels of user satisfaction based on personal factors and environmental barriers. Five kinds of measurements were applied as objective-oriented and subjective-oriented tests. Objective-oriented evaluations were performed on the Help Desk web page for students with disability, included in the website of a large Italian state university. Subjective-oriented tests were administered to 19 students labeled as disabled on the basis of their own declaration at the University enrolment: 13 students were tested by means of the SUMI test and six students by means of the ‘Cooperative evaluation’. Objective-oriented and subjective-oriented methods highlighted different and sometimes conflicting results. Both methods have pointed out much more consistency regarding levels of accessibility than of usability. Since usability is largely affected by individual differences in user's own (dis)abilities, subjective-oriented measures underscored the fact that blind students encountered much more web surfing difficulties.     

Dopamine-containing neurons are silenced by energy deprivation: a defensive response or beginning of cell death?

Metabolic stress associated to mitochondrial dysfunction has been put forward as an important factor causing degeneration of mesencephalic dopamine-containing neurons in Parkinson's disease (PD). Here we overview how these neurons react to acute hypoxia or hypoglycemia, that are conditions of energy deprivation causing a reduced production of ATP by mitochondria. These neurons, which show a tonic firing discharge under normal condition, undergo into membrane hyperpolarization during hypoxia or hypoglycemia that silence their spontaneous activity. We outline the cellular mechanisms causing membrane hyperpolarization and the accompanied disturbances of intracellular calcium and sodium homeostasis. A better understanding of the changes occurring during transient energy deprivation might contribute to understand the physiopathology of these neurons that derives from mitochondrial dysfunction.