Phosphorylation of factor Va and factor VIIIa by activated platelets

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.     

Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation

Initial attachment of leukocytes to the vessel wall at sites of inflammation is supported by a family of carbohydrate-binding adhesion molecules called the selectins. Selectin ligands include sialyl-Lewis x (sLex, Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc--) and related structures. We report here that defined heparin oligosaccharides interact with the selectins. Heparin chains containing four or more monosaccharide residues inhibited the function of L- and P-selectin, but not E-selectin, in vitro. In a competition enzyme-linked immunosorbent assay measuring inhibition of solution-phase selectin-Ig fusion proteins (selectin-Ig) binding to immobilized bovine serum albumin-sLex neoglycoprotein, a heparin-derived tetrasaccharide mixture inhibited 50% of L- and P-selectin-Ig binding (IC50) at 200 +/- 40 mumol/L and 850 +/- 110 mumol/L, respectively. A single hexasulfated tetrasaccharide (delta UA2S alpha 1-4GlcNS6S alpha 1-4IdoA2S alpha 1- 4GlcNS6S) was particularly active against L- and P-selectin-Ig (IC50 = 46 +/- 5 mumol/L and 341 +/- 24 mumol/L). By comparison, the tetrasaccharide sLex was not inhibitory at concentrations up to 1 mmol/L. In cell adhesion assays, heparin tetrasaccharides reduced binding of neutrophils to COS cells expressing P-selectin but not to COS cells expressing E-selectin. They also blocked colon cancer cell adhesion to L- and P-selectin but not E-selectin. In a model of acute inflammation, intravenously administered heparin tetrasaccharides diminished influx of neutrophils into the peritoneal cavities of thioglycollate-treated mice. We conclude that heparin oligosaccharides, including non-anticoagulant tetrasaccharides, are effective L- and P- selectin inhibitors in vitro and have anti-inflammatory activity in vivo.     

Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.     

Inhibition of hepatic sulfatase-2 in vivo: a novel strategy to correct diabetic dyslipidemia

Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial triglyceride-rich lipoproteins (TRLs). We recently reported that livers from T2DM db/db mice markedly overexpress the heparan sulfate glucosamine-6-O-endosulfatase-2 (SULF2), an enzyme that removes 6-O sulfate groups from heparan sulfate proteoglycans (HSPGs) and suppresses uptake of TRLs by cultured hepatocytes. In the present study, we evaluated whether Sulf2 inhibition in T2DM mice in vivo could correct their postprandial dyslipidemia. Selective second-generation antisense oligonucleotides (ASOs) targeting Sulf2 were identified. Db/db mice were treated for 5 weeks with Sulf2 ASO (20 or 50 mg/kg per week), nontarget (NT) ASO, or phosphate-buffered saline (PBS). Administration of Sulf2 ASO to db/db mice suppressed hepatic Sulf2 messenger RNA expression by 70%-80% (i.e., down to levels in nondiabetic db/m mice) and increased the ratio of tri- to disulfated disaccharides in hepatic HSPGs (P < 0.05). Hepatocytes isolated from db/db mice on NT ASO exhibited a significant impairment in very-low-density lipoprotein (VLDL) binding that was entirely corrected in db/db mice on Sulf2 ASO. Sulf2 ASO lowered the random, nonfasting plasma triglyceride (TG) levels by 50%, achieving nondiabetic values. Most important, Sulf2 ASO treatment flattened the plasma TG excursions in db/db mice after corn-oil gavage (iAUC, 1,500 ± 470 mg/dL·h for NT ASO versus 160 ± 40 mg/dL · h for Sulf2 ASO\P < 0.01). CONCLUSIONS: Despite extensive metabolic derangements in T2DM mice, inhibition of a single dys-regulated molecule, SULF2, normalizes the VLDL-binding capacity of their hepatocytes and abolishes postprandial hypertriglyceridemia. These findings provide a key proof of concept in vivo to support Sulf2 inhibition as an attractive strategy to improve metabolic dyslipidemia.     

Are quality of family life and disease severity related in childhood atopic dermatitis?

BACKGROUND: Atopic dermatitis (AD) can be traumatizing to family life. Little is known about the relationship between quality of life in AD and disease severity. OBJECTIVE: To document family quality of life and relate this to severity of AD in children, for a 6-month period from a given point in time. STUDY DESIGN: These data are part of a longitudinal study conducted in two parts of the UK to investigate risk factors for AD severity and its impact on quality of life. SUBJECTS: and methods Thetargetedpopulation comprised children with AD aged 5-10 years in a primary-care setting. The general practitioners identified potential subjects and the UK diagnostic criteria for AD were used to verify the diagnosis. Both the children and their parents were interviewed. Eczema severity was assessed using a modified form of the SCORAD (SCORe Atopic Dermatitis) Index (SCORAD-D) from which parents' score of itching and sleep loss were excluded. The quality of family life was quantified by the Dermatitis Family Impact (DFI) questionnaire. These two parameters were evaluated on two occasions 6 months apart. ANALYSIS: Multiple regression analysis was used to investigate the relationship between the quality of family life and the severity of the AD in the children, at a specific point in time and over the following 6-month period. RESULTS: Of the 116 children attending the first visit, mean age 8 years, 106 attended the second visit (91%) and were included in the analysis. Quality of family life was shown to be significantly affected in 48 (45%) cases at the first visit and 38 (36%) cases at the second visit. The initial means of the DFI and SCORAD-D were 2.4 and 8.2, respectively. Six months later the mean final DFI and SCORAD-D were 1.9 and 7.7, respectively. Using multiple regression on the first and second visits, each unit increase in SCORAD-D was associated with 0.21 [95% confidence interval (CI) 0.06-0.37 P = 0.008] and 0.37 (95% CI 0.15-0.59, P = 0.001) units increase in quality of family life, respectively. This relationship remained significant even after adjustment for potential confounders (black skin, social class, sex, child's age, family size and location) each unit increase in SCORAD-D led to a 0.25 unit (95% CI 0.11-0.4, P = 0.001) and 0.23 unit (95% CI 0.05-0.42, P = 0.014) increase in DFI on the first and second visits, respectively. Changes in the DFI scores were significantly related to changes in the SCORAD-D scores (regression coefficient; 0.17 (95% CI 0.06-0.29, P = 0.002). CONCLUSIONS: We show that quality of family life is related to the severity of AD in children. This confirms the importance of parental assessment of the impact of the disease in the management of AD, because the disease affects the entire family. Also, these results show the response of DFI to change predictably with disease severity. This may imply that the DFI questionnaire could be used as an extra measure of outcome in everyday clinical practice as well as in research studies.     

Computed tomography perfusion imaging in acute stroke

The development of thrombolytic and neuroprotective agents for the treatment of acute stroke has created an imperative for improved imaging techniques in the assessment of acute stroke. Five cases are presented to illustrate the value of perfusion CT in the evaluation of suspected acute stroke. To obtain the perfusion data, a rapid series of images was acquired without table movement following a bolus of contrast medium. Cerebral blood flow, cerebral blood volume and mean transit time were determined by mathematically modelling the temporal changes in contrast enhancement in the brain and vascular system. Pixel‐by‐pixel analysis allowed generation of perfusion maps. In two cases, CT‐perfusion imaging usefully excluded acute stroke, including one patient in whom a low‐density area on conventional CT was subsequently proven to be tumour. Cerebral ischaemia was confirmed in three cases, one with an old and a new infarction, one with a large conventional CT abnormality but only a small perfusion defect, and one demonstrating infarct and penumbra. Perfusion CT offers the ability to positively identify patients with non‐haemorrhagic stroke in the presence of a normal conventional CT, to select those cases where thrombolysis is appropriate, and to provide an indication for prognosis.     

Severe electrolyte disorders following cardiac surgery: a prospective controlled observational study

Introduction

Electrolyte disorders are an important cause of ventricular and supraventricular arrhythmias as well as various other complications in the intensive care unit. Patients undergoing cardiac surgery are at risk for development of tachyarrhythmias, especially in the period during and immediately after surgical intervention. Preventing electrolyte disorders is thus an important goal of therapy in such patients. However, although levels of potassium are usually measured regularly in these patients, other electrolytes such as magnesium, phosphate and calcium are measured far less frequently. We hypothesized that patients undergoing cardiac surgical procedures might be at risk for electrolyte depletion, and we therefore conducted the present study to assess electrolyte levels in such patients.

Methods

Levels of magnesium, phosphate, potassium, calcium and sodium were measured in 500 consecutive patients undergoing various cardiac surgical procedures who required extracorporeal circulation (group 1). A total of 250 patients admitted to the intensive care unit following other major surgical procedures served as control individuals (group 2). Urine electrolyte excretion was measured in a subgroup of 50 patients in both groups.

Results

All cardiac patients received 1 l cardioplegia solution containing 16 mmol potassium and 16 mmol magnesium. In addition, intravenous potassium supplementation was greater in cardiac surgery patients (mean ± standard error: 10.2 ± 4.8 mmol/hour in cardiac surgery patients versus 1.3 ± 1.0 in control individuals; P < 0.01), and most (76% versus 2%; P < 0.01) received one or more doses of magnesium (on average 2.1 g) for clinical reasons, mostly intraoperative arrhythmia. Despite these differences in supplementation, electrolyte levels decreased significantly in cardiac surgery patients, most of whom (88% of cardiac surgery patients versus 20% of control individuals; P < 0.001) met criteria for clinical deficiency in one or more electrolytes. Electrolyte levels were as follows (mmol/l [mean ± standard error]; cardiac patients versus control individuals): phosphate 0.43 ± 0.22 versus 0.92 ± 0.32 (P < 0.001); magnesium 0.62 ± 0.24 versus 0.95 ± 0.27 (P < 0.001); calcium 1.96 ± 0.41 versus 2.12 ± 0.33 (P < 0.001); and potassium 3.6 ± 0.70 versus 3.9 ± 0.63 (P < 0.01). Magnesium levels in patients who had not received supplementation were 0.47 ± 0.16 mmol/l in group 1 and 0.95 ± 0.26 mmol/l in group 2 (P < 0.001). Urinary excretion of potassium, magnesium and phosphate was high in group 1 (data not shown), but this alone could not completely account for the observed electrolyte depletion.

Conclusion

Patients undergoing cardiac surgery with extracorporeal circulation are at high risk for electrolyte depletion, despite supplementation of some electrolytes, such as potassium. The probable mechanism is a combination of increased urinary excretion and intracellular shift induced by a combination of extracorporeal circulation and decreased body temperature during surgery (hypothermia induced diuresis). Our findings may partly explain the high risk of tachyarrhythmia in patients who have undergone cardiac surgery. Prophylactic supplementation of potassium, magnesium and phosphate should be seriously considered in all patients undergoing cardiac surgical procedures, both during surgery and in the immediate postoperative period. Levels of these electrolytes should be monitored frequently in such patients.