Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C.

Introduction: Cell surface thiol isomerase enzymes, principally protein disulphide isomerase (PDI), have emerged as important regulators of platelet function and tissue factor activation via their action on allosteric disulphide bonds. Allosteric disulphides are present in other haemostasis-related proteins, and we have therefore investigated whether thiol isomerase inhibition has any influence on two endothelial activities relevant to haemostatic regulation, namely activation of protein C and activation of plasminogen, with subsequent fibrinolysis.

Materials And Methods: The study was performed using the human microvascular endothelial cell line HMEC-1.

An investigation of the protective effect of alpha+-thalassaemia against severe Plasmodium falciparum amongst children in Kumasi, Ghana.

Introduction: Several factors influence the severity of Plasmodium falciparum; here, we investigate the impact of alpha+-thalassaemia genotype on P. falciparum parasitemia and prevalence of severe anaemia amongst microcytic children from Kumasi, Ghana.

Methods: Seven hundred and thirty-two children (≤10 years) with P.

Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.

S-nitrosothiols as selective antithrombotic agents - possible mechanisms.

S-nitrosothiols have a number of potential clinical applications, among which their use as antithrombotic agents has been emphasized. This is largely because of their well-documented platelet inhibitory effects, which show a degree of platelet selectivity, although the mechanism of this remains undefined. Recent progress in understanding how nitric oxide (NO)-related signalling is delivered into cells from stable S-nitrosothiol compounds has revealed a variety of pathways, in particular denitrosation by enzymes located at the cell surface, and transport of intact S-nitrosocysteine via the amino acid transporter system-L (L-AT).

Protein disulfide-isomerase mediates delivery of nitric oxide redox derivatives into platelets.

S-nitrosothiol compounds are important mediators of NO signalling and can give rise to various redox derivatives of NO: nitrosonium cation (NO+), nitroxyl anion (NO-) and NO* radical. Several enzymes and transporters have been implicated in the intracellular delivery of NO from S-nitrosothiols. In the present study we have investigated the role of GPx (glutathione peroxidase), the L-AT (L-amino acid transporter) system and PDI (protein disulfide-isomerase) in the delivery of NO redox derivatives into human platelets.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry).

Rapid S-nitrosothiol metabolism by platelets and megakaryocytes.

RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs.

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells.

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress.

Uremic concentrations of guanidino compounds inhibit neutrophil superoxide production.

Background: In uremia, diminished reactive oxygen intermediate (ROI) production is an important consequence of impaired neutrophil function. We have studied the effect of guanidino compounds, known uremic toxins, on neutrophil ROI production in vitro.

Methods: Neutrophils from healthy volunteers were exposed for three hours to individual or mixed guanidino compounds (GCmix) at concentrations encountered in uremic plasma.

Inhibition of neutrophil superoxide production by uremic concentrations of guanidino compounds.

In uremia, diminished reactive oxygen intermediate production is an important consequence of impaired neutrophil function. The effects of guanidino compounds, which are known uremic toxins, on neutrophil reactive oxygen intermediate production in vitro were studied. Neutrophils from healthy volunteers were exposed for 3 h to individual guanidino compounds or mixed guanidino compounds (GCmix), at concentrations observed in uremic plasma.

S-nitrosothiols are stored by platelets and released during platelet-neutrophil interactions.

Interaction between platelets and neutrophils is important in vascular injury. We have investigated the storage and release of nitric oxide (NO) by platelets interacting with neutrophils. Shear-activated platelets were added to neutrophils in suspension and both superoxide and peroxynitrite formations monitored by lucigenin- and luminol-enhanced chemiluminescence.

Inhibition of NADPH supply by 6-aminonicotinamide: effect on glutathione, nitric oxide and superoxide in J774 cells.

We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6-aminonicotinamide. In resting cells 6-phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6-aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon-lambda decreased glutathione by approximately 50% and synchronous 6-aminonicotinamide treatment exacerbated this to 31.

Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione.

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin.

Interaction of macrophage-migration-inhibitory factor with haematin.

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione-agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Aman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med.

How cytotoxic is nitric oxide?

Nitric oxide (NO) shows an unusual divergence of action, being utilised both as a physiological signalling molecule, and as a toxic mediator. NO-mediated cellular injury may arise by a variety of mechanisms, including disruption of mitochondrial respiration, enzyme inhibition, lipid peroxidation and genetic mutation. Toxicity is largely mediated via intermediates such as N2O3 and peroxynitrite, arising from the reaction of NO with either molecular oxygen or reactive oxygen species.

Cell-mediated biotransformation of S-nitrosoglutathione.

Spontaneous release of nitric oxide (NO) from S-nitrosothiols cannot explain their bioactivity, suggesting a role for cellular metabolism or receptors. Using immortalised cells and human platelets, we have identified a cell-mediated mechanism for the biotransformation of the physiological S-nitrosothiol compound S-nitrosoglutathione (GSNO) into nitrite. We suggest the name "GSNO lyase" for this activity.

Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.

1. S-nitrosoglutathione (GSNO) is a potent and selective anti-platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti-aggregatory action of GSNO.

The hepatocyte growth factor/scatter factor (HGF/SF) receptor, met, transduces a morphogenetic signal in renal glomerular fibromuscular mesangial cells.

Previous studies have demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is secreted by mesenchymal cells and that it elicits motility, morphogenesis and proliferation of epithelia expressing the met receptor. We now report that HGF/SF may act as an autocrine factor in fibromuscular renal mesangial cells. These cells mechanically support glomerular endothelia, control the rate of plasma ultrafiltration and are implicated in the pathogenesis of a variety of chronic renal diseases.

Copper chelation-induced reduction of the biological activity of S-nitrosothiols.

1. The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells. 2.

Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum.

Background And Objective: Degradation of AVP by placental vasopressinase may precipitate gestational diabetes insipidus, which in some cases is accompanied by pre-eclampsia. Abnormally elevated vasopressinase has also been reported in pre-eclampsia without diabetes insipidus. This association between excessive vasopressinase production and pre-eclampsia might be explained if the products of AVP degradation by vasopressinase retained pressor activity even after anti-diuretic activity had been destroyed.

Serum Vasopressinase and Platelet Responses to Arginine Vasopressin in Normal Pregnancy, Pregnancy-induced Hypertension and Pre-eclampsia.

Pregnancy is marked by the placental production of vasopressinase, an enzyme which accelerates the metabolic clearance of arginine vasopressin (AVP), and serum vasopressinase is reported to be abnormally elevated in hypertensive pregnancy. Since platelet AVP binding capacity is influenced by the plasma AVP concentration, we sought to determine whether alterations in vasopressinase and AVP concentrations affect platelet responsiveness to AVP in normotensive and hypertensive pregnancy. Four groups of 10 women were studied: non-pregnant subjects, normotensive pregnancy, hypertensive pregnancy, and pre-eclampsia (PE).

Platelets from patients on haemodialysis show impaired responses to nitric oxide.

1. Platelets from patients on haemodialysis showed a loss of sensitivity to nitric oxide, reflected by a reduction in the ability of nitric oxide both to inhibit thrombin-induced aggregation and to increase intraplatelet cyclic GMP levels. Responses of platelets from patients on continuous ambulatory peritoneal dialysis were slightly, but not significantly, impaired.