Uwe Till

Prothrombinase activity of human platelets is inhibited by β2-glycoprotein-I

Abstract In the present paper the influence of β 2 -glycoprotein-I, also known as apolipoprotein H, upon the prothrombinase activity of platelets and phospholipid vesicles was investigated. The results can be summarized as follows. 1. The prothrombinase activity of resting, non-activated platelets, lysed platelets and vesicles composed of phosphatidylserine and phosphatidylcholine at different molar ratios is inhibited by β 2 -glycoprotein-I in a dose-dependent manner. The concentration of glycoprotein which produces marked inhibition is within the physiological plasma concentration range of β 2 -glycoprotein-I. 2. The time dependence of this inhibition is a relatively slow process, which is not fully expressed before 1 h or incubation. 3. The effect of the glycoprotein is not due to a direct interaction with the components of the prothrombinase complex, i.e. factors X a , V a , Ca 2+ or prothrombin, nor is the inhibitory action abolished by increasing concentrations of coagulation factors X a and V a . This suggests that β 2 -glycoprotein-I causes a reduction of the prothrombinase binding sites of these coagulation factors to platelets or phospholipid vesicles. 4. The prothrombinase activity of platelets stimulated with ionophore A23187 or with collagen plus thrombin is also inhibited by β 2 -glycoprotein-I in a manner similar to that oberved for phospholipid vesicles or for lysed platelets. These findings suggest a regulatory role for β 2 -glycoprotein-I in the pathway of blood coagulation.

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether l-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1–100 μm ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 μm) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 μm and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor moleculel-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate l-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.

Nitrovasodilators inhibit thrombin-induced platelet-activating factor synthesis in human endothelial cells

In response to inflammatory agents such as thrombin, cultured endothelial cells produce platelet-activating factor (PAF), which has been linked with most inflammatory and immune processes, and is a potent coronary constrictor. Sodium nitroprusside (SNP) and SIN-1 (3-morpholinosydnonimine), which spontaneously release the free radical nitric oxide (NO), cause direct relaxation of blood vessels and inhibition of platelet aggregation by activating soluble guanylate cyclase. In the present study we report that in human umbilical vein endothelial cells (HUVEC) these compounds stimulate the production of cGMP and inhibit thrombin-induced PAF synthesis in a concentration-dependent manner. 8-bromo-cGMP, a permeant non-hydrolysable analogue of cGMP, mimics the inhibitory effect of NO-generating vasodilators. PAF synthesis requires phospholipase A2-mediated hydrolysis of membrane precursors to lyso-PAF, which is in turn converted into PAF by an acetyltransferase. The thrombin-elicited activation of both enzymes is inhibited in a dose-dependent way in HUVEC pretreated with SNP and SIN-1. The inhibitory effect of SNP and SIN-1 on the thrombin-mediated PAF synthesis suggests a new mechanism of action whereby the endogenous NO can affect vascular tone and endothelium-dependent intercellular adhesion. Moreover, PAF production in endothelial cells appears to be an important target for the pharmacological action of nitrovasodilators.

Flow cytometric determination of E‐selectin, vascular cell adhesion molecule‐1, and intercellular cell adhesion molecule‐1 in formaldehyde‐fixed endothelial cell monolayers

BACKGROUND Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.

[4] Purification of commercial NADH

Publisher Summary This chapter describes the study demonstrating the purification of commercial NADH. For the purification of 5 g of commercial nicotinamide adenine dinucleotide (NAD)H (Arzneimittelwerk Dresden, G.D.R.) a 5×100 cm column is used. The separation is carried out at 4° in the dark. DEAE-Sephadex A-25, particle size 40-120/μm (Pharmacia, Uppsala, Sweden), is prepared, according to the manufacturers instructions. It is transformed into the bicarbonate form, by washing, with a 10-fold volume of l M KHCO3, on a sintered-glass funnel layed out with nylon net. The gel is then rinsed, with twice-distilled water, till neutral. The neutral gel is suspended in 50 mM KHCO3 and fine particles are removed by repeated decantations. The prepared gel is poured into the column, with care, taken to avoid turbulences and inclusion of air bubbles. The column outlet is adjusted so that the hydrostatic pressure amounts to 150 cm of water. The gel bed is allowed to stabilize, by running 4 l of 50 mM KHCO3, through the column. The chromatography takes from 5 to 10 days. This chapter discusses the purification of NADH by thin-layer chromatography. Equipment for the removal of the KHCO3 from eluate is described in the chapter. Characterization of purified NADH is also done.

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. I. Relationships between Ca2+-loading, ATP-consumption and glycolytic flux

Abstract Human red cells (RBC) were loaded with moderate amounts of Ca 2+ by the ionophore A23187. Quantitative relationships between Ca 2+ -loading, ATP consumption and glycolytic flux were established. 1. Ca 2+ -loading is accompanied by ATP depletion. A maximum ATP consumption of approximately 10 mmoles/l RBC/h was estimated. 2. There is a positive correlation between lactate formation and Ca 2+ -loading. This is linear from 1.4 to about 4 mmoles lactate/l RBC/h. 3. Ca 2+ -induced glycolytic stimulation seems not to be mediated by adenine nucleotides. A wide range of energy charges and very different adenine nucleotide patterns were associated with the same stimulation of lactate production. 4. The turnover of the (Ca 2+ -Mg 2+ )-ATPase and its share in the Ca 2+ -stimulated ATP consuming processes were estimated with inhibitors. 1 mM La 3+ inhibited both Ca 2+ -outward transport and ATP consumption by 80%. The remaining 20% of the ATP consumption was accounted for by the (Na + -K + )-ATPase. 5. A Ca 2+ extrusion to ATP consumption molar ratio of 2:1 was found. However, when ATP consumption was due to the breakdown of previously accumulated glycolytic intermediates, the ratio dropped to about 1.

Human platelet electrorotation change induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca2+-loading

Abstract Human red cells (RBC) respond to moderate Ca2+-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca2+-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase ( GAPDH PGK )-steps . 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca2+-extrusion rate and not to the loaded Ca2+. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca2+-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the GAPDH PGK equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. NAD NADH ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.