Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria.

The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and pre-concentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic anti-lipopolysaccharide peptides (SALP). For the proof-of-concept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.

MAIT cell activation is reduced by direct and microbiota-mediated exposure to bisphenols.

Oral uptake is the primary route of human bisphenol exposure, resulting in an exposure of the intestinal microbiota and intestine-associated immune cells. Therefore, we compared the impact of bisphenol A (BPA), bisphenol F (BPF) and bisphenol S (BPS) on (i) intestinal microbiota, (ii) microbiota-mediated immunomodulatory effects and (iii) direct effects on mucosal-associated invariant T (MAIT) cells in vitro. We acutely exposed human fecal microbiota, Bacteroides thetaiotaomicron and Escherichia coli to BPA and its analogues BPF and BPS referring to the European tolerable daily intake (TDI), i.e. 2.3 µg/mL, 28.3 µg/mL and 354.0 µg/mL. Growth and viability of E. coli was most susceptible to BPF, whereas B.thetaiotaomicron and fecal microbiota were affected by BPA > BPF > BPS. At 354.0 µg/mL bisphenols altered microbial diversity in compound-specific manner and modulated microbial metabolism, with BPA already acting on metabolism at 28.3 µg/mL. Microbiota-mediated effects on MAIT cells were observed for the individual bacteria at 354.0 µg/mL only. However, BPA and BPF directly modulated MAIT cell responses at low concentrations, whereby bisphenols at concentrations equivalent for the current TDI had no modulatory effects for microbiota or for MAIT cells. Our findings indicate that acute bisphenol exposure may alter microbial metabolism and impact directly on immune cells.

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers.

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody.

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an alpha-L-fucose and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of extracellular fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H greater than A2 greater than A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1-2 microM. 10 microM DIDS, 10 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 20 microM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.

Accelerated maximal velocity of the red blood cell Na+/K+ pump in hyperlipidemia is related to increase in 1-palmitoyl,2-arachidonoyl-plasmalogen phosphatidylethanolamine.

Among several phospholipid classes and molecular species of phosphatidylcholine and phosphatidylethanolamine (PE) analyzed, only the percentage of the molecular species 1-palmitoyl,2-arachidonoyl (16: 0/20: 4)-plasmalogen-(alkenylacyl)-PE showed positive relations to the maximal activity and to the dissociation constant of the red blood cell Na+/K+ pump for Na+ in normo- and hyperlipidemic donors. A preferential interaction of this molecular species with the Na+/K+ pump is proposed.

Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. II. Increases in distinct molecular species of phosphatidylethanolamine and phosphatidylcholine containing arachidonic acid.

The molecular species composition of red blood cell diacyl-phosphatidylcholine (PC), diacyl-phosphatidylethanolamine (PE) and alkenylacyl-PE (plasmalogen PE) has been analyzed in normolipidemic and hyperlipidemic donors. In all three phospholipid subclasses the percentages of the species 16:0/20:4 were increased in hyperlipidemic patients. In diacyl-PE, 18:1/20:4 was also elevated. No changes were observed in the other quantitatively important molecular species containing arachidonic acid at sn-2, namely 18:0/20:4. The rise in 16:0/20:4 in diacyl-PC and diacyl-PE of hyperlipidemic donors was accompanied by a fall in molecular species with linoleic acid (18:2) at sn-2 (in particular 18:1/18:2). In alkenylacyl-PE the elevation of 16:0/20:4 was compensated by a decrease in species with docosatetraenoic acid (22:4) at sn-2 in particular by a fall in 16:0/22:4. Among all donors, the percentages of 16:0/20:4 in diacyl-PC and PE were positively associated with plasma total cholesterol levels. The changes in molecular species composition of PC and PE in hyperlipidemia are expected to alter the function of erythrocyte membrane transport proteins and--if present also in other cell types--to affect eicosanoid metabolism.

Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. I. Increased phosphatidylcholine and reduced sphingomyelin in patients with elevated levels of triacylglycerol-rich lipoproteins.

The composition of red blood cell membrane and plasma phospholipids has been analyzed in patients with hyperlipidemias. In red cells of patients with elevated levels of triacylglycerol-rich lipoproteins, phosphatidylcholine (PC) was raised and sphingomyelin (SM) reduced, resulting in a 20% increase of the membrane PC/SM ratio. In plasma phospholipids of these patients PC and SM levels were also higher and lower, respectively and the plasma PC/SM ratio was elevated by more than 50%. Close positive correlations between plasma and membrane phospholipids were obtained for PC, SM and the PC/SM ratio in normolipidemic and hyperlipidemic donors. Plasmalogen phosphatidylethanolamine (PE), a supposed endogenous protector against lipid oxidation, was reduced by about 20% in red cell membrane lipids in hyperlipidemic patients. Also plasmalogen-PE in plasma tended to be reduced in hyperlipidemic donors. Plasma HDL levels were positively related to the content of plasmalogen PE in the red cell membrane. In conclusion, there are closely related increases in PC/SM ratios in plasma and the red cell membrane in patients with elevated levels of triacylglycerol-rich lipoproteins. It is speculated that decreases in red cell membrane plasmalogen-PE in hyperlipidemic patients could be related to impaired antioxidant protection, possibly as a consequence of reductions in plasma HDL levels.

Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis. Evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane.

In order to evaluate whether acute changes in fatty acids bound to phospholipids in plasma are transmitted into red blood cell membrane (RBCM) phospholipids, molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were analyzed after reduction of apo B containing lipoproteins through low density lipoprotein (LDL) apheresis in patients with severe hypercholesterolemia. As compared to the control, increases and decreases in molecular species with arachidonic acid (20:4) and with linoleic acid (18:2), respectively, at sn-2 of plasma diacyl-PC were seen in the patients before the apheresis. Directly after the procedure, the sum of species of plasma and RBCM PC plus PE with 20:4 were reduced. Two days after apheresis major species of plasma diacyl-PC reapproached preapheresis values while, in contrast, the composition of plasma alkenylacyl(plasmalogen)-PE was distinctly altered. In plasmalogen-PE of RBCM similar modifications were induced by the apheresis as in the same subgroup in plasma. In vitro experiments using vesicles with plasmalogen-PE labeled at sn-2 with either [14C]20:4 or a fluorescent pyrenedecanoyl residue indicated fast incorporation of the subgroup into the RBCM. In contrast, diacyl-PE was not taken up by the RBCM. In conclusion, apo B containing lipoproteins are partially responsible for the supply of phospholipids with arachidonic acid to RBCM, in particular by means of the fast incorporation of plasmalogen-PE. The transmission of changes induced by apheresis in plasma into those of the RBCM suggest that erythrocytes play an important role in the homeostasis of fatty acids bound to plasma phospholipids in vivo.

Phosphatidylethanolamine participates in the stimulation of the contact system of coagulation by very-low-density lipoproteins.

We have analyzed the influence of plasma lipoproteins on the activation of the contact pathway of blood coagulation in platelet-rich plasma (PRP). The formation of thrombin in PRP incubated in vitro was abolished by the factor XIIa antagonist corn trypsin inhibitor and by severe factor XII deficiency, indicating mediation by the contact system. Addition of VLDL to the PRP shortened the lag period and increased the generation of thrombin. There was no effect of HDL and LDL. In whole blood, VLDL accelerated the rate of fibrin formation, the procoagulant effect being prevented by factor XII deficiency and by corn trypsin inhibitor. The thrombin formation in the PRP was strongly increased by microemulsions of the VLDL lipids while it was reduced by the aqueous phase of the particles. Separation of the VLDL lipids indicated the phospholipid component as the major activating principle. Vesicles supplemented with all VLDL phospholipids but lacking specifically the fraction containing phosphatidylethanolamine (PE) prolonged the lag time. The PE containing fraction alone as well as vesicles enriched with egg PE shortened the lag period. In summary, VLDL stimulates the contact pathway of blood coagulation, ethanolamine phospholipids being the most active components of the particles.

Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes.

OBJECTIVE: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. METHODS: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. RESULTS: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. CONCLUSIONS: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.

Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids.

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol.

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.

Relations of sodium-lithium countertransport kinetics to plasma and red cell membrane phospholipids in hyperlipidemia.

As compared to 7 normolipidemic donors, the maximal velocity of sodium-lithium countertransport was accelerated by nearly 70% in 10 patients with elevated levels of triglyceride-rich lipoproteins and tended to be stimulated also in 5 patients with hypercholesterolemia. No significant differences were observed between normolipidemia and both hyperlipidemic groups for the apparent affinities of the transport system for intracellular sodium and extracellular lithium. Strong positive relations of the maximal activity of sodium-lithium countertransport to the percentages of red cell membrane phosphatidylcholine (r = 0.85, 2P < 0.001), the phosphatidylcholine/sphingomyelin (r = 0.82, 2P < 0.001) and the phosphatidylcholine/phosphatidylethanolamine ratio (r = 0.81, 2P < 0.001) were seen in all donors. A negative correlation was found to membrane sphingomyelin (r = -0.72, 2P < 0.001). Also plasma phosphatidylcholine and sphingomyelin exhibited positive and negative associations, respectively, to the maximal activity of sodium-lithium countertransport (r = 0.66, 2P < 0.01 and r = -0.78, 2P < 0.001). Among several plasma lipoprotein parameters investigated, total triglycerides or VLDL cholesterol levels showed independent relations to both the plasma and the membrane phosphatidylcholine/sphingomyelin ratio as well as to the maximal velocity of sodium-lithium countertransport. The results indicate that an increase in red cell membrane phosphatidylcholine and a concomitant fall in sphingomyelin are closely associated with the acceleration of sodium-lithium countertransport in hyperlipidemia.

Arachidonic acid of platelet phospholipids is decreased after extracorporeal removal of plasma low density lipoproteins in patients with familial hypercholesterolemia.

Platelet phospholipid composition was analyzed before and after extracorporeal removal of low density lipoproteins (LDL) by LDL apheresis in six patients with familial hypercholesterolemia. Elevated levels of total plasma cholesterol and the portion of plasma cholesterol carried by LDL were reduced by 56 and 66% after LDL apheresis. Platelet cholesterol contents remained unaffected. While the phosphatidylcholine (PC):sphingomyelin (SM) ratio in plasma lipoproteins was increased by 22% following apheresis, the same parameter was lowered by 14% in platelets. LDL apheresis induced decreases in the percentages of distinct molecular species containing arachidonic acid in platelet diacyl subgroups of PC, phosphatidylinositol (PI) and phosphatidylserine (PS) as well as in alkenylacyl (plasmalogen) phosphatidylethanolamine (PE). Directly after apheresis, the percentages of molecular species with arachidonic acid of diacyl PC, diacyl PI and alkenylacyl PE were reduced by 20, 23 and 8%, respectively. Two days after the procedure, total arachidonic acid of diacyl PC, diacyl PS and alkenylacyl PE was lowered by 11, 20 and 8%. Overall, the amount of phospholipid bound arachidonic acid was reduced by 16% after apheresis (from 79.1 to 66.4 nmol/10(8) platelets). The results are thus in agreement with previous data indicating decreased phospholipid bound arachidonic acid in red blood cells after apheresis (Engelman B. Bräutigam C, Kulschar R et al. Biochim Biophys Acta 1994:1196:154). Urinary 2,3-dinor thromboxane B2, an estimate of platelet thromboxane A2 (TXA2) production, tended to be decreased following the procedure. The percentage change in the TXA2 metabolite was positively related to the magnitude of change induced by apheresis in phospholipid bound arachidonic acid. In summary, the results suggest that in patients with hypercholesterolemia, the level of plasma LDL is an important determinant of the arachidonic acid content of several platelet phospholipids.

Plasmalogen phospholipids in plasma lipoproteins of normolipidemic donors and patients with hypercholesterolemia treated by LDL apheresis.

Recent evidence indicates that plasmalogen phospholipids are particularly sensitive to oxidation and may possess antioxidative properties. Approximately 4.4%-5.5% of phosphatidylcholine (PC), and 53%-60% of phosphatidylethanolamine (PE) consisted of the plasmalogen phospholipids, plasmenylcholine and plasmenylethanolamine, respectively, in whole plasma, low density lipoprotein (LDL) and high density lipoprotein (HDL) of 11 normolipidemic donors. Of total plasmalogen phospholipids in plasma, slightly more was associated with LDL particles (about 42%) than with HDL (36%). Plasmalogen phospholipid levels were analyzed in 12 patients with familial hypercholesterolemia (FH) regularly treated by LDL apheresis, of whom 6 were supplemented with vitamin E (alpha tocopherol, 400 IU/day), the remaining 6 not receiving the antioxidant. Before apheresis (pre), total plasmalogen phospholipid levels in plasma and LDL (expressed as mumol/mmol cholesterol of compartment) decreased as follows: patients receiving vitamin E > normolipidemia > patients not receiving vitamin E. In both hypercholesterolemic groups, the contents of plasmalogen phospholipids in whole plasma and LDL were 3-5-fold higher than those of vitamin E. Directly after apheresis (post), plasmalogen phospholipid levels in plasma were raised by about 50% in the two hypercholesterolemic groups, mostly due to increases in plasmenylethanolamine levels. Two days after apheresis (48 h post), plasmalogen contents were still elevated in plasma and red blood cell membranes of patients receiving vitamin E, while they had already reached pre-apheresis values in those not supplemented with alpha tocopherol. Molecular species of plasma diacyl phospholipids containing polyunsaturated fatty acids were elevated at pre in patients receiving vitamin E as compared to patients without supplementation. At 48 h post, LDL apheresis induced an increase in these molecular species only in patients receiving vitamin E. In conclusion, the contents of plasmalogen phospholipids in plasma lipoproteins are at least three times higher than those of vitamin E. LDL apheresis raises the level of plasmalogen phospholipids in plasma, the increase persisting longer in patients supplemented with vitamin E. Supplementation with vitamin E appears to protect plasmalogen phospholipids in plasma lipoproteins against oxidative degradation.

Differential antiplatelet effects of various glycoprotein IIb-IIIa antagonists.

The blockade of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) was recently introduced as a new antiplatelet strategy. At present, various GPIIb-IIIa inhibitors are available to treat patients with acute coronary syndrome or when undergoing percutaneous coronary interventions. The current study systematically evaluates the antiplatelet effects of GPIIb-IIIa inhibitors in clinical use. Using conformation-dependent monoclonal antibodies [ligand-induced binding sites (LIBS-1), PMI-1] and flow cytometry, we showed that the GPIIb-IIIa antagonists abciximab, integrelin, lamifiban, and tirofiban, but not EMD 122347 or YM 337, induced LIBS activity of platelet GPIIb-IIIa. The LIBS activity of GPIIb-IIIa antagonists correlates with a proaggregatory response of fixed platelets pretreated with GPIIb-IIIa antagonists (intrinsic activity). All tested GPIIb-IIIa antagonists completely inhibit concentration-dependent ADP (20 micromol/l)-induced aggregation. In contrast, substantial TRAP (25 micromol/l)-induced platelet aggregation still occurs even at high inhibitor concentrations of the tested GPIIb-IIIa antagonists. In addition, we show that GPIIb-IIIa antagonists are poor inhibitors of platelet release reaction (ATP and P-selectin secretion) especially when strong agonists such as TRAP are used to activate platelets. Inhibition of platelet procoagulant activity (thrombin generation) by GPIIb-IIIa antagonists is dependent on the type and concentration of antagonists and on the strength of stimulus (thrombin, tissue factor) used to induce platelet-dependent thrombin generation. The present data show that significant pharmacological differences exist between GPIIb-IIIa antagonists that may have consequences for antithrombotic strategies and for future drug development.

Total intracellular calcium content and sodium transport in erythrocytes of essential hypertensive patients.

To estimate the amount of Ca bound to the inner aspect of the membrane of human erythrocytes, a method was developed to determine the total intracellular calcium content (Cai) using flameless atomic absorption. The mean Cai was 1.65 +/- 0.34 mumol/l cells in normotensive individuals (range 1.1-2.4). In untreated essential hypertensive patients Cai was slightly reduced, the difference being not significant. Antihypertensive treatment possibly affects Cai. Plasma ionized Ca and total plasma Ca tended to be lower and higher in the hypertensive patients, respectively. The activities of red cell Na-K co-transport, Na-Li exchange and the Na-K pump were inversely correlated to Cai in normotensives (P less than 0.05). The relations of Cai to Na-K co-transport and Na-Li exchange were displaced to higher values in untreated essential hypertensive patients.

Crossroads of coagulation and innate immunity: the case of deep vein thrombosis.

Deep vein thrombosis (DVT) is a common condition characterized by the formation of an occlusive blood clot in the venous vascular system, potentially complicated by detachment and embolization of thrombi into the lung. Recent evidence from mouse models has shed light on the sequence of events and on the cellular (innate immune cells, platelets) and molecular (hematopoietic tissue factor, nucleic acids) components involved. In response to decreased blood flow, circulating neutrophils and monocytes adhere to the activated endothelium within hours. They initiate and propagate DVT by interacting with platelets and by the exposure and activation of circulating tissue factor and FXII. Intravascular blood coagulation is also induced by extracellular nucleosomes released mainly from activated neutrophils. Interestingly, these mechanisms are closely linked to an evolutionary conserved immune defense mechanism activated in response to infections. In this review, we will give an overview of DVT and the role of innate immune pathways supporting this process. While the latter are aimed at preserving tissue integrity and function, uncontrolled blood coagulation and activation of immune cells may result in pathological thrombus formation and vascular occlusion. Understanding the molecular and cellular players triggering occlusion of large veins, and their distinction from physiological hemostasis, is important for the development of strategies to prevent and treat DVT.

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin ulex europaeus agglutinin I.

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.