Catherine Calzada

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets.

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.

Pro- and antioxidant activities of docosahexaenoic acid on human blood platelets.

Summary.u2002 nu2003−u20033 polyunsaturated fatty acids may protect against vascular diseases, however, their high accumulation in membranes may increase lipid peroxidation and subsequently induce deleterious effects in patients suffering from oxidative stress. This led us to investigate in vitro the dose‐dependent effect of docosahexaenoic acid (DHA) on the redox status of human platelets. We have compared the effect of different DHA concentrations (0.5, 5 and 50u2003µmolu2003L−1) corresponding to DHA/albumin ratios of 0.01, 0.1 and 1. At the highest concentration, DHA elicited a marked oxidative stress, as evidenced by high malondialdehyde and low vitamin E levels whereas the lowest DHA concentration significantly decreased the malondialdehyde formation, with no change in vitamin E. The proportion of DHA was only increased in plasmalogen phosphatidylethanolamine at low concentration to rise in all phosphatidyl‐choline and ‐ethanolamine subclasses at high concentration. Thus, the results show a biphasic effect of DHA with antioxidant and prooxidant effects at low and high concentrations, respectively, with a possible relationship with the phospholipid subclass in which it accumulates.

Interactions between arachidonic and eicosapentaenoic acids during their dioxygenase-dependent peroxidation

Eicosapentaenoic acid (EPA), a major polyunsaturated fatty acid of fish has been widely proposed as a potential nutrient for decreasing platelet-endothelial cell interactions and the subsequent atherogenesis and thrombogenesis. This is mainly based upon the decrease of arachidonic acid (AA) oxygenation into bioactive molecules like thromboxane A2. In addition, EPA may be oxygenated into its own active derivatives via cell dioxygenases. We report evidence for the requirement of specific peroxides, adequately provided by AA, to allow EPA to be oxygenated into its bioactive products like prostaglandin I3, a prostacyclin mimetic. On the other hand, we present some data that argue for a decreased basal AA dioxygenation (specific peroxidation) by small concentrations of EPA. The interactions between AA and EPA are then dual, EPA being able to counteract AA oxygenation whereas EPA requires AA to be efficiently oxygenated.

Pathophysiologic role of redox status in blood platelet activation. Influence of docosahexaenoic acid

Decrease of platelet glutathione peroxidase activity results in increased life span of lipid hydroperoxides, especially the 12-lipoxygenase product of arachidonic acid, 12-HpETE. Phospholipase A2 activity is subsequently enhanced with the release of arachidonic acid, which results in higher thromboxane formation and platelet function. Docosahexaenoic acid may either potentiate platelet lipid peroxidation or lower it when used at high or low concentrations, respectively. In the case of slowing down lipid peroxidation, docosahexaenoic acid was specifically incorporated in plasmalogen ethanolamine phospholipids. This could have a relevant pathophysiologic role in atherothrombosis.

Involvement of lipid peroxidation in platelet signalling

A well-known signalling pathway in blood platelets consists in the release of arachidonic acid (AA) from membrane phospholipids and its specific oxygenation into bioactive derivatives. In particular, cyclic prostaglandin endoperoxides and thromboxane A2 are potent inducers of platelet functions and are produced in greater amounts when the level of lipid hydroperoxides is higher than normal, as physiological concentrations of such peroxides activate the cyclooxygenation of AA. In this context, a lower activity of platelet glutathione peroxidase (GPx), the key-enzyme for the degradation of lipid hydroperoxides, has been reported in aging, which will ensure a longer life span to those peroxides. Accordingly, the biosynthesis of pro-aggregatory prostanoids is elevated in platelets from the elderly. On the other hand, fatty acids from marine origin have been recognized as inhibitors of platelet functions, and they may alter the redox status of cells. They may for instance increase the platelet GPx activity, an effect that can be prevented by antioxidants. Overall, these data point out the relevance of the redox status in platelet functions.

Stimulation of platelet aggregation in response to arachidonic acid hydroperoxidevia phospholipase activation

Considerable interest exists concerning the role played by lipid peroxides in cell damage and in the development of cardiovascular diseases. Indeed, an exacerbated platelet activation, as observed in aging, was associated with an increased overall lipid peroxidation and an increased peroxidation of arachidonic acid (AA). The platelet antioxidant defenses were altered, namely, the vitamin E level and the activity of glutathione-peroxidase (GSH-Px) (1), a key enzyme reducing lipid hydroperoxides. A transient accumulation of the 12lipoxygenase-derived product of AA, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), resulting from the decreased GSH-Px activity, is likely to activate the dioxygenases and therefore to lead to platelet hyperactivation. Considering that the biological functions of lipid hydroperoxides are not well-defined, it is of interest to determine whether low concentrations of 12-HPETE may induce platelet aggregation. Human platelets were incubated in the presence or absence of 12-HPETE for 1 rain at 37°C. A subthreshold concentration (STC) of collagen--defined as the highest concentration of collagen that did not induce any platelet aggregation--was then added to the platelet suspension for another minute. Although 12-HPETE alone had no effect on platelet aggregation, the addition of nanomolar concentrations of 12-HPETE (from 10 to 100 nM) together with an STC of collagen significantly induced platelet aggregation. It was closely associated with an increased formation of thromboxane B2, the stable catabolite of thromboxane A 2. Notably the concentration of 12-HPETE required to potentiate platelet aggregation was 50-fold lower than the one required to prime platelet aggregation in response to AA via an activation of the cyclo-oxygenase (2). As the activation of platelets by collagen involves the release of AA from platelet phospholipids, the concentration of unesterified AA was determined by gas chromatography. The addition of 12-HPETE to platelets co-incubated with an STC of collagen resulted in a threefold increase in the amount of unesterified AA. As it is Lipids 34,

Oxygenation of polyunsaturated fatty acids and oxidative stress within blood platelets

295 (1999). *To whom correspondence should be addressed at INSERM U 352, Biochimie et Pharmacologie, INSA-Lyon, B~timent 406, 20 avenue Albert Einstein, 69621 Villeurbanne Cedex, France. E-mail: Catherine,Calzada @insa-lyon.fr Abbreviations : AA, arachidonic acid; AACOCF3, arachidonoyl trifluoromethylketone; cPLA2, cytosolic phospholipase A2; GPC, glycerophosphocholine; GSH-Px, glutathione-peroxidase; HPLC, high-performance liquid chromatography; 12-HPETE, 12-hydroperoxy-eicosatetraenoic acid; STC, sub-threshold concentration; TLC, thin-layer chromatography. known that the cytosolic phospholipase A 2 (cPLA 2) of 85 kDa is involved in the release of AA from membrane phospholipids, platelets were preincubated with a potent and selective inhibitor of the cPLA 2, arachidonoyl trifluoromethyl ketone (AACOCF3). AACOCF 3 prevented the increase in the amount of unesterified AA induced by 12-HPETE and an STC of collagen, suggesting the involvement of the cPLA 2. In order to determine the origin of released AA, the molecular species of platelet phospholipid subclasses were measured by reversed-phase high-performance liquid chromatography (HPLC) (3). After lipid extraction, phospholipid classes were separated by thin-layer chromatography (TLC), hydrolyzed by phospholipase C, and corresponding diradylglycerols were derivatized with 3,5-dinitrobenzoyl chloride. Phospholipid subclasses were separated by TLC, and various molecular species were quantified by HPLC with ultraviolet detection at 240 nm. Among the arachidonoyl-containing molecular species of 1,2-diacyl-glycerophosphocholine (GPC), 16:0/AA and 18:0/AA species decreased in platelets incubated with 12-HPETE whereas 18: l/AA did not change significantly. Concerning the molecular species of the l-alkyl-2acyl-GPC subclass, 18:I/AA, 16:0/AA, and 18:0/AA tended to decrease in response to 12-HPETE. On the contrary, no difference was observed between control platelets and 12HPETE-treated platelets in the 1,2-diacyl-glycerophosphoethanolamine pool. In conclusion, physiologically relevant concentrations of 12-HPETE stimulate the aggregation of platelets co-incubated with nonaggregating concentrations of collagen which could be mediated via an activation of the cPLA 2. These results enlighten the physiological importance of 12-HPETE in controlling the unesterified AA level and therefore the formation of eicosanoids.

P237 Marqueurs spécifiques de peroxydation lipidique dans les lipoprotéines de faible densité de diabétiques de type-2

The oxygenation metabolism of arachidonic acid (ArA) has been early described in blood platelets, in particular with its conversion into the potent labile thromboxane A2 that induces platelet aggregation and vascular smooth muscle cells contraction. In addition, the primary prostaglandins D2 and E2 have been mainly reported as inhibitors of platelet function. The platelet 12-lipoxygenase (12-LOX) product, i.e. the hydroperoxide 12-HpETE, appears to stimulate platelet ArA metabolism at the level of its release from membrane phospholipids through phospholipase A2 (cPLA2) and cyclooxygenase (COX-1) activities, the first enzymes in prostanoid production cascade. Also, 12-HpETE may regulate the oxygenation of other polyunsaturated fatty acids (PUFA) by platelets, especially that of eicosapentaenoic acid (EPA). On the other hand, the reduced product of 12-HpETE, 12-HETE, is able to antagonize TxA2 action. This is even more obvious for the 12-LOX end-products from docosahexaenoic acid (DHA), 11- and 14-HDoHE. In addition, 12-HpETE plays a key role in platelet oxidative stress as observed in pathophysiological conditions, but may be regulated by DHA with a bimodal way according to its concentration. Other oxygenated products of PUFA, especially omega-3 PUFA, produced outside platelets may affect platelet functions as well.

P86 Activation des plaquettes sanguines humaines par les lipoprotéines de faible densité glycoxydées in vitro et isolées de diabétiques de type 2

Introduction Le diabete de type-2 (DT-2) est associe a diverses maladies cardiovasculaires ainsi qu’a un stress oxydant accru. Les lipoproteines de faible densite (LDL) oxydees jouent un role dans l’atherosclerose. Or les LDL de DT-2 subissent des modifications oxydatives, encore partiellement caracterisees. Notre objectif est de definir le profil detaille des LDL en acides gras et peroxydes lipidiques chez des DT-2 et des volontaires sains. Materiels et Methodes Apres avoir isole les LDL du plasma par ultracentrifugations sequentielles a temps court, nous avons etabli leurs profils lipidomiques. La composition en acides gras des differentes classes lipidiques a ete determinee par chromatographie en phase gazeuse. Differents marqueurs stables de peroxydation lipidique, notamment le dialdehyde malonique et les derives monohydroxyles des acides gras polyinsatures, ont ete separes et doses par HPLC. Resultats Le profil lipidique detaille des LDL de DT-2 revele d’abord une nette diminution, dans toutes les classes lipidiques, du principal acide gras polyinsature constitutif des LDL : l’acide linoleique. Les acides gras de la sous-classe des plasmalogenes a phosphatidylethanolamine, sensible au stress oxydant, sont egalement diminues. D’autre part, la concentration en dialdehyde malonique, marqueur global reconnu de peroxydation lipidique, est augmentee. Les LDL contiennent les acides hydroxy-octadecadienoiques (HODE) et hydroxy-eicosatetraenoiques (HETE) derivant respectivement des acides linoleique et arachidonique. Le rapport HODE/acide linoleique est nettement augmente dans les LDL de DT-2. Conclusion Nos resultats mettent en evidence une augmentation des concentrations de peroxydes lipidiques dans les LDL de DT-2. Les acides gras monohydroxyles et les plasmalogenes alteres pourraient representer des marqueurs originaux du stress oxydant chez les DT-2.

Low concentrations of lipid hydroperoxides prime human platelet aggregation specifically via cyclo-oxygenase activation

Introduction L’hyperactivation plaquettaire et la dyslipidemie contribuent a un risque atherothrombotique plus eleve lors du diabete. Les plaquettes de diabetiques de type 2 presentent un stress oxydant accru et les lipoproteines de faible densite (LDL) sont exposees a la fois a l’hyperglycemie et au stress oxydant. Notre objectif a ete de determiner si des LDL glycoxydees in vitro et in vivo sont susceptibles d’activer les plaquettes. Materiels et methodes Suite a l’interaction des LDL avec des plaquettes de volontaires sains, la phosphorylation de la p38 MAPK, kinase de stress impliquee dans l’activation de la phospholipase A2 cytosolique, et la concentration de thromboxane B2 (TxB2), catabolite stable du thromboxane A2, puissant agent pro-agregant, ont ete determinees dans les plaquettes. Les concentrations de dialdehyde malonique (MDA), marqueur de peroxydation lipidique, et des isomeres de tocopherol dans les LDL, ont ete determinees par HPLC. Resultats Des LDL glycoxydees in vitro par 50 mM de glucose et 1 μm de sulfate de cuivre activent les plaquettes via une phosphorylation accrue de la p38 MAPK (x 3, p Conclusion Nos resultats indiquent que les LDL glycoxydees peuvent jouer un role important dans l’hyperactivation des plaquettes chez les diabetiques de type 2.