Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.     

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.     

Hemin-induced platelet activation and ferroptosis is mediated through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin

Reactive oxygen species (ROS) are capable of inducing cell death or apoptosis. Recently, we demonstrated that lipid-ROS can mediate ferroptosis and activation of human platelets. Ferroptosis is an intracellular iron-mediated cell death, distinct from classical apoptosis and necrosis, which is mediated through the accumulation of ROS, lipid peroxides and depletion of cellular GSH. Lately, we demonstrated that hemoglobin degradation product hemin induces ferroptosis in platelets via ROS and lipid peroxidation. In this study, we demonstrate that hemin-induced ferroptosis in platelets is mediated through ROS-driven proteasome activity and inflammasome activation, which were mitigated by Melatonin (MLT). Although inflammasome activation is linked with pyroptosis, it is still not clear whether ferroptosis is associated with inflammasome activation. Our study for the first time demonstrates an association of platelet activation/ferroptosis with proteasome activity and inflammasome activation. Although, high-throughput screening has recognized ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent ferroptosis inhibitors, having an endogenous antioxidant such as MLT as ferroptosis inhibitor is of high interest. MLT is a well-known chronobiotic hormone that regulates the circadian rhythms in vertebrates. It also exhibits potent antioxidant and ROS quenching capabilities. MLT can regulate fundamental cellular functions by exhibiting cytoprotective, oncostatic, antiaging, anti-venom, and immunomodulatory activities. The ROS scavenging capacity of MLT is key for its cytoprotective and anti-apoptotic properties. Considering the anti-ferroptotic and anti-apoptotic potentials of MLT, it could be a promising clinical application to treat hemolytic, thrombotic and thrombocytopenic conditions. Therefore, we propose MLT as a pharmacological and therapeutic agent to inhibit ferroptosis and platelet activation.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

Effect of functional electrical stimulation on the effort and walking speed,surface electromyography activity,and metabolic responses in stroke subjects

ObjectiveTo investigate the effects of functional electrical stimulation (FES) combined with conventional rehabilitation program on the effort and speed of walking, the surface electromyographic (sEMG) activity and metabolic responses in the management of drop foot in stroke subjects.MethodsFifteen patients with a drop foot resulting from stroke at least 3 months prior to the start of the trial took part in this study. All subjects were treated 1 h a day, 5 days a week, for 12 weeks, including conventional stroke rehabilitation program and received 30 min of FES to the tibialis anterior (TA) muscle of the paretic leg in clinical settings. Baseline and post-treatment measurements were made for temporal and spectral EMG parameters of TA muscle, walking speed, the effort of walking as measured by physiological cost index (PCI) and metabolic responses.ResultsThe experimental results showed a significant improvement in mean-absolute-value (21.7%), root-mean-square (66.3%) and median frequency (10.6%) of TA muscle EMG signal, which reflects increased muscle strength. Mean increase in walking speed was 38.7%, and a reduction in PCI of 34.6% between the beginning and at end of the trial. Improvements were also found in cardiorespiratory responses with reduction in oxygen consumption (24.3%), carbon dioxide production (19.9%), heart rate (7.8%) and energy cost (22.5%) while walking with FES device.ConclusionsThe results indicate that the FES may be a useful therapeutic tool combined with conventional rehabilitation program to improve the muscle strength, walking ability and metabolic responses in the management of drop foot with stroke patients.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.     

Differential effects of 15-HPETE on arachidonic acid metabolism in collagen-stimulated human platelets

The 15-hydroperoxyeicosatetraenoic acid (15-HPETE) has been shown to affect platelet aggregation induced by collagen, arachidonic acid (AA), and PGH2-analogue. Furthermore, it also inhibits the platelet cyclooxygenase and lipoxygenase enzymes, and prostacyclin synthase. The present study was designed to test the effect of 15-HPETE on the mobilization of endogenous AA in collagen-stimulated human platelets. For this purpose, human platelets pretreated with BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase) were stimulated with collagen in the presence of varied concentrations of 15-HPETE. We observed a significant inhibition of oxygenases at all concentrations of 15-HPETE. In contrast, our results indicate that 15-HPETE at lower concentrations (10 microM and 30 microM) significantly stimulated the collagen-induced release of AA from phospholipid sources. Although higher concentrations of 15-HPETE (50 microM and 100 microM) caused some inhibition of AA accumulation in the free fatty acid fraction (25% and 60%), the degree of inhibition was significantly lower than the inhibition observed for the oxygenases (65% and 88% for cyclooxygenase and 77% and 94% for lipoxygenase respectively). These results provide support that hydroperoxides also regulate phospholipases presumably by a different mechanism, which may be important in the detoxification of phospholipid peroxides.     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.