1,721,011 research outputs found
The Ca2+/calmodulin kinase/AMP-activated protein kinase pathway regulates the lectin phaseolus vulgaris agglutinin induced NO production in human platelets
No full textPreviously it was shown that wheat germ agglutinin (WGA) and, at a minor extent, phaseolus vulgaris agglutinin (PHA), are able to induce platelet activation. Since the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO)/soluble guanylyl cyclase/cGMP/cGMP-dependent protein kinase (PKG) pathway is one of the major antiaggregating mechanism present in platelets, we tested the WGA or PHA effect on this pathway. It has been shown that platelets treated with WGA did not produce NO, while PHA stimulated NO production in a dose and time dependent manner. It has been found that the increased NO formation induced by PHA was dependent on eNOS phosphorylation/activation. The Ca2+/calmodulin-dependent kinase kinase/AMP activated protein kinase pathway seems to be greatly involved as STO-609 and Compound C, Ca2+/calmodulin protein kinase kinase/AMP kinase inhibitors respectively, cancelled eNOS phosphorylation induced by PHA. One crucial effect of NO and cGMP elevation is the activation of PKG, that can phosphorylate vasodilator-stimulated phosphoprotein (VASP). It was found that NO and cGMP elevation and VASP phosphorylation both on ser239 and thr278 were greatly stimulated by PHA and strongly inhibited by STO-609 and Compound C and by the eNOS inhibitor L-NAME. Thus, the CaMKK/AMPK pathway activated by PHA can regulate platelet activation stimulating the eNOS/NO/cGMP/PKG signalling pathwa
Anandamide Induces Platelet Nitric Oxide Synthase through AMP-Activated Protein Kinase
No full textThe objective of this study was to determine whether adenosine 5' monophosphate (AMP)-activated protein kinase (AMPK) is activated by anandamide (AEA) and is involved in endothelial nitric oxide synthase (eNOS) activation. We found that AEA stimulates and activates AMPKα through a Ca2+ -dependent/Calmodulin (CaM)-dependent pathway as the specific inhibitor of the Ca2+ /Calmodulin kinase kinase β (CaMKKβ) STO-609 abolishes the AMPK phosphorylation/activation. The same inhibiting effect is shown in platelets pretreated with LY294002, an inhibitor of phosphatidylinositol 3 kinase (PI3K), or with MK2206, an inhibitor of protein kinase B (AKT), suggesting that AMPK is downstream of the PI3K/AKT pathway. Moreover, the AEA-induced eNOS activation and the consequent nitric oxide (NO) and guanosine 3'-5' cyclic monophosphate (cGMP) increase are mediated by the CaMKKβ/AMPKα pathway as STO-609 significantly inhibits these parameters. In contrast, liver kinase B1 (LKB1) seems to be very poorly involved. One crucial effect of NO and cGMP elevation is the activation of protein kinase G that can phosphorylate the vasodilator-stimulated phosphoprotein (VASP). We have demonstrated that AEA stimulates VASP phosphorylation on both thr278 and ser239 that is strongly inhibited by STO-609, LY294002, and MK2206. Finally, AMPK phosphorylation/activation and VASP phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid receptor (CB1). SR144528, an antagonist of type 2 cannabinoid receptor (CB2), has a less-potent effect, suggesting that the CB1 receptor is overall involved in the AEA effect. In conclusion, we show that the CaMKKβ/AMPKα pathway, downstream of the PI3K/AKT pathway, is activated by AEA in human platelets and leads to increase NO levels producing beneficial effects during ischemic conditions and contributing to extend platelet survival
Effect of 2-arachidonoylglycerol on myosin light chain phosphorylation and platelet activation: The role of phosphatidylinositol 3 kinase/AKT pathway
No full textThe endocannabinoid 2-arachidonoylglycerol (2-AG) can be considered a true agonist as it is able to activate human platelets stimulating arachidonic acid release, thromboxane B2 formation and calcium intracellular elevation. Recently we have shown that 2-AG induces a rapid myosin light chain (MLC) phosphorylation/activation, early mediated by RhoA kinase (ROCK) signalling pathway and later by myosin light chain kinase. The aim of the present study was to investigate the role of phosphatidylinositol 3 kinase (PI3K)/AKT pathway in MLC phosphorylation and some downstream events such as actin polymerization, ATP secretion and aggregation. We demonstrated that PI3K in particular the isoforms alfa and beta and AKT have a role in MLC phosphorylation. The stimulation of PI3K/AKT pathway activates ROCK. ROCK is directly involved in the early phase of MLC activation stimulating thr18 phosphorylation. MLC activation is strengthened through the MLC phosphatase inhibition, that is accomplished through the phosphorylation of MYPT1, catalytic subunit of MLC phosphatase, overall mediated by ROCK. In addition we have found that the PI3Kalfa/beta isoforms and AKT are involved in the downstream mechanisms leading to actin polymerization, ATP secretion and aggregation of human platelets stimulated by 2-AG
The arachidonic acid effect on platelet nitric oxide level
No full textArachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A(2) mimetic U46619 behaved in a similar way. The Arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A(2) receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that Arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by Arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, Arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely Arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power
Activation of p38 MAPKinase / cPLA2 pathway in homocysteine treated platelets
No full textHyperhomocysteinaemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine supports athereothrombosis is still unknown and may be multifactorial. Earlier “in vitro” studies demonstrated that homocysteine induced arachidonic acid release and increased thromboxane B2 formation. In this work we found that homocysteine stimulated the rapid and sustained phosphorylation of platelet p38 mitogen-activated protein kinase (p38 MAPK). The effect was time and dose-dependent. The homocysteine effect on p38 MAPK phosphorylation was prevented by N-acetyl-L-cysteine and iloprost and was partially inhibited by nordihydroguaiaretic acid. Moreover the incubation of platelets with homocysteine led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition homocysteine promoted cPLA2 activation, assessed as arachidonic acid release. The cPLA2 phosphorylation and activation were both impaired by the inhibition of p38 MAPK through SB203580. This effect was not complete, reaching at the most the 50 % of the total. In FURA 2-loaded platelets homocysteine induced a dose-dependent intracellular calcium rise suggesting that the calcium elevation promoted by homocysteine could participate in the cPLA2 activation, leading to arachidonic acid release and thromboxane B2 formation. In conclusion our data provide insight into the mechanisms of platelet activation induced by homocysteine, suggesting that the p38 MAPK / cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia
A role for PLCgamma2 in platelet activation by homocysteine
No full textThe aim of this study was to examine the homocysteine effect on phospholipase C gamma 2 (PLC gamma 2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLC gamma 2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor-mediated tyrosine kinase p60src:, inducing phosphorylation and activation of p72syk. The antioxidant N-acetyl-L-cysteine prevented the activation of these kinases. The phosphorylation and activation of PLC gamma 2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLC gamma 2, suggesting that thromboxane A(2) Could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N-acetyl-L-cysteine of 35% or 50% respectively, while the PLC gamma 2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLC gamma 2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A(2)
The L-arginine/NO pathway in the early phases of platelet stimulation by collagen
No full textNitric oxide production, L-arginine transport and intracellular [Ca2+] changes in human platelets stimulated without stirring by low doses of collagen have been evaluated. Collagen decreased in a dose-dependent manner the nitric oxide formation. A reduction of about 30% of the basal level was produced by 5 mug/mL. Aspirin did not change the collagen effect. The inhibition was reversed by EGTA. Moreover collagen reduced L-arginine uptake. The exposure of platelets to 5 mug/mL collagen diminished of about 30% L-arginine transport. The specific involvement of the system y(+) is suggested. In addition in FURA 2-loaded platelets collagen induced a dose-dependent slow sustained [Ca2+] rise that was almost completely cancelled by EGTA. Finally the treatment of whole platelets with collagen affected in a dose-dependent manner the maximal nitric oxide formation, suggesting a direct effect at the level of nitric oxide synthase enzyme. The phosphorylation of specific serine/threonine residues regulated by protein kinase C could be involved. In conclusion during the early phases of platelet stimulation with collagen nitric oxide formation is diminished. This reduction can be due to a lower availability of L-arginine for cytosolic nitric oxide synthase and/or to a decreased activity related to modifications of the enzyme
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