Nitrolinoleate Inhibits Platelet Activation by Attenuating Calcium Mobilization and Inducing Phosphorylation of Vasodilator-stimulated Phosphoprotein through Elevation of cAMP

Barbara Coles,Allison Bloodsworth,Jason P Eiserich,Marcus J Coffey,Rachel M Mcloughlin,John C Giddings,Malcolm J Lewis,Richard J Haslam,Bruce A Freeman,Valerie B O'Donnell

doi:10.1074/jbc.m105209200

Barbara Coles, Allison Bloodsworth + Show 8 more

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https://doi.org/10.1074/jbc.m105209200

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Abstract

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO(2)). The effect of LNO(2) on human platelets was examined to define how nitrated lipids might behave in vivo. LNO(2), but not LA or 3-nitrotyrosine, dose dependently (0.5-10 microm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO(2) caused slight mobilization of calcium (Ca(2+)) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca(2+) elevations. LNO(2) did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO(2) treatment of platelets, and the adenylyl cyclase inhibitors 2',5'-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO(2) significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO(2). The platelet inhibitory actions of LNO(2) indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.

Highlights

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO2)
The effect of LNO2 on human platelets was examined to define how nitrated lipids might behave in vivo
Similar concentrations of LA or 3-NT added in the same volumes of ethanol were without effect, indicating that this is specific for the nitrated lipid (Fig. 1B)

Summary

Introduction

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO2). N2O3 rapidly hydrolyzes to form nitrite (NO2Ϫ), the major decomposition product of NO in aqueous buffers (Equation 2) This species is stable at physiological pH, acidification yields nitrous acid (HONO), which in turn generates a complex mixture of oxidizing and nitrating/nitrosating intermediates (e.g. N2O3, NO, NO2, and N2O4). The major stable NO-derived end product of oxidation is nitrate (NO3Ϫ), generally assumed to form through the reaction of NO with oxyhemoglobin (oxyHb). In vascular diseases such as hypertension and atherosclerosis, the reaction of NO with superoxide (O2.), forming the nitrating/nitrosating peroxynitrite (ONOOϪ), may account for NO3Ϫ formation and removal of a significant proportion of NO [1, 2]. This occurs at diffusion-limited rates (k ϭ 1.9 ϫ 1010 MϪ1 sϪ1) [3], kinetically outcompeting the reaction of O2. with superoxide dismutase

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