Homocysteine decreases platelet NO level via protein kinase C activation.
Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47(phox) and p67(phox), were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cgamma2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.
Signorello
Maria Grazia
MG
Department of Experimental Medicine, University of Genoa, Genoa, Italy.
Segantin
Alessia
A
Passalacqua
Mario
M
Leoncini
Giuliana
G
D000704:Analysis of Variance; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002956:Citrulline / Q000097:blood; D006152:Cyclic GMP / Q000097:blood; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006710:Homocysteine / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000097:blood; D009569:Nitric Oxide / Q000097:blood*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000097:blood*; D013481:Superoxides / Q000097:blood; D014443:Tyrosine / Q000031:analogs & derivatives / Q000097:blood
D004791:Enzyme Inhibitors; D006710:Homocysteine; D013481:Superoxides; D002956:Citrulline; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D011493:Protein Kinase C; D006152:Cyclic GMP