A role for PLCgamma2 in platelet activation by homocysteine.
The aim of this study was to examine the homocysteine effect on phospholipase Cgamma2 (PLCgamma2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCgamma2. 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 PLCgamma2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCgamma2, 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 PLCgamma2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCgamma2 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).
Leoncini
Giuliana
G
Department of Experimental Medicine, Biochemistry Section, University of Genoa, Viale Benedetto XV 1, 16132 Genova, Italy. leoncini@unige.it
Bruzzese
Debora
D
Signorello
Maria Grazia
MG
D002118:Calcium / Q000378:metabolism; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D018384:Oxidative Stress; D051966:Phospholipase C gamma / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D006710:Homocysteine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); C000606896:SYK protein, human; D000072377:Syk Kinase; D010744:Phosphoric Monoester Hydrolases; D051966:Phospholipase C gamma; D002118:Calcium