Activation of p38 MAPKinase/cPLA2 pathway in homocysteine-treated platelets.
Hyperhomocysteinemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine (HCy) supports atherothrombosis is still unknown and may be multifactorial. Earlier in vitro studies demonstrated that HCy induced arachidonic acid release and increased thromboxane B2 (TXB2) formation. In this work, we found that HCy stimulated the rapid and sustained phosphorylation of platelet p38 mitogen-activated protein kinase (p38 MAPK). The effect was time- and dose-dependent. The HCy 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 HCy led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition HCy 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, HCy induced a dose-dependent intracellular calcium rise suggesting that the calcium elevation promoted by HCy could participate in the cPLA2 activation, leading to arachidonic acid release and TXB2 formation. In conclusion, our data provide insight into the mechanisms of platelet activation induced by HCy, suggesting that the p38 MAPK/cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia.
Leoncini
G
G
Department of Experimental Medicine, Biochemistry Section, University of Genoa, Genova, Italy. leoncini@unige.it
Bruzzese
D
D
Signorello
M G
MG
D016824:Antigens, Human Platelet / Q000378:metabolism*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D016824:Antigens, Human Platelet; C083451:human platelet antigen 1b; D006710:Homocysteine; D048051:p38 Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2