Activation of p38 MAPKinase / cPLA2 pathway in homocysteine treated platelets

Hyperhomocysteinaemia 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.