Activation of p38 MAPKinase/cPLA2 pathway in homocysteine‐treated platelets

Abstract

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.

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

“…29,30 In platelets, phosphorylation of cPLA 2 has been documented to occur in response to several different agonists, including thrombin, and to be mainly mediated by p38 MAPK rather than ERK1/2. [31][32][33][34] In the present study, we confirmed that thrombin-induced phosphorylation of cPLA 2 in mouse platelets is prevented by the specific p38 MAPK inhibitor SB203580 (data not shown) and also documented that cPLA 2 phosphorylation is significantly reduced in platelets lacking Pyk2. In addition we found that in thrombin-stimulated platelets, the phosphorylation of p38 MAPK, but not ERK1/2, was reduced in the absence of Pyk2.…”

“…[29][30] In platelets, cPLA 2 phosphorylation is mainly mediated by p38 MAPK. [31][32][33][34] Figure 6A shows that thrombin-induced cPLA 2 phosphorylation on Ser505 was significantly reduced in Pyk2-deficient platelets compared with those from WT littermates. In agreement with previous findings, we confirmed that cPLA 2 phosphorylation in thrombin-stimulated platelets was mediated by p38 MAPK, because it was prevented by the specific inhibitor SB203580 (data not shown).…”

Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels

“…Hcys increases intracellular levels of Ca 2ϩ in platelets, dorsal root ganglion cells, motor neurons, and vascular smooth muscle cells (Mujumdar et al, 2000;Adalbert et al, 2002;Tjiattas et al, 2004;Leoncini et al, 2006). Hence, the effects of Hcys on cell-cell and cell-substrate interactions could be due to an effect of Hcys on intracellular Ca 2ϩ signaling.…”

(S)-Tetrahydroisoquinoline Alkaloid Inhibits LPS-Induced Arachidonic Acid Release through Downregulation of cPLA2 Expression

“…Several studies showed that MAPKs, including p38 MAPK (Leoncini et al, 2006), ERK (Su et al, 2004), and JNK (Casas et al, 2009), also induce cPLA 2 phosphorylation. However, our results indicate that phosphorylation of cPLA 2 was not observed after 2 h of stimulation with LPS (data not shown).…”