Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA2 metabolism ETYA affect both these parameters. Thus we aimed to define the role of p38MAPK/cytosolic phospholipase A2 (cPLA2) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA2 activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B2 formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover the incubation of platelets with 2-AG led to the phosphorylation of cPLA2 and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK abolished both cPLA2 phosphorylation and activation. In addition SR141716 strongly impaired cPLA2 phosphorylation, arachidonic acid release and thromboxane B2 formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA2 pathway could play a relevant role in this complicated process.