Evidence for a role for phospholipase C, but not phospholipase A2, in platelet activation in response to low concentrations of collagen

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 mug/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 mug/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A(2), and p38(MAPK) phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38(MAPK) phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38(MAPK) phosphorylation. Pretreatment with a cytosolic phospholipase A(2) inhibitor, AACOCF(3), blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38(MAPK) phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Ro31-8220 inhibited collagen-induced aggregation, did not affect p38(MAPK) phosphorylation hut significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C gamma2 which was inhibited by pretreatment with U73122, unaffected by AACOCF(3) and enhanced by Ro31-8220. These results suggest that cytosolic phospholipase A(2) plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C gamma2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.