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 microg/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 microg/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK 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 p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase Cgamma2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Ro31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase Cgamma2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.