Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3^bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A₂ (PLA₂) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation in-duced by CD69 stimulation. Thromboxane A₂ (TXA₂) synthetase inhibitor and TXA₂R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA₂ was the main mediator of the response. CD69-induced arachidonic acid release and TXA₂ production were essentially PLA₂ dependent because they could be blocked by the PLA₂ inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA₂ release and TXA₂R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA₂-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA₂ in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA₂-mediated activation pathways in transmembrane receptor signaling.