Investigation of the Relocation of Cytosolic Phospholipase A and Annexin V in Activated Platelets

Cytosolic phospholipase A₂ is a Ca²⁺-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A₂. Annexin V is a Ca²⁺-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A₂. Here, we have studied the association of cytosolic phospholipase A₂ and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A₂ and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A₂ to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A₂ or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A₂ remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A₂ with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A₂ can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A₂ copurify annexin V and cytosolic phospholipase A₂ from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A₂ and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A₂ and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A₂ into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.