Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+‐ionophore activation. The time‐dependence of annexin V incorporation into the detergent‐extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re‐associated with the actin filaments when re‐polymerization was inducedin vitro. Using a bifunctional crosslinking reagent we have identified an 85‐kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin‐stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F‐actin to physiological levels. We also show that GST–annexin V mimics the physiological binding of annexin V to membranes, and that this GST–annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and γ‐ but not β‐actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely γ‐actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to γ‐actin in an activation‐dependent manner forms a strong association with the platelet cytoskeleton.