Assays of Platelet SNARE-actin Interactions

The actin cytoskeleton serves an important, but poorly characterized, role in controlling granule exocytosis. The dynamic nature of actin remodeling allows it to act both as a barrier to prevent indiscriminate granule release and as a facilitator of membrane fusion. In its capacity to promote exocytosis, filamentous actin binds to components of the exocytotic machinery through actin binding proteins, but also through direct interactions with SNAREs. The platelet is an excellent cellular model to evaluate SNARE-actin interactions because of the marked reorganization of its actin cytoskeleton that occurs with activation and because of its abundance of secretory granules. This chapter will describe methods to evaluate SNARE-actin interactions in platelets using isolated platelet actin cytoskeleton, granule-enriched membrane fractions in a cell-free secretory system, and purified actin and recombinant SNAREs.