Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release

Abstract

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant -SNAP and inhibited by a dominant negative -SNAP–L294A mutant or by anti–-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti–syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.