To understand the molecular basis of granule release from platelets, we examined the role of vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in alpha-granule secretion. A vesicle-associated membrane protein, SNAP-23, and syntaxin 4 were detected in platelet lysate. These proteins form a SDS-resistant complex that disassembles upon platelet activation. To determine whether these proteins are involved in alpha-granule secretion, we developed a streptolysin O-permeabilized platelet model of alpha-granule secretion. Streptolysin O-permeabilized platelets released alpha-granules, as measured by surface expression of P-selectin, in response to Ca2+ up to 120 min after permeabilization. Incubation of streptolysin O-permeabilized platelets with an antibody directed against vesicle-associated membrane protein completely inhibited Ca2+-induced alpha-granule release. Tetanus toxin cleaved platelet vesicle-associated membrane protein and inhibited Ca2+-induced alpha-granule secretion from streptolysin O-permeabilized platelets. An antibody to syntaxin 4 also inhibited Ca2+-induced alpha-granule release by approximately 75% in this system. These results show that vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form a heterotrimeric complex in platelets that disassembles with activation and demonstrate that alpha-granule release is dependent on vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions.