Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release.
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 alpha-SNAP and inhibited by a dominant negative alpha-SNAP-L294A mutant or by anti-alpha-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.
Chen
D
D
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Bernstein
A M
AM
Lemons
P P
PP
Whiteheart
S W
SW
D000954:Antigens, Surface / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005799:Genes, Dominant; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D017830:Octoxynol; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000954:Antigens, Surface; D002352:Carrier Proteins; D003902:Detergents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010982:Platelet-Derived Growth Factor; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D012701:Serotonin; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D007770:L-Lactate Dehydrogenase; D001619:beta-N-Acetylhexosaminidases; D002118:Calcium