Molecular mechanisms of platelet exocytosis: requirements for alpha-granule release.
Platelets function by secreting components necessary for primary clot formation. This report describes an in vitro assay that measures alpha-granule secretion. Using permeabilized platelets, it is possible to recreate Ca(2+)-stimulated release of platelet factor 4 (PF4) that is ATP- and temperature-dependent. Though other divalent cations can replace Ca(2+) (i.e., Sr(2+), Mn(2+), Zn(2+)), there is no effect of Ba(2+). Analysis by electron microscopy indicates that the in vitro assay also mimics the cytoskeletal rearrangements and granule centralization that occurs upon platelet activation in vivo. Antibody inhibition studies show that PF4 release requires the general membrane fusion protein N-ethylmaleimide-sensitive factor (NSF) and well as the target membrane SNAP receptors (t-SNAREs), syntaxin 2, 4, and SNAP-23. As shown by electron microscopy, the anti-t-SNARE antibodies block granule to target membrane fusion. This finding is unique in that it is the first report of a role for two syntaxins in the same exocytosis event.
Lemons
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PP
Department of Biochemistry, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky, 40536, USA.
Chen
D
D
Whiteheart
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SW
D000255:Adenosine Triphosphate / Q000097:blood; D000954:Antigens, Surface / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000097:blood; D002413:Cations, Divalent / Q000494:pharmacology; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000097:blood; D010978:Platelet Factor 4 / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; D050827:Syntaxin 1; D013696:Temperature; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D010978:Platelet Factor 4; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; D002118:Calcium