Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2.
Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.
Feng
Dian
D
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Crane
Katharine
K
Rozenvayn
Nataliya
N
Dvorak
Ann M
AM
Flaumenhaft
Robert
R
D000906:Antibodies; D000954:Antigens, Surface / Q000097:blood*; D001792:Blood Platelets / Q000737:chemistry / Q000648:ultrastructure*; D002352:Carrier Proteins / Q000097:blood*; D002463:Cell Membrane Permeability; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000097:blood*; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000097:blood*; D019007:P-Selectin / Q000097:blood / Q000235:genetics; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D022142:Secretory Vesicles / Q000502:physiology; D013347:Subcellular Fractions / Q000648:ultrastructure; D050827:Syntaxin 1; D051040:Vesicle-Associated Membrane Protein 3
D000906:Antibodies; D000954:Antigens, Surface; D002352:Carrier Proteins; D000939:Epitopes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D019007:P-Selectin; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050827:Syntaxin 1; D051040:Vesicle-Associated Membrane Protein 3