| Platelet secretion is critical to hemostasis. Release of granule cargo is mediated by soluble NSF attachment protein receptors (SNAREs), and despite consensus on t-SNAREs usage, it was unclear which Vesicle Associated Membrane Protein (VAMP: syntaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, or endobrevin/VAMP-8) is required. Here, data is presented to demonstrate that endobrevin/VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. Consistent with this result, anti-VAMP-8 antibody but not anti-VAMP-7 antibody or tetanus toxin impairs secretion from permeabilized human platelets. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets show no secretion defect. Tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. These data suggest that VAMP-8 but not VAMP-2, -3, or -7 is required for platelet secretion.;Tetanus toxin does block the residual release from permeabilized VAMP-8 -/- platelets, suggesting a secondary role for VAMP-2 and/or -3. Consistent with this result, combined treatment of anti-VAMP-8 antibody and tetanus toxin yielded a more severe inhibition than inclusion of VAMP-8 antibody alone or with VAMP-7 antibody in the permeabilized human platelet secretion assay. These data imply a ranked redundancy of v-SNARE usage in human platelets. Endobrevin/VAMP-8 is the primary v-SNARE for platelet secretion while VAMP-2 and/or -3 but not VAMP-7 can play a secondary role in a less efficient manner.;Semi-quantative western blotting analysis suggests that VAMP-8 is the most abundant v-SNARE of the four tested VAMPs in human and mouse platelets. Using immunofluorescence microscopy technique, subcellular fractionation and organelle immunoprecipitation methods, endobrevin/VAMP-8 is shown to localize onto the platelet dense core granules, alpha granules, and lysosomes. VAMP-8 forms endogenous complexes with functionally relevant platelet t-SNAREs in vivo. These data support the conclusion that endobrevin/VAMP-8 is the major v-SNARE protein for platelet exocytosis.;Apart from studying the core secretory machinery, I also investigated the role of the regulatory molecule, Munc13-4, in platelet exocytosis. Recombinant full-length Munc13-4 enhances secretion from dense core granules, alpha granules, and lysosomes. The C-terminal C2 domain of Munc13-4 is important for its proper function as deletion of this C2 domain produced a mutant protein that potently inhibits secretion from all three granules. In addition, I also identified two Munc13-4 containing complexes in vivo: Doc2alpha/Munc13-4 and Rab27a/Munc13-4. These Munc13-4-interacting proteins provide future candidates to probe the functional roles of Munc13-4 in membrane fusion. In summary, these results suggest that Munc13-4 plays an important regulatory role in platelet secretion.;KEYWORDS: platelet, SNARE, VAMP, endobrevin, VAMP-8, exocytosis... |
