Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that 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. In contrast, VAMP-2 ؉/؊ , VAMP-3 ؊/؊ , and VAMP-2 ؉/؊ /VAMP-3 ؊/؊ platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3 ؊/؊ platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8 ؊/؊ platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8 ؊/؊ mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. Regulated exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from intracellular stores into the extracellular space (1). One common characteristic of regulated exocytosis, whether it be from neurons, cytotoxic lymphocytes, adipocytes, or mast cells, is that the cells response to this stimulus results in pre-formed intracellular granules moving toward and fusing with the plasma membrane. Secretory granule/plasma membrane fusion is the essence of regulated exocytosis, and there is an intense effort underway to identify the molecular mechanisms regulating this process in the hopes of identifying ways to modulate exocytosis.The RBL-2H3 1 mast cell line has been extensively studied as a model not only for mast cell biology but also as a paradigm for regulated exocytosis from non-neuronal cells (2). Stimulation of the high affinity IgE receptor, Fc⑀RI, on these cells by crosslinking initiates a signal transduction cascade that culminates in secretory granule fusion with the plasma membrane, thereby liberating a variety of inflammatory mediators (3, 4). Numerous proteins necessary for the tethering, docking, and fusion steps between various membrane compartments in eukaryotic cells have been described. Among those, SNAREs (soluble NSF-attachment protein receptors) are a large family of membrane-associated proteins essential for membrane-membrane fusion (5, 6). These proteins include members of the vesicle-associated synaptobrevin/VAMP family as well as members of the syntaxin and SNAP-23 families of "target" membrane SNAREs. The current model proposes that while vesicles are docked on the target membrane, SNAREs from the donor or vesicle membrane (v-SNAREs) form trans-SNARE complexes with their cognate SNARE partners on the opposing target membrane. Structurally, the exocytic SNARE complex is a trimolecular protein complex containing one member of the VAMP, syntaxin, and SNAP-23 family, each contributing to the formation of a four-helix coiled-coil bundle (7, 8) whose formation is sufficient for in vitro membrane fusion (9).Given that SNAREs play a central role in the membrane fusion process, it is likely that their function is modulated in vivo. In particular, protein kinases, which have been extensively associated with the regulation of exocytosis (10), could participate in SNARE function by phosphorylating residues essential in SNARE complex assembly or the binding of SNARE regulatory proteins (6,11,12). Members of the syntax...

It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [ 3 H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice. (Blood. 2003;

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.