In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.

Neuronal communication is mediated by Ca2+-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca2+ sensor that regulates exocytosis, whereas soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca2+ trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca2+, drive rapid, highly efficient lipid mixing. Ca2+-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca2+; we demonstrate that Ca2+–synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca2+ converts synaptotagmin from a clamp to a trigger for exocytosis.

Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimidesensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13d Jinx mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from ␣-granules and lysosomes was severely compromised. Unc13d Jinx platelets showed attenuated aggregation and, consequently, Unc13d Jinx mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13d Jinx platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13d Jinx platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis. (Blood. 2010;116(6):869-877) IntroductionPlatelets play a key role in hemostasis through their ability to respond to vascular injury. Damage to endothelial cells causes the exposure of agonists, for example, collagen and von Willebrand factor (VWF), which initiate platelet adhesion and activation. Platelet activation is marked by a rapid rise in intracellular [Ca 2ϩ ] i that triggers secretion from 3 types of internal granule stores: dense granules, ␣-granules, and lysosomes. 1,2 Each granule type carries specific molecules that promote hemostatic plug formation and the sequelae that are required to maintain a pressurized vasculature. Dense granules contain small molecules and ions such as adenosine triphosphate (ATP), adenosine 5Ј-diphosphate (ADP), serotonin, and Ca 2ϩ which are important for thrombogenesis. 3 Although few (3ϳ8/platelet), dense granule content is released much more rapidly than content from ␣-granules or lysosomes. 4 ␣-Granules are the most abundant granules in platelets (40ϳ60/platelet) and their cargo is diverse, ranging from growth factors (eg, plateletderived growth factor [PDGF]) and chemokines (eg, platelet factor IV [PF4]) to adhesive molecules (eg, VWF and fibrinogen). 5 These factors are not only important for clot stabilization but also play a role in wound repair. Release of lysosomal cargo (eg, ␤-hexosaminidase) is thought to be involved in clot remodeling. 1,2 Much like neurons and endocrine cells, platelet exocytosis is dependent on [Ca 2ϩ ] i and mediated by soluble N-ethylmaleimidesensitive fusion protein attachment protein receptors (...