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.

Summary Background Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. Objectives To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. Subjects/methods Two hundred and eighty-eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n = 18) and hyporeactivity (n =11) was used to screen the human transcriptome. Results Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v-SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q = 0.0275). Validation studies of microarray results showed 4.8-fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P = 0.0023). VAMP8 protein levels varied 13-fold among platelets from these normal subjects, and were 2.5-fold higher in hyperreactive platelets (P = 0.05).Among our cohort of 288 subjects, a VAMP8 single-nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age-dependent manner (P < 0.003). MicroRNA-96 was predicted to bind to the 3′-untranslated region of VAMP8 mRNA and was detected in platelets. Overexpression of microRNA-96 in VAMP8-expressing cell lines caused a dose-dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. Conclusions These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA-96 in the regulation of VAMP8 expression.

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 (...

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.

Individuals whose platelets lack dense or ␣-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/ VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and ␣-granule contents; thus, VAMP-8 ؊/؊ mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8 ؊/؊ and rubyeye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.