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 (...

The platelet release reaction plays a critical role in thrombosis and contributes to the events that follow hemostasis. Previous studies have shown that platelet secretion is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins from granule and plasma membranes. The SNAREs form transmembrane complexes that mediate membrane fusion and granule cargo release. Although VAMP-8 (v-SNARE) and SNAP-23 (a t-SNARE class) are important for platelet secretion, the identity of the functional syntaxin (another t-SNARE class) has been controversial. Previous studies using anti-syntaxin Abs in permeabilized platelets have suggested roles for both syntaxin-2 and syntaxin-4. In the present study, we tested these conclusions using platelets from syntaxin-knockout mouse strains and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient. Platelets from syntaxin-2 and syntaxin-4 single-or double-knockout mice had no secretion defect. Platelets from a FHL4 patient deficient in syntaxin-11 had a robust defect in agonistinduced secretion although their morphology, activation, and cargo levels appeared normal. Semiquantitative Western blotting showed that syntaxin-11 is the more abundant syntaxin in both human and murine platelets. Coimmunoprecipitation experiments showed that syntaxin-11 can form SNARE complexes with both VAMP-8 and SNAP-23. The results of the present study indicate that syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet exocytosis. (Blood. 2012;120(12):2484-2492) IntroductionPlatelets are important for hemostasis because they respond to vascular damage by secreting components that promote thrombosis and its sequelae. Hypoactive secretion leads to bleeding, 1-4 whereas hyperactive platelet secretion may be linked to increased thrombosis. Agonists exposed at a damaged site initiate platelet adhesion and activation. Platelet activation is marked by a rapid increase in the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), which triggers release from 3 granular stores: dense granules, ␣-granules, and lysosomes. 5,6 Dense granules contain small molecules and ions such as ADP, serotonin, and Ca 2ϩ , which are important for thrombogenesis. 7 ␣-ganules are the most abundant granules in platelets (40-60/platelet) and their cargos are diverse, ranging from growth factors (eg, PDGF) and chemokines (eg, platelet factor 4 [PF4]) to adhesive molecules (eg, VWF and fibrinogen). 8 These factors are not only important for clot stabilization, but also play roles in wound repair. Release of lysosomal cargo (eg, ␤-hexosaminidase) is thought to be involved in clot remodeling. 5,6 Given its central role, the platelet secretory machinery could be a valuable target for antithrombotic therapy.As with other regulated secretory systems, platelet exocytosis is mediated by Soluble NSF Attachment Protein Receptors (SNAREs) present on the granule/vesicle membranes (v-SNAREs) and on plasma/target membranes (t-SNAREs). Cognate v-SNAREs and t-SNAREs interact to form a trans-bilayer complex that juxt...

Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilumoccupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or -APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors.

Obligate vacuolar pathogens produce proteins that localize to the host cell-derived membranes of the vacuoles in which they reside, yielding unique organelles that are optimally suited for pathogen survival. Anaplasma phagocytophilum is an obligate vacuolar bacterium that infects neutrophils and causes the emerging and potentially fatal disease human granulocytic anaplasmosis. Here we identified APH_1387 as the first A. phagocytophilum-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_1387, also referred to as P100, is a 61.4-kDa acidic protein that migrates with an apparent molecular weight of 115 kDa on SDS-PAGE gels. It carries 3 tandem direct repeats that comprise 58% of the protein. Each APH_1387 repeat carries a bilobed hydrophobic alpha-helix domain, which is a structural characteristic that is consistent with the structure of chlamydia-derived proteins that traverse inclusion membranes. APH_1387 is not detectable on the surfaces of A. phagocytophilum dense core organisms bound at the HL-60 cell surface, but abundant APH_1387 is detected on the surfaces of intravacuolar reticulate cell and dense core organisms. APH_1387 accumulates on the AVM throughout infection. It associates with the AVM in human HL-60, THP-1, and HMEC-1 cells and tick ISE6 cells. APH_1387 is expressed and localizes to the AVM in neutrophils recovered from A. phagocytophilum-infected mice. This paper presents the first direct evidence that A. phagocytophilum actively modifies its host cell-derived vacuole.

Purpose of the Review-In response to agonists produced at vascular lesions, platelets release a host of components from their three granules: dense core, alpha, and lysosome. This releasate activates other platelets, promotes wound repair, and initiates inflammatory responses. While widely accepted, the specific mechanisms underlying platelet secretion are only now coming to light. This review focuses on the core machinery required for platelet secretion.Recent Findings-Proteomic analyses have provided a catalogue of the components released from activated platelets. Experiments using a combination of in vitro secretion assays and knockout mice have lead to assignments of both v-and t-SNAREs (Soluble NSF Attachment Protein Receptors) to each of the three secretion events. SNARE knockout mice are also proving to be useful models for probing the role of platelet exocytosis in vivo. Other studies are beginning to identify SNARE regulators which control when and where SNAREs interact during platelet activation.Summary-A complex set of protein-protein interactions control the membrane fusion events required for the platelet release reaction. SNARE proteins are the core elements but the proteins that control SNARE interactions represent key points at which platelet signaling cascades could affect secretion and thrombosis.