VAMP-7 Interacts With The Actin Cytoskeleton To Control Platelet Spreading and Mediate α-Granule Exocytosis During Thrombus Formation (original) (raw)
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VAMP-7 links granule exocytosis to actin reorganization during platelet activation
Blood, 2015
Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein-7 (VAMP-7) is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α-Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-...
The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in α-Granule Secretion
Biochemistry, 2010
Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activationinduced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for αgranule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system and purified platelet actin could substitute for platelet cytosol to support α-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release. The role of the actin cytoskeleton in granule exocytosis is enigmatic. It has been demonstrated to act both as a physical barrier that limits granule secretion and as a positive regulator of membrane fusion and cargo release. The ability of the resting actin cytoskeleton to serve as a barrier to granule secretion has been demonstrated in neutrophils, neurons, chromaffin cells, melanotrophs, pancreatic beta cells, and acinar cells (1-6). We have previously demonstrated that platelet granules are coated with actin and that the actin cytoskeleton impedes platelet dense granule and α-granule release (7). Partial disruption of this barrier results in augmented and more rapid release of granule contents from platelets. This actin cytoskeletal barrier may help prevent unregulated release of thrombogenic substances into the circulation (7). Yet accumulating evidence indicates that actin polymerization can promote membrane fusion. Actin polymerization contributes to homotypic fusion of yeast vacuoles (8), fusion of phagosomes with endocytotic organelles (9) as well as secretion of granules from neuroendocrine cells (6,10,11), and mast cells (12). In some cells, actomyosin contraction and/ † Supported by NIH grants HL63250 and HL87203 (R.F.) and T32 HL07917 (K.
British Journal of Haematology, 2010
The critical role of the platelet is to sense vascular damage and respond by secreting components that promote primary haemostasis and clot formation. Activated platelets initiate signalling cascades that lead to cytoskeletal reorganization , centralization of secretory granules, and exocytosis of small molecules and proteins from three classes of granules: dense core and a-granules and secretory lysosomes (Rendu & Brohard-Bohn, 2001). Platelet granules are the most prominent structural features, and upon activation, they coalesce in the centre of the platelet and fuse with the open canalicular system (OCS). The OCS represents a membrane reservoir that is evaginated onto the platelet surface during interaction with surfaces (Stenberg et al, 1984; Escolar et al, 1989), fusing with the plasma membrane (Ginsberg et al, 1980). The release of the granule contents into the OCS and their diffusion into the extracellular environment exert a paracrine role to activate other platelets in the immediate area that are critical to the formation of the haemostatic thrombus (Escolar & White, 1991; White & Escolar, 1991). Dense core granules mainly contain small molecules, such as adenosine diphosphate (ADP), serotonin and calcium, which are critical for further platelet activation and vasoconstriction. a-Granules represent the storage sites for a diverse set of proteins, such as platelet factor 4, von Willebrand factor, platelet-derived growth factor and P-selectin, which play roles in clot formation and initiating wound healing. Platelets also release lysosomal enzymes, such as cathepsins and hexosaminidase, which may play a role in clot remodelling or in further platelet activation (Anitua et al, 2004). To date, more than 300 proteins and small molecules have been
To cite this article: Calaminus SDJ, Thomas S, McCarty OJT, Machesky LM, Watson SP. Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. J Thromb Haemost 2008; 6: 1944-52.
SNARE-dependent membrane fusion initiates α-granule matrix decondensation in mouse platelets
Blood Advances, 2018
Platelet α-granule cargo release is fundamental to both hemostasis and thrombosis. Granule matrix hydration is a key regulated step in this process, yet its mechanism is poorly understood. In endothelial cells, there is evidence for 2 modes of cargo release: a jack-in-the-box mechanism of hydration-dependent protein phase transitions and an actin-driven granule constriction/extrusion mechanism. The third alternative considered is a prefusion, channel-mediated granule swelling, analogous to the membrane “ballooning” seen in procoagulant platelets. Using thrombin-stimulated platelets from a set of secretion-deficient, soluble N-ethylmaleimide factor attachment protein receptor (SNARE) mutant mice and various ultrastructural approaches, we tested predictions of these mechanisms to distinguish which best explains the α-granule release process. We found that the granule decondensation/hydration required for cargo expulsion was (1) blocked in fusion-protein-deficient platelets; (2) charac...
The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps that can be reproducible in vitro. The actin dynamics that underlie these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.
Identification and functional characterisation of novel SNARE proteins in platelets
University of Bristol Theses, 2014
Platelet secretion not only drives thrombosis and haemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelets. Although several platelet SNAREs have been identified, further members of the SNARE family may help fine-tune platelet secretion. In this study I identified expression of t-SNAREs VTI1A, VTI1B (Qb SNAREs) and STX8 (Qc SNARE) in human and mouse platelets. Those ‘novel’ SNAREs were able to interact with each other and previously reported SNAREs VAMP8 (R-SNARE) and STX11 (Qa SNARE), thus suggesting existence of a secondary SNARE complex in addition to the widely accepted SNAP23-VAMP8-STX11 complex. In following mouse studies, whereas neither gene was found to be essential for α-granule or lysosome secretion, Stx8-/- platelets showed a significant defect in dense granule secretion and aggregation, that was most pronounced at intermediate concentrations of agonists. Addition of exogenous ADP could rescue the aggregation defect but failed to restore dense granule secretion, suggesting the defect lies in the ‘primary’ secretory pathway. Pretreatment with P2Y receptors antagonists reduced secretion and aggregation to the same extent in WT and Stx8-/- platelets, suggesting that the ADP-driven positive feedback mechanism was not defective in Stx8-/- platelets. In addition, STX8 was found to play a role in regulating pro-coagulant activity suggesting novel roles for SNAREs in platelets. Neither Vti1a-/- nor Vti1b-/- showed any significant defects, suggesting complementarity between those homologues in platelets. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in differentially regulating granule release from platelets. Taken together, data presented in this Thesis not only provides first evidence of an additional SNARE complex present in platelets but also suggests novel roles for SNAREs in regulation of thrombosis and haemostasis
Endobrevin/VAMP-8 Is the Primary v-SNARE for the Platelet Release Reaction
Molecular Biology of the Cell, 2006
(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.