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Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation

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Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Apollo THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 3, pp. 1536–1545, January 16, 2015 Author’s Choice © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability*□S Received for publication, August 4, 2014, and in revised form, November 7, 2014 Published, JBC Papers in Press, November 17, 2014, DOI 10.1074/jbc.M114.602615 Ewelina M. Golebiewska‡, Matthew T. Harper‡, Christopher M. Williams‡, Joshua S. Savage§, Robert Goggs¶, Gabriele Fischer von Mollardʈ, and Alastair W. Poole‡1 From the ‡School of Physiology and Pharmacology, Medical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom, the §School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, the ¶Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, and the ʈFakultät für Chemie, Biochemie III, Universität Bielefeld, Postfach 100131, 33501 Bielefeld, Germany Background: The molecular machinery controlling exocytosis of the three secretable granules types in platelets is not fully elucidated. Results: ATP secretion, aggregation, and thrombus stability are defective in Stx8Ϫ/Ϫ mouse platelets. Conclusion: STX8 is involved in platelet dense granule secretion, and the STX8-mediated pathway contributes to thrombus stabilization. Significance: Identification of the novel functional SNARE STX8 suggests alternative mechanisms of granule secretion exist in platelets. Platelet secretion not only drives thrombosis and hemostasis, regulatory proteins including small GTPases (5–7), MUNC pro- but also mediates a variety of other physiological and patholog- teins (8, 9), and calcium sensors (10, 11) contribute to regulation of ical processes. The ubiquitous SNARE machinery and a number platelet secretion alongside the “core” SNARE complex. of accessory proteins have been implicated in regulating secre- The functional core SNARE complex invariably consists of 4 tion in platelet. Although several platelet SNAREs have been SNARE domains: three t-SNARE domains (residing on the tar- identified, further members of the SNARE family may be needed get membrane) and a v-SNARE on the vesicle form a tight het- to fine-tune platelet secretion. In this study we identified erotetrameric complex that drives membrane fusion (12). The expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in majority of mammalian SNAREs, including all syntaxins and mouse and human platelets. In mouse studies, whereas STX8 SNAP23 homologues, are classified as t-SNAREs, whereas ؊ ؊ was not essential for ␣-granule or lysosome secretion, Stx8 / VAMPs function as v-SNAREs. Although SNAREs are func- platelets showed a significant defect in dense granule secretion tionally classified as v-SNAREs or t-SNAREs, they can also be in response to thrombin and CRP. This was most pronounced at structurally distinguished as Qabc or R types according to the intermediate concentrations of agonists. They also showed an amino acid residue (glutamine and arginine) located in the cen- aggregation defect that could be rescued with exogenous ADP ter of the functional complex (13). Both the functional organi- ؊ ؊ and increased embolization in Stx8 / mice in vivo consistent zation and the structure of the central SNARE complex are with an important autocrine and paracrine role for ADP in remarkably conserved between cells, and even species (14). aggregation and thrombus stabilization. STX8 therefore specif- The currently accepted platelet secretion machinery is the ically contributes to dense granule secretion and represents complex of STX112 (a Qa SNARE), SNAP23 (Qbc), and another member of a growing family of genes that play distinct VAMP8 (an R SNARE); however, in light of the level of tempo- roles in regulating granule release from platelets and thus plate- ral and spatial regulation required in platelet secretion, the “one let function in thrombosis and hemostasis. size fits all” model does not seem to be plausible. In addition, even loss of STX11 (in FHL4 patients) (15) or knock-out of VAMP8 coupled with tetanus neurotoxin (TnT-LC) treatment Platelet secretory granules, ␣-granules, dense granules, and (16) does not lead to full ablation of secretion, suggesting lysosomes contain a plethora of active substances that regulate ranked redundancy and compensation mechanisms (16). The thrombosis and hemostasis, and increasingly found to contrib- evidence supporting SNAP23 is even more indirect and reliant ute to other physiological and pathological processes, such as upon treatment of SLO-permeabilized platelets with functional inflammation (1), angiogenesis (2), and malignancy (3). blocking antibodies and the correlative evidence of phosphor- Our understanding of platelet secretion has developed tremen- ylation of SNAP23 occurring at a similar rate to secretion dously since the first description of the ubiquitous SNARE secre- (17–20). tory machinery in platelets by Lemons et al. (4). We now know that There are numerous examples of SNARE redundancy and specialization in secretory cells, for example, neutrophils (21). However, in those cells approaches such as protein overexpres- * This work was supported by grants from the British Heart Foundation to A. W. P. (RG/10/006/28299, PG/12/25/29488, FS/09/009/26444). Author’s Choice—Final version full access. □S This article contains supplemental Videos S1 and S2. 2 The abbreviations used are: STX, syntaxin; ANOVA, analysis of variance; 1 To whom correspondence should be addressed. Tel.: 44-117-331-1435; Fax: 5-HT, 5-hydroxytryptamine; CRP, collagen-related peptide; GP, glycopro- 44-117-331-2288; E-mail: [email protected]. tein; PE, phycoerythrin. 1536 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 3•JANUARY 16, 2015 Syntaxin 8 in Platelet Secretion and Function sion or targeted introduction of specific antibodies by elec- minimize signaling contributions from receptors other than the tropermeabilization (21) allow for easier characterization of primary receptor being activated. SNARE function in secretion. Characterization of similar pro- Protein Electrophoresis and Western Blotting—Washed platelet cesses in platelets remains more challenging. lysates were separated on SDS-PAGE, and PVDF membranes We hypothesized that other SNARE proteins may help “fine- were probed with specific primary antibodies: VAMP8, tune” secretion alongside the VAMP8-STX11-SNAP23 com- SNAP23, STX11, VTI1B (all rabbit IgG, Synaptic Systems plex. There is substantial expression evidence that additional GmBH, Göttingen, Germany), STX8 (sheep IgG, R&D Systems, SNAREs are present in platelets, but currently there are several Abingdon, UK), GAPDH (mouse IgG, Santa Cruz Biotechnol- of these that have not been investigated (22–25). We decided to ogy, Heidelberg, Germany), ␣-tubulin (mouse IgG), and FcR␥ prioritize characterization of a syntaxin STX8, an abundant Qc (rabbit IgG, both Abcam, Cambridge, UK). Membranes were SNARE, which could partially substitute for SNAP23 (which in washed in excess TBS-T and incubated with appropriate HRP- the current model contributes both Qb and Qc domains) in conjugated secondary antibodies (mouse and rabbit, GE platelet SNARE complexes (26). Healthcare Life Sciences; sheep, Jackson ImmunoResearch, Here we report that syntaxin 8 (STX8) (Qc) is expressed in Stratech, Newmarket, UK). Protein levels were quantified using mouse and human platelets, and localizes to the membrane ImageJ analysis software and compared using ANOVA. fraction of platelets. We have also found that it forms a complex Digitonin Fractionation of Human Platelets—Human plate- with STX11 in human platelets, which unlike the activation-de- lets were incubated with an equal volume of 0.1% digitonin in pendent STX11-SNAP23-VAMP8 complex, is present in rest- phosphate-buffered saline (PBS) supplemented with protease ing platelets. We also show a novel role for STX8 in dense gran- inhibitor mixtures for 120 s on ice with intermittent agitation. The ule secretion in mouse platelets in response to intermediate samples were centrifuged at 1,800 ϫgfor 10 min at 4 °C to separate agonist stimulation in vitro, and impairment of aggregation sec- the soluble (cytosolic) fraction from the digitonin-insoluble mem- ondary to this defect. Most importantly, however, we show that brane-bound fraction. The supernatants were extracted with 4ϫ the embolization rate from thrombi in vivo was significantly SDS sample buffer. The remaining insoluble pellet was washed increased in the absence of STX8, whereas the total size of the twice with ice-cold PBS and solubilized with 0.1% Triton X-100 in thrombus and rate of adhesion as measured by traditional PBS for 5 min and centrifuged as before. The supernatant was methods remained unchanged. This suggested that the rela- extracted as before and membrane and cytosol fractions were sep- tively subtle deficit in dense granule secretion can result in a arated using SDS-PAGE, and localization was confirmed with substantial physiological defect downstream. Western blotting relative to FcR␥ (membrane fraction marker) This study is the first to suggest a role for STX8 in platelet and GAPDH (cytosolic fraction marker). secretion and function, which opens up the possibility of other Co-immunoprecipitation—Double-washed human platelets, SNAREs playing secondary roles to the “main”
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