Platelet exocytosis is integral to hemostasis and vascular integrity. Conversely, inhibiting exocytosis is an important target for clinically managing improper thrombosis. Thus, understanding the molecular events required for platelet exocytosis will broaden our ability to control platelet function. From previously funded work, it is clear that specific Soluble NSF Attachment Protein Receptors (v- and t-SNAREs) are required for platelet secretion. With this established, our goals have now expanded to address how this exocytosis machinery is regulated. We hypothesize that platelet exocytosis is governed by proteins that control SNARE- SNARE interactions.
Three specific aims are proposed:
Aim 1) To determine which v-SNARE proteins are required for platelet granule release. Our work demonstrates that VAMP-8/endobrevin is required for platelet exocytosis. The goals of Aim 1 are to complete the characterization of VAMP-8's role, to identify which VAMP (2 or 3) serves as the secondary v-SNARE and to determine when this mechanism is used.
Aim 2) To define the pathway of interactions required for t-SNARE function in platelet exocytosis. Previous work assigned roles for SNAP-23, syntaxin 2 and 4.
This aim focuses on proteins that regulate these t-SNAREs to promote granule release. The syntaxin chaperones of the Munc18 family and their effectors, Munc13S and Doc2s, will be examined using a combination of immunological and biochemical techniques to determine how these proteins interact during exocytosis. These data will expand our understanding of how the syntaxin t-SNAREs are regulated to pair with appropriate SNAREs and to mediate membrane fusion.
Aim 3) To determine how the small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), participates in platelet activation. Members of the Arf family are associated with membrane trafficking, specifically exocytosis. Our work shows that Arf6 is critical to platelet activation, secretion, and actin dynamics.
This aim will directly address how changes in Arf6-nucleotide state affect actin cytoskeleton and secretion, and will identify the Arf6 effectors required for those functions. These data will yield insight into the central role of this small GTP- binding protein in activated platelets. Overall the information gained from this proposal will help to identify new therapeutic targets that will be useful for modulating hyperactive platelets, and to provide diagnostic reagents for determining how hypoactive platelets are defective.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056652-13
Application #
7848332
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
1996-08-20
Project End
2012-03-31
Budget Start
2010-06-01
Budget End
2012-03-31
Support Year
13
Fiscal Year
2010
Total Cost
$293,000
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Whiteheart, Sidney W (2017) Fueling Platelets: Where Does the Glucose Come From? Arterioscler Thromb Vasc Biol 37:1592-1594
Joshi, Smita; Whiteheart, Sidney W (2017) The nuts and bolts of the platelet release reaction. Platelets 28:129-137
Banerjee, Meenakshi; Whiteheart, Sidney W (2017) The ins and outs of endocytic trafficking in platelet functions. Curr Opin Hematol 24:467-474
Huang, Yunjie; Joshi, Smita; Xiang, Binggang et al. (2016) Arf6 controls platelet spreading and clot retraction via integrin ?IIb?3 trafficking. Blood 127:1459-67
Jones, Mark B; Oswald, Douglas M; Joshi, Smita et al. (2016) B-cell-independent sialylation of IgG. Proc Natl Acad Sci U S A 113:7207-12
Chicka, Michael C; Ren, Qiansheng; Richards, David et al. (2016) Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion. Biochem J 473:627-39
Banerjee, Meenakshi; Whiteheart, Sidney W (2016) How Does Protein Disulfide Isomerase Get Into a Thrombus? Arterioscler Thromb Vasc Biol 36:1056-7
Xiang, Binggang; Zhang, Guoying; Ye, Shaojing et al. (2015) Characterization of a Novel Integrin Binding Protein, VPS33B, Which Is Important for Platelet Activation and In Vivo Thrombosis and Hemostasis. Circulation 132:2334-44
Ouseph, Madhu M; Huang, Yunjie; Banerjee, Meenakshi et al. (2015) Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood 126:1224-33
Nair-Gupta, Priyanka; Baccarini, Alessia; Tung, Navpreet et al. (2014) TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell 158:506-21

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