Platelet-rich, arterial thrombi mediate tissue infarction in myocardial infarction, stroke, and peripheral vascular disease and, thus, represent the most common cause of morbity and mortality in the United States. During thrombus formation, platelets secrete their granule contents. The most abundant platelet granule, the alpha-granule, contains adhesion molecules, coagulation factors, and vasoactive factors thought to contribute to thrombus formation and platelet recruitment. Such observations suggest that platelet alpha-granule secretion may be a useful target in interfering with propagation of mural platelet thrombosis. However, little is known about the molecular mechanisms that direct alpha-granule secretion. In contrast, molecular studies of vesicle secretion from nucleated cells has lead to the discovery of a superfamily of proteins, termed SNARE proteins, that mediate membrane fusion between vesicle and plasma membrane in nucleated cells. We have developed a novel permeabilized platelet model of alpha-granule secretion to demonstrate that SNARE proteins mediate alpha-granule secretion. Using this model, we will define the role of SNARE proteins in alpha-granule secretion. Experiments described in the Specific Aim 1 of this proposal will determine the subcellular localization of SNARE proteins within the platelet using subcellular fractionation followed by immunoprecipitation to purify alpha-granules and surface-connected membranes. These membranes will then be analyzed for SNARE protein content by flow cytometry and immunoblotting. The subcellular localization will also be determined by immunofluorescence light microscopy and immunogold electron microscopy. Studies described in the Specific Aim 2 are based on the preliminary findings that ATP-gamma-S, an inhibitor of NEM-sensitive fusion protein, stimulates alpha-granule secretion in permeabilized platelets. The role of NEM-sensitive fusion protein in platelet secretion will be determined using other specific inhibitors of NSF in the permeabilized model of alpha-granule secretion. This permeabilized platelet model will also be used in experiments described in Specific Aim 3 to determine whether Rab GTPases and phosphatidylinositol (4,5)-bisphosphate mediate alpha-granule secretion. Experiments described in Specific Aim 4 will use protein fractionation to purify proteins that reconstitute alpha- granule secretion in permeabilized platelets. These studies are of fundamental importance as they will define the molecular basis of alpha-granule secretion, a phenomenon that has previously been described only morphologically.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL063250-01A1
Application #
6127440
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
2000-05-01
Project End
2004-03-31
Budget Start
2000-05-01
Budget End
2001-03-31
Support Year
1
Fiscal Year
2000
Total Cost
$261,000
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
Flaumenhaft, Robert (2015) A new story ARC for ?-granule formation. Blood 126:123-4
Dowal, Louisa; Sim, Derek S; Dilks, James R et al. (2011) Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1. Proc Natl Acad Sci U S A 108:2951-6
Woronowicz, Kamil; Dilks, James R; Rozenvayn, Nataliya et al. (2010) The platelet actin cytoskeleton associates with SNAREs and participates in alpha-granule secretion. Biochemistry 49:4533-42
Flaumenhaft, Robert; Dilks, James R; Richardson, Jennifer et al. (2009) Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood 113:1112-21
Tanaka, Eiichi; Chen, Frederick Y; Flaumenhaft, Robert et al. (2009) Real-time assessment of cardiac perfusion, coronary angiography, and acute intravascular thrombi using dual-channel near-infrared fluorescence imaging. J Thorac Cardiovasc Surg 138:133-40
Graham, Gwenda J; Ren, Qiansheng; Dilks, James R et al. (2009) Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo. Blood 114:1083-90
Blair, Price; Flaumenhaft, Robert (2009) Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 23:177-89
Flaumenhaft, R; Dilks, J R (2008) Discovery-based strategies for studying platelet function. Mini Rev Med Chem 8:350-7
Flaumenhaft, Robert; Rozenvayn, Nataliya; Feng, Dian et al. (2007) SNAP-23 and syntaxin-2 localize to the extracellular surface of the platelet plasma membrane. Blood 110:1492-501
Sim, Derek S; Dilks, James R; Flaumenhaft, Robert (2007) Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation. Arterioscler Thromb Vasc Biol 27:1478-85

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