Granule exocytosis is an essential platelet function, contributing not only to hemostasis and thrombosis, but also to inflammation, wound healing, malignancy, and angiogenesis. Previous efforts to understand the molecular mechanisms of granule exocytosis have identified the SNARE machinery and chaperone proteins required for membrane fusion. Studies described in this proposal will evaluate the premise that this machinery serves a role in sorting specific cargos to distinct subpopulations of ?-granules and enabling differential release of granule contents upon platelet activation. We will study granule segregation and secretion in mice lacking VAMP-3, VAMP-5, VAMP-7, and VAMP-8. Cytoskeleton dynamics that occur during platelet spreading can influence SNARE interactions and modify granule exocytosis. We will evaluate granule exocytosis during platelet spreading. These studies will also assess the role of exocytosis in providing membrane for growing actin structures at the periphery of adherent platelets and for directed spreading and movement on micropatterned matrices of adhesion proteins. Use of high-speed intravital microscopy will be used to monitor release of granules from individual platelets during thrombus formation in vivo. These approaches will allow us to evaluate the hypothesis that the mechanism of granule secretion is altered in adherent platelets compared to suspension platelets. We will also evaluate the role of dynamins in controlling fusion pore dynamics, demonstrating a level of control of platelet exocytosis that occurs following SNARE-mediated membrane fusion. These studies will use single-cell amperometry to monitor the release of individual granules from single platelets. The role of dynamins in controlling platelet exocytosis during thrombus formation in vivo will evaluate the hypothesis that the platelet fusion pore can be regulated to control thrombus formation. These experiments will address poorly understood aspects of platelet granule exocytosis and provide a foundation for targeting select platelet membrane fusion events during thrombosis.

Public Health Relevance

Arterial clot formation causes myocardial infarction, strokes, and vascular disease and is the single most common cause of morbidity and mortality in the U.S. Platelets serve a central role in arterial thrombosis. During incorporation into thrombi, platelets secrete factors that propagate thrombus formation. We will identify platelet proteins responsible for platelet granule secretion and, thereby, identify new targets for inhibiting arteril occlusive disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL112809-01A1
Application #
8436082
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2013-02-01
Project End
2017-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
1
Fiscal Year
2013
Total Cost
$435,000
Indirect Cost
$185,000
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Schulman, Sol; Bendapudi, Pavan; Sharda, Anish et al. (2016) Extracellular Thiol Isomerases and Their Role in Thrombus Formation. Antioxid Redox Signal 24:1-15
Flaumenhaft, Robert; Furie, Bruce (2016) Vascular thiol isomerases. Blood 128:893-901
Bekendam, Roelof H; Bendapudi, Pavan K; Lin, Lin et al. (2016) A substrate-driven allosteric switch that enhances PDI catalytic activity. Nat Commun 7:12579
Flaumenhaft, Robert (2015) A new story ARC for α-granule formation. Blood 126:123-4
Flaumenhaft, Robert; Furie, Bruce; Zwicker, Jeffrey I (2015) Therapeutic implications of protein disulfide isomerase inhibition in thrombotic disease. Arterioscler Thromb Vasc Biol 35:16-23
Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L et al. (2015) VAMP-7 links granule exocytosis to actin reorganization during platelet activation. Blood 126:651-60
Sakurai, Yumiko; Fitch-Tewfik, Jennifer L; Qiu, Yongzhi et al. (2015) Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition. Blood 126:531-8
Sharda, Anish; Kim, Sarah H; Jasuja, Reema et al. (2015) Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome. Blood 125:1633-42
Aisiku, Omozuanvbo; Peters, Christian G; De Ceunynck, Karen et al. (2015) Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar. Blood 125:1976-85
Flaumenhaft, Robert (2014) Bioengineering in platelet biology. Thromb Res 133:523-4

Showing the most recent 10 out of 15 publications