Extracellular nucleotides (ATP, ADP and UTP) are known to activate type-2 purinergic (P2Y and P2X) receptors on platelets, endothelium and immune cells to induce vascular inflammation and thrombosis. Cellular ectonucleotidases hydrolyze P2-mediators to down regulate P2 receptor-signaling responses. CD39 is the dominant vascular, T regulatory and Natural Killer (NK)T cell ectonucleotidase/NTPDase and plays a crucial role in the ectonucleotidase cascade, involving CD73, to generate adenosine. This nucleoside has important antithrombotic and anti-inflammatory properties. The roles of CD39L1 (a preferential ecto-ATPase), expressed by adventitial cells, and CD39L3 (functionally akin to CD39) expressed by vascular endothelium, on thrombus formation are completely unexplored in vivo.
Specific Aim 1 : Study functional impacts of the three vascular CD39/NTPDases on thrombus formation in vivo. a) Evaluate and compare accumulation of CD39, CD39L1 and CD39L3 in generated thrombi, with platelet sequestration, associated tissue factor and fibrin deposition. b) Contrast the impact of vascular Cd39L1 and/or Cd39L3 deletion vs. global absence of Cd39 on arteriolar thrombus formation and platelet activation in mutant mice.
Specific Aim 2 : Thromboregulatory functions of immune cells in vivo a) Perform adoptive transfer of T regulatory cells expressing Cd39 to determine the role of immune regulatory cells on platelet activation and thrombus formation in vivo. b) Test mice following adoptive transfer of isolated NKT cells expressing Cd39 and Cd73 to determine impact on thrombus formation in vivo.
Specific Aim 3 : Association of CD39 with membrane proteins and lipids. Palmitoylation at the N-terminus targets CD39 to lipid rafts within cell membranes that are linked to microparticle formation. The C-terminus of CD39 binds apo-lipoprotein A2 and incorporates CD39 into High Density Lipoproteins (HDL). a) Explore structural and functional interactions of CD39 within lipid rafts. b) Examine how palmitoylation and other post-translational modifications of CD39 impact association with cholesterol and sphingomyelin. c) Determine effects of activation of acid sphingomyelinase and ceramide generation on CD39 bioactivity in endothelial cells, lymphocytes and their derived microparticles. d) Study thrombus formation in hyperlipidemic Cd39 mutant mice. This work will provide insights into the central thromboregulatory mechanisms impacting platelet activation and vascular injury in vivo.

Public Health Relevance

The generation of a blood clot causes or provokes ischemia in inflammatory cardiovascular diseases and stroke, which are the leading causes of morbidity and mortality in North America. Our laboratories and others have generated data to suggest that there are purinergic mechanisms, related to the effects of extracellular nucleotides/ nucleosides, common to vascular inflammation and thrombosis. The ultimate goal of this application will be to develop novel therapeutic agents for better management of inflammatory and thrombotic vascular disorders in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL094400-01
Application #
7563342
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Kindzelski, Andrei L
Project Start
2009-07-01
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
1
Fiscal Year
2009
Total Cost
$737,500
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
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Covarrubias, Roman; Chepurko, Elena; Reynolds, Adam et al. (2016) Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice. Arterioscler Thromb Vasc Biol 36:1809-20
Jiang, Z Gordon; Feldbrügge, L; Tapper, E B et al. (2016) Aspirin use is associated with lower indices of liver fibrosis among adults in the United States. Aliment Pharmacol Ther 43:734-43
Zeiser, R; Robson, S C; Vaikunthanathan, T et al. (2016) Unlocking the Potential of Purinergic Signaling in Transplantation. Am J Transplant 16:2781-2794

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