? Circulating platelets generate intracellular signals when exposed to extracellular matrix proteins and soluble agonists, resulting in rapid activation of adhesion receptors such as integrin alphallb-beta3, platelet aggregation, secretion of granular contents and increased procoagulant activity. These changes contribute not only to normal hemostasis but also to thrombotic events such as myocardial infarction and stroke. Platelets are anucleate and therefore not amenable to direct molecular approaches, thus limiting our understanding of the specific components of these activation-dependent signaling pathways. The Parise laboratory has become proficient in the use of knockdown and overexpression approaches in megakaryocytes, which are platelet precursors, to dissect signaling pathways relevant to platelet function. Here we propose to join forces with Dr. Benjamin Cravatt of The Scripps Research Institute, who has developed a novel natural products inspired spiroepoxide probe library that has been used previously to identify enzymes of importance in transformed cells. Probes from this library bind covalently to target enzymes in cell-based screens, thus affecting specific cellular readouts. By use of new technology termed in situ proteome profiling, covalently bound probes are then labeled with a biotin and fluorescent tag by use of """"""""click chemistry"""""""", thus facilitating rapid identification of targets via mass spectrometry. However, this library has never been applied to platelets. Therefore, we propose to 1) identify molecular targets necessary for essential platelet functions by screening human platelets with the spiroepoxide probe library and performing in situ proteome reactivity profiling, and 2) validate molecular targets in megakaryocytes and platelets by use of independent counter screens involving overexpression and knock down, as well as pharmacological inhibition and enzyme activity assays, when possible, of candidate targets. Platelet-based screening and proteome reactivity profiling with this library, together with the ability to modulate protein expression levels in megakaryocytes provides a powerful combination of approaches in the platelet field for rapidly identifying and verifying new therapeutic targets for modulating platelet function. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HL086361-02
Application #
7295727
Study Section
Special Emphasis Panel (ZHL1-CSR-I (S1))
Program Officer
Mondoro, Traci
Project Start
2006-09-01
Project End
2008-07-31
Budget Start
2007-08-01
Budget End
2008-07-31
Support Year
2
Fiscal Year
2007
Total Cost
$232,020
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599