Arterial thrombosis mediates tissue infarction in coronary artery disease, cerebrovascular disease, and peripheral vascular disease, and thus is the single most common cause of morbidity and mortality in the United States. The high rate of recurrence following heart attacks and strokes despite current therapies indicates a need to identify new targets with improved efficacy for inhibiting arterial thrombosis. We and others have determined that protein disulfide isomerase (PDI) is released onto the extracellular surface of endothelial cells and platelets following vascular injury and serves a critical role in thrombus formation. PDI is the founding member of a large family of oxidoreductases that catalyze posttranslational disulfide exchange. Antibodies directed at PDI inhibit arterial thrombosis in animal models. However, there are presently no known potent and selective small molecule inhibitors of PDI to test in these systems. In an effort to identify PDI inhibitors, we have developed an insulin-based turbidometric assay for high throughput screening. We have performed a pilot screen of 5000 compounds from the Known Bioactives Collection at the Institute of Chemistry and Cell Biology. This assay demonstrated a Z'factor of 0.89 and a coefficient of variance of 4.9%. Twenty new PDI inhibitors were identified during this pilot screen. Among them was the quercetin flavonol, rutin. Rutin inhibited PDI with an IC50 of approximately 1 Aim 1 of this project is to transfer the insulin-based turbidometric assay to the MLPCN collaborator, who will conduct a screen of 300,000-500,000 compounds.
Aim 2 is to validate active compounds using a fluorescence-based assay of insulin aggregation, remove non-specific oxidase inhibitors using a glutathione peroxidase assay, and determine the specificity of inhibitors using alternative oxidoreductases, including ERp5, ERp46, ERp57, and ERp72, in the insulin-based turbidometric assay.
Aim 3 is to use a platelet-based assay to identify biologically active compounds and determine reversibility of compounds that inhibit platelet activation. Active compounds will be tested in vivo in a mouse model of thrombus formation. These studies will establish PDI as a valid target for antithrombotic therapy and provide lead compounds for development of PDI-targeted antithrombotics.

Public Health Relevance

Protein disulfide isomerase is absolutely required for thrombus formation in animal models of arterial thrombosis. We will develop inhibitors to protein disulfide isomerase to study the role of thiol isomerases in the regulation of thrombus formation and to interfere with atherothrombosis, which causes heart attacks and stroke and thus is the most prevalent cause of morbidity and mortality in the United States.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Small Research Grants (R03)
Project #
5R03DA032476-02
Application #
8233397
Study Section
Special Emphasis Panel (ZRG1-BST-F (50))
Program Officer
Purohit, Vishnudutt
Project Start
2011-03-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2014-02-28
Support Year
2
Fiscal Year
2012
Total Cost
$43,500
Indirect Cost
$18,500
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Furie, Bruce; Flaumenhaft, Robert (2014) Thiol isomerases in thrombus formation. Circ Res 114:1162-73