Inhibition of protein disulfide isomerase (PDI) using antibodies prevents both platelet accumulation and fibrin formation in murine models of thrombus formation. This observation indicates that inhibition of PDI could represent a viable strategy for control of pathological thrombus formation. However, potent, selective small molecule inhibitors to test this hypothesis are not presently available. We have begun high throughput screening to identify compounds that inhibit PDI. A preliminary screen of ~5000 compounds identified PDI inhibitors with a hit rate of 0.3%. Among the active compounds were several flavonoids, including the widely used nutritional supplement quercetin-3-rutinoside. Quercetin-3-rutinoside was markedly antithrombotic in murine models. The fact that this PDI inhibitor is well-tolerated and potently antithrombotic in vivo supports the feasibility of inhibition of PDI for antithrombotic therapy. However, more selective, potent compounds with improved bioavailability are required. We will perform a large scale high throughput screen to identify novel PDI inhibitors. The objective of this project is to characterize a set of potent and selective PDI inhibitors as probes to study the role of PDI in thrombus formation and identify lead compounds that could be developed as antithrombotics. PDI demonstrates multiple functions in the vasculature including oxidoreductase/isomerase, chaperone, and denitrosation activities.
In Aim 1, we will characterize PDI inhibitors on the basis of their ability to block these different activities. Studies performed in Aim 2 will use NMR spectroscopy to determine the structural basis of PDI inhibitor activity. The effect of PDI inhibitors on platelet activation and endothelial cell function will be detennined in Aim 3. Select compounds will then be tested for their inhibitory activity in a mouse model of thrombus formation using intravital microscopy (Aim 4). Evaluation of PDI inhibitors in enzymatic and cell-based assays will enable the identification of characteristics that are essential for the antithrombotic activity of PDI inhibitors. Such information will be critical for further development of PDI inhibitors as a novel class of antithrombotics.

Public Health Relevance

Arterial thrombosis resulting in heart attack and stroke as well as venous thromboembolism resulting in deep vein thrombosis and pulmonary embolism remain most the common causes of mortality in the United States.There is a need forthe development of therapies targeting alternative components of the blood clotting mechanism, based on new knowledge about the mechanisms of thrombus formation. Studies described in this application will determine whether PDI is a tractable target for antithrombotic therapy

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
1U54HL112302-01
Application #
8401640
Study Section
Special Emphasis Panel (ZHL1-CSR-C (F1))
Project Start
Project End
Budget Start
2012-09-01
Budget End
2013-04-30
Support Year
1
Fiscal Year
2012
Total Cost
$445,854
Indirect Cost
$189,616
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Galinski, Christine N; Zwicker, Jeffrey I; Kennedy, Daniel R (2016) Revisiting the mechanistic basis of the French Paradox: Red wine inhibits the activity of protein disulfide isomerase in vitro. Thromb Res 137:169-73
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
Lin, Lin; Gopal, Srila; Sharda, Anish et al. (2015) Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain. J Biol Chem 290:23543-52
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
Schulman, Sol; Furie, Bruce (2015) How I treat poisoning with vitamin K antagonists. Blood 125:438-42
Passam, Freda H; Lin, Lin; Gopal, Srila et al. (2015) Both platelet- and endothelial cell-derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis. Blood 125:2276-85
Landeta, Cristina; Blazyk, Jessica L; Hatahet, Feras et al. (2015) Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria. Nat Chem Biol 11:292-8
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

Showing the most recent 10 out of 14 publications