Thiol isomerases serve a critical role in thrombus formation, as demonstrated in several independent models of thrombus formation using genetic deletion of thiol isomerases, inhibitory antibodies, and small molecule antagonists. A therapy targeting protein disulfide isomerase (PDI) is currently in phase II/III studies to evaluate its efficacy and safety as an antithrombotic. Yet despite compelling evidence that thiol isomerases function in thrombus formation, little is known about how these enzymes contribute to thrombosis. Vascular thiol isomerases - PDI, ERp5, and ERp57 - control the formation and cleavage of allosteric disulfide bonds and modify protein function. The nature of the modifications and how they impact protein function are largely unstudied. Using molecular, cellular and whole animal studies of thrombus formation, we will address critical questions related to the role of thiol isomerases in thrombus formation.
In Aim 1, we will determine how thiol isomerases escape retention in the endoplasmic reticulum, how thiol isomerases are organized and packaged into granules in platelets and endothelial cells, and the mechanisms that regulate the exocytosis of thiol isomerases in these cells.
In Aim 2, we will identify the pathways and mechanisms that link vascular thiol isomerases to platelet thrombus formation and fibrin generation. Protein components of this initiation pathway will be identified using mechanism-based kinetic trapping to identify substrates from platelets, plasma, and endothelial cells.
In Aim 3, we will evaluate the regulation of thiol isomerases by nitric oxide and test the ability of thiol isomerases to control nitric oxide during thrombus formation. We will study how platelet receptors are activated by thiol isomerase-mediated denitrosylation. We will also image nitric oxide and reactive oxygen species in live mice to assess the control of nitric oxide and oxidative stress by thiol isomerases. This project will provide new fundamental knowledge of how thiol isomerases are released following vascular injury, how they modify vascular protein substrates, and how they are regulated. Given the paucity of knowledge of extracellular thiol isomerase- mediated pathways, these studies will provide essential information for beginning to decrypt this essential, yet largely unexplored, layer of thrombus formation.

Public Health Relevance

We have shown that a group of enzymes called thiol isomerases promote blood clotting when released into the bloodstream. The current proposal focuses on understanding the mechanisms by which these thiol isomerases control clot formation. Successful completion of these studies will enable the development of new diagnostic and therapeutic agents for diseases such as heart attacks, strokes, and venous clots.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL136394-01
Application #
9285480
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
2017-04-01
Project End
2021-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
1
Fiscal Year
2017
Total Cost
$848,706
Indirect Cost
$202,506
Name
Beth Israel Deaconess Medical Center
Department
Type
Independent Hospitals
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
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Stopa, Jack D; Zwicker, Jeffrey I (2018) The intersection of protein disulfide isomerase and cancer associated thrombosis. Thromb Res 164 Suppl 1:S130-S135
Stopa, Jack D; Neuberg, Donna; Puligandla, Maneka et al. (2017) Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation. JCI Insight 2:e89373
Schulman, Sol; Bendapudi, Pavan; Sharda, Anish et al. (2016) Extracellular Thiol Isomerases and Their Role in Thrombus Formation. Antioxid Redox Signal 24:1-15
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; Furie, Bruce; Zwicker, Jeffrey I (2015) Therapeutic implications of protein disulfide isomerase inhibition in thrombotic disease. Arterioscler Thromb Vasc Biol 35:16-23
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
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
Furie, Bruce; Flaumenhaft, Robert (2014) Thiol isomerases in thrombus formation. Circ Res 114:1162-73
Flaumenhaft, Robert (2013) Protein disulfide isomerase as an antithrombotic target. Trends Cardiovasc Med 23:264-8

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