To determine whether extracellular PDl plays a role in thrombus formation, PDl expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice following laser-induced arteriolar injury. A time-dependent increase in PDl antigen was observed in the thrombus following injury. Infusion of bacitracin or a blocking monoclonal antibody to PDl into the circulation completely inhibited platelet thrombus formation and fibrin generation. These results indicate that PDl is required in vivo for both fibrin generation and platelet thrombus formation. The objective in this project is to determine whether inhibitors of PDl represent a new class of antithrombotic agents.
In Aim #1, the current project proposes to Identify the substrates of PDl that participate in the initiation of thrombus formation by the expression of mutant forms of thiol isomerases into experimental thrombi ex vivo and the trapping of disulfide-linked complexes of thiol isomerases and their substrates.
In Aim #2, the xray crystal structure of human PDl and PDl in complex will be determined. These structures will include PDl and PDl bound to an anfi-PDI Fab, PDl complexed to quercetin 3-rutinoside and to quercetin, and PDl complexed to beta3 integrin.
In Aim #3, antithrombotic properties of quercetin will be tested in mice. The pharmacology of quercetin as an antithrombotic agent will be initially studied by direct visualization of thrombus inhibifion using the laserinduced thrombus formation model in the cremaster muscle of the mouse using intravital microscopy. The inhibition of thrombosis-initiated fetal/neonatal demise by quercetin will be tested in two mouse models: perinatal thrombosis with homozygous Factor V Leiden and heterozygous TFPI deficiency, and consumptive coagulopathy with homozygous protein C deficiency. If quercetin is an active antithrombotic, we will determine whether quercetin, which inhibits both platelet aggregation and fibrin generation, is superior to standard antithrombotic agents. PDl inhibitors will be compared to conventional antithrombotic agents in mouse models of thrombosis. These will include death or paralysis induced by pulmonary embolism, and by intravital study of thrombus formation associated with acquired anti-phospholipid syndrome.
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 to develop new classes of antithrombotic therapies. Studies in this project will determine whether PDl is a novel target for antithrombotic therapy.
|Bowley, Sheryl R; Fang, Chao; Merrill-Skoloff, Glenn et al. (2017) Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation. Nat Commun 8:14151|
|Stopa, Jack D; Baker, Katherine M; Grover, Steven P et al. (2017) Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates. J Biol Chem 292:9063-9074|
|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|
|Flaumenhaft, R (2016) Probing for thiol isomerase activity in thrombi. J Thromb Haemost 14:1067-9|
|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|
|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|
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