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.
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
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