Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents
Reema Jasuja, Freda H. Passam, Daniel R. Kennedy, Sarah H. Kim, Lotte van Hessem, Lin Lin, Sheryl R. Bowley, Sucharit S. Joshi, James R. Dilks, Bruce Furie, Barbara C. Furie, Robert Flaumenhaft
Reema Jasuja, Freda H. Passam, Daniel R. Kennedy, Sarah H. Kim, Lotte van Hessem, Lin Lin, Sheryl R. Bowley, Sucharit S. Joshi, James R. Dilks, Bruce Furie, Barbara C. Furie, Robert Flaumenhaft
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Research Article Hematology

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Abstract

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell–mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

Authors

Reema Jasuja, Freda H. Passam, Daniel R. Kennedy, Sarah H. Kim, Lotte van Hessem, Lin Lin, Sheryl R. Bowley, Sucharit S. Joshi, James R. Dilks, Bruce Furie, Barbara C. Furie, Robert Flaumenhaft

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Figure 1

Identification of quercetin-3-rutinoside as a PDI inhibitor.

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Identification of quercetin-3-rutinoside as a PDI inhibitor. (A) Data fr...
(A) Data from representative 384-well plates, including samples with no PDI (green); 3 mM bacitracin, a nonspecific inhibitor of the PDI family of oxidoreductases (blue); or test compounds (red). Duplicate readings are plotted, and an arrow indicates the inhibitory compound identified as quercetin-3-rutinoside. Each symbol represents an individual reading. (B) Effect of the indicated concentrations of quercetin-3-rutinoside on PDI activity, determined using the insulin reduction assay (mean ± SD). (C) Sensorgram of binding of quercetin-3-rutinoside to recombinant PDI. The thin arrow indicates the time of injection of quercetin-3-rutinoside onto the PDI-coated sensor chip, and the thick arrow indicates the time of injection of regeneration buffer. Black lines represent the fitted curves for duplicates of the varying concentrations of quercetin-3-rutinoside analyte at 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μM.