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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.
Introduction
Protein disulfide isomerase (PDI) is the prototypical member of an extended family of oxidoreductases, best known as endoplasmic reticulum-resident enzymes. These enzymes catalyze posttranslational disulfide bond formation and exchange and serve as chaperones during protein folding (1). Despite having a C-terminal endoplasmic reticulum retention sequence, PDI has been identified at many diverse subcellular locations outside the endoplasmic reticulum. It has biological functions on the cell surfaces of lymphocytes, hepatocytes, platelets, and endothelial cells (2-6). Platelets are a rich source of extracellular PDI, expressing this protein on their surface and also secreting PDI in response to thrombin stimulation (5, 7). Endothelial cells also express PDI upon agonist stimulation or when challenged by a vascular injury (3, 8).
We have previously shown that PDI is rapidly secreted from both endothelial cells and platelets during thrombus formation in vivo (7, 8). Inhibition of PDI using neutralizing antibodies blocks thrombus formation in several thrombosis models (refs. 6-9 and L. Bellido-Martin, B. Furie, B.C. Furie, unpublished observations). Inhibition of PDI in these models abrogates not only platelet accumulation at...