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 6

Quercetin-3-rutinoside inhibits thrombus formation and fibrin generation in vivo.

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Quercetin-3-rutinoside inhibits thrombus formation and fibrin generation...
Platelet-specific anti-CD42b antibody conjugated to Dylight 649 (0.1 μg/g body weight) and fibrin-specific mouse anti-human fibrin II β-chain monoclonal antibody conjugated to Alexa Fluor 488 (0.5 μg/g body weight) were infused into the mice. At varying doses, quercetin-3-rutinoside was subsequently infused intravenously 5 minutes prior to the initial laser injury. Representative binarized images of the appearance of fluorescence signals associated with fibrin (green) and platelets (red) over the 180 seconds after laser-induced vessel wall injury in wild-type mice are shown in A–D, with mice infused with (A) vehicle only; (B) quercetin-3-rutinoside at 0.1 mg/kg body weight; (C) quercetin-3-rutinoside at 0.3 mg/kg body weight; and (D) quercetin-3-rutinoside at 0.5 mg/kg body weight shown. (E) Median integrated platelet fluorescence intensity and (F) median integrated fibrin fluorescence intensity at the injury site are plotted versus time, with mice infused with vehicle only (black); quercetin-3-rutinoside at 0.1 mg/kg body weight (green); quercetin-3-rutinoside at 0.3 mg/kg body weight (blue); and quercetin-3-rutinoside at 0.5 mg/kg body weight shown (red). Data are from 30 thrombi in 3 mice for each condition. RFU, relative fluorescence units. (G) Quercetin-3-rutinoside at 0.5 mg/kg was infused 5 minutes prior to FeCl3 injury of cremaster arterioles. Data points represent time to occlusion, as determined by cessation of flow in control mice infused with vehicle or mice infused with quercetin-3-rutinoside as indicated. Horizontal bars denotes median values. Original magnification, ×60. Scale bars: 10 μm.