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 4

Quercetin-3-rutinoside inhibits platelet aggregation.

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Quercetin-3-rutinoside inhibits platelet aggregation. (A) Washed human p...
(A) Washed human platelets (2 × 108 platelets/ml) were incubated with vehicle alone (black), 30 μM quercetin-3-rutinoside (blue), 45 μM quercetin-3-rutinoside (red), or anti-PDI antibody, RL90 (green), for 15 minutes and subsequently stimulated with 50 μM PAR4 peptide AYPGKF. (B) Washed human platelets (2 × 108 platelets/ml) were incubated with either vehicle (black) or 60 μM quercetin-3-rutinoside (red) for 15 minutes or incubated with vehicle followed by a wash (dark blue) or 60 μM quercetin-3-rutinoside followed by a wash (light blue) and subsequent stimulation with 50 μM PAR4 peptide AYPGKF. (C) Quercetin-3-rutinoside was infused into mice intravenously at 0.5 mg/kg. Five minutes after infusion, blood was obtained by cardiac puncture, and platelet-rich plasma was isolated. Platelet-rich plasma from quercetin-3-rutinoside–treated mice (red) or vehicle control (black) was stimulated with 200 μM AYPGKF.