Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Lin Lin,Srila Gopal,Anish Sharda,Freda Passam,Sheryl R Bowley,Jack Stopa,Guangpu Xue,Cai Yuan,Barbara C Furie,Robert Flaumenhaft,Mingdong Huang,Bruce Furie

doi:10.1074/jbc.m115.666180

Abstract

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

Highlights

Quercetin-3-rutinoside is an inhibitor of protein disulfide isomerase, a potential target for antithrombotic therapy
We further found that quercetin-3-rutinoside inhibits both platelet thrombus formation and fibrin generation in a dose-dependent manner via inhibition of protein disulfide isomerase (PDI) in a mouse thrombosis model, and have raised the possibility that PDI be considered as a target for antithrombotic therapy [11]
Platelets were monitored in vivo using an anti-CD42b antibody conjugated to DyLight 649, and fibrin generation was monitored using a fibrin-specific monoclonal antibody conjugated to Alexa Fluor 488

Summary

Background

Quercetin-3-rutinoside is an inhibitor of protein disulfide isomerase, a potential target for antithrombotic therapy. Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. The infusion of the b؅x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. We further found that quercetin-3-rutinoside inhibits both platelet thrombus formation and fibrin generation in a dose-dependent manner via inhibition of PDI in a mouse thrombosis model, and have raised the possibility that PDI be considered as a target for antithrombotic therapy [11]. Based on these findings, we demonstrate that fragment bЈx of PDI reverses quercetin-3rutinoside-induced inhibition of thrombus formation in vivo using a mouse thrombosis model

Experimental Procedures

Results

Discussion