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
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 bx 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
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