The identification of quercetin-3-rutinoside as an antagonist of PDI and an inhibitor of thrombus formation validates PDI as a drug target for antithrombotic therapy. Inhibition of PDI using neutralizing antibodies blocks thrombus formation in vivo after carotid artery ligation (9
), ferric chloride exposure (unpublished observations, L. Bellido-Martin, B. Furie, and B.C. Furie), or laser injury (7
), demonstrating an essential role for PDI in thrombus formation in multiple models. This observation raises the question of whether small molecule inhibition of PDI could be used to control thrombus formation in vivo, particularly given the advantage that both platelet accumulation and fibrin generation are blocked following inhibition of PDI. Several observations indicated that PDI is the relevant molecular target of quercetin-3-rutinoside in our thrombus formation studies. Quercetin-3-rutinoside selectively inhibited PDI, without significant inhibition of other thiol isomerases that may have been present in the vasculature during thrombus formation. A structurally related analog of quercetin-3-rutinoside, diosmetin, which does not inhibit PDI, failed to inhibit thrombus formation. In addition, the antithrombotic activity of quercetin-3-rutinoside was entirely reversed after infusion of recombinant PDI. Thus, although quercetin-3-rutinoside may have other physiologic properties, the dominant effect of quercetin-3-rutinoside in thrombus formation is to inhibit extracellular PDI function, thereby preventing thrombi from forming after vascular injury.
PDI inhibition prevents both platelet accumulation and fibrin generation during thrombus formation. Although the substrates activated by PDI during thrombus formation remain unknown, candidates have been proposed. PDI has been implicated in αIIb
-mediated platelet aggregation (2
). Antibodies directed at PDI inhibit platelet aggregation in vitro (4
), and this effect has been attributed in part to the influence of PDI on αIIb
). Glycoprotein 1bα
contains free thiols and is modified by PDI (5
). PDI catalyzed disulfide exchange also influences adhesion of collagen to α2
). Quercetin-3-rutinoside inhibited platelet aggregation in vitro. Moreover, platelet-rich plasma from mice infused with quercetin-3-rutinoside demonstrated impaired aggregation compared with platelet-rich plasma from control mice when tested ex vivo (Figure C), indicating that the compound directly impairs platelet aggregation. The direct effect of quercetin-3-rutinoside on platelet receptors in vivo, however, remains to be determined.
Fibrin formation after laser-induced injury of arterioles occurs in a platelet-independent manner, and the endothelium is an important source of PDI during thrombus formation (8
). Previous studies using cultured endothelial cells demonstrate that fibrin formation is dependent on endothelial cell–derived PDI (8
). We found that quercetin-3-rutinoside inhibited fibrin formation on cultured endothelial cells with approximately the same potency as it inhibited purified PDI in the insulin reductase assay. Quercetin-3-rutinoside failed to inhibit endothelial cell activation (Supplemental Figure 2, A–C), and it did not inhibit fibrin formation induced by thrombin (Supplemental Figure 2D). These observations are consistent with an effect of quercetin-3-rutinoside on PDI. The relevant PDI substrates on endothelium are not known. Thiol isomerase–catalyzed oxidation of an allosteric disulfide bond in tissue factor may convert it from a noncoagulant cryptic state to a procoagulant decrypted state by PDI-mediated oxidation (28
). However, the role of PDI in tissue factor deencryption is controversial and indirect pathways have been proposed (31
is also a putative PDI substrate on endothelial cells (32
), though its role in thrombus formation is not known. Alternatively, extracellular PDI has been invoked in a transnitrosation reaction, enabling delivery of nitric oxide from extracellular to intracellular environments (33
The observation that a commonly ingested flavonol and its metabolites are potent inhibitors of PDI indicates the feasibility of targeting PDI without substantial toxicity. Genetic deletion of PDI is toxic to cells (10
). The fundamental role of PDI in disulfide bond formation and protein folding raises the question of whether PDI inhibition can be well tolerated. We found that quercetin-3-rutinoside demonstrates no toxicity in cultured endothelial cells for at least 72 hours at concentrations as high as 100 μM quercetin-3-rutinoside (data not shown). Quercetin-3-rutinoside may lack toxicity because the same glycosidic linkage that is required for inhibition of PDI activity impairs cell permeability. Thus, flavonols with 3-O-glycosidic linkages could preferentially target extracellular PDI.
Plasma concentrations of quercetin-3-rutinoside achieved after infusion during thrombus formation studies were substantially lower than concentrations required for inhibition of PDI in vitro. Quercetin-3-rutinoside concentrations detected in vivo were also lower than those required for inhibition of platelet aggregation or fibrin generation on endothelial cells. Several considerations limit the strength of conclusions that can be drawn from a comparison of in vitro and in vivo findings. Foremost among these is the extensive metabolism of quercetin-3-rutinoside in vivo. Exposure to quercetin-3-rutinoside results in the generation of more than 60 metabolites (35
). Many major metabolites, such as quercetin-3-glucuronide, possess a 3-O-glycosidic linkage and are active against PDI, as demonstrated by structure activity relationships (Figure ). In addition, rutinosides are known to bind to the blood vessel wall (36
), where they may maintain antithrombotic activity but not be detected in plasma. Also limiting comparisons between in vitro studies of purified PDI and in vivo studies is the fact that in vitro studies rely on reduction of insulin by PDI while the relevant PDI substrates during thrombus formation in vivo remain to be determined. Thus, while these data are consistent with the possibility that quercetin-3-rutinoside is more potent in vivo than in vitro, it is difficult to draw firm conclusions regarding such a comparison.
Quercetin-3-rutinoside and the other 3-O-glycoside–linked flavonols identified as PDI inhibitors are found in high concentrations in tea, fruits, berries, and buckwheat (38
). Multiple studies demonstrate that chronic administration of dietary flavonols, at concentrations as high as 3,000 mg/kg, has no significant toxicity in animal studies (39
). Dietary flavonols used in clinical trials have also been well tolerated (41
). Epidemiologic studies evaluating the effect of flavonol ingestion on cardiovascular events demonstrate protection from myocardial infarction and stroke with increased intake (42
In summary, we identify quercetin-3-rutinoside as an inhibitor of PDI and show that inhibition of PDI potently blocks thrombus formation in vivo. These observations provide proof of principle for targeting extracellular PDI for inhibition of thrombus formation. Other agents that inhibit PDI function, such as juniferdin (14
) or bacitracin (45
), also inhibit thrombus formation in vivo (data not shown and ref. 7
). However, these agents are either cytotoxic or nonselective (12
). The fact that quercetin-3-rutinoside is antithrombotic at flavonol concentrations that are well tolerated, based on extensive animal and human clinical literature, indicates that inhibition of extracellular PDI is a safe strategy for inhibition of thrombus formation. Pharmacological regulation of PDI enzymatic activity could prevent thrombosis in the setting of coronary artery disease, stroke, or venous thromboembolism.