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Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b′ domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

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Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b′ domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b′ domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 μM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b′ substrate-binding pocket and the a′ domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b′ domain is a critical domain for inhibitor development. Besides, we also report an innovative highthroughput screening method for the rapid discovery of PDI b′ targeted inhibitors.

Web Services is a platform which enables the applications interoperate on the Internet. It is widely used in designing and building systems in open and dynamic distributed environments such as EAI (Enterprise Application Integration) and B2B (Business to Business). As the development of framework technology, it is convenient and standardized to use framework to develop web applications. For Web Services, the frameworks Axis, Axis2, XFire and CXF are widely used. By the performance testing of the four frameworks, this paper not only introduces the four framework but also analyzes the differences of the four frameworks, and then makes some suggestions for developers to choose the appropriate one. The framework can simplify the development process and decrease the development time. However, because of the differences of the frameworks, the interoperation between different web service's server and client may cause incompatible problems. This paper analyzes the reasons of this incompatible problems and finally presents a mechanism based on unified invoking component to solve this problems. By parsing and repacking the SOAP messages, the incompatible problems between client and server can be solved successfully.