Naringin binds and activates protein C inhibitor for enhanced inhibition of thrombin and activated protein C with implications in coagulation control

Antithrombotic drugs are associated with drawbacks however they continue to be used globally for thrombus control. A majority of these anticoagulants activate antithrombin for enhanced inhibition of coagulation proteases like thrombin and factor Xa. Protein C Inhibitor (PCI) is able to inhibit both thrombin and activated protein C to regulate procoagulant and anticoagulant pathways. It is a potential drug lead in treating septic shock due to its strong anticoagulant and anti-inflammatory properties. A comprehensive screening of natural compounds that can bind PCI show a high affinity for naringin (-9.4 kcal/mol). Naringin is a flavonoid that is commonly found in the citrus fruit and has been shown to impart protection against cardiovascular diseases. Interestingly, naringin binds to the shutter region of the PCI away from the heparin binding site. A molecular dynamic simulation of PCI conformation showed exposure of the reactive center loop on binding naringin, a region that binds and catalyses the inhibition of the target proteases. Consequently, addition of naringin (50 µM) resulted in a significant decrease in the coagulation rates in the extrinsic and common pathways, however an increased rate was observed in the intrinsic pathway. A UV-Vis spectroscopic, fluorometric and circular dichroism based structural assessment showed PCI-naringin binding stoichiometry to be 1.0, with ground state-static binding and largely conserved secondary structure. Interestingly, the study reveals that naringin imparts its affect on PCI through enhancing the rate of inhibition of thrombin and activated protein C by 22-fold and 21-fold respectively, which might indicate an alternate basis of cardiovascular protection.