Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity

Chen W, et al., 13(2):248-261, J Thromb Haemost, 2014

Standard anticoagulant drugs are commonly associated with a higher risk of excessive bleeding. Alternatives that can target intrinsic coagulation factors are constantly being pursued in order to slowdown coagulation cascade in the event of an intravascular thrombosis while lowering the bleeding risks during an injury. Described herein is the identification of a novel factor XIa (FXIa) specific inhibitor, Fasxiator, which was isolated from the venom of the banded krait snake, Bungarus fasciatus. Furthermore, the Fasxiator was subjected to a systematic site-specific mutagenesis in order to design a series of variants with an enhanced potency and selectivity. FasxiatorN17R,L19E mutant demonstrated the best balance in-between the potency and selectivity. Binding interactions of rFasxiator with FXIa and chymotrypsin was studied by Surface Plasmon Resonance (SPR) spectroscopy. A Pall ForteBio Pioneer system equipped with COOH5 sensor chip was used for the SPR assays. By using standard amine coupling chemistry, rFasxiator was immobilized onto the COOH5 sensor chip surface. Subsequently, different concentrations of FXIa or chymotrypsin diluted in running buffer were injected. The sensor chip surface was regenerated with glycine/HCl buffer. KD values were determined for the binding interactions assuming a simple 1:1 stoichiometry and one-step binding. Overall results of this investigation predict Fasxiator as a promising anticoagulant drug candidate.

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