Open Access Article
This Open Access Article is licensed under a Creative Commons Attribution-Non Commercial 3.0 Unported LicenceEngineering Ultrapotent Trivalent Anticoagulants Through Hybridisation of Salivary Peptides from Multiple Haematophagous Organisms
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Joshua W C Maxwell , Jorge Ripoll-Rozada , Angus S Mackay , Imala Alwis , Daniel J. Ford , Cameron B. J. Trought , Joana A. Santos , Rhyll E. Smythe , Joanna S.T. Liu , Zack Zuccolotto , Simone M. Schoenwaelder , Shaun P. Jackson , Pedro José Barbosa Pereira and Richard James Payne
First published on 1st September 2025
Abstract
Haematophagous organisms are a rich source of salivary anticoagulant polypeptides that exert their activity by blocking the catalytic site and one of two positively charged exosites on the host protease thrombin. Here, we describe a molecular engineering approach to hybridise post-translationally sulfated polypeptides from different blood-feeding organisms to enhance anticoagulant activity. This led to the discovery of a triply sulfated hybrid anticoagulant, XChimera, possessing fragments from flea, leech, and fly salivary polypeptides that exhibits femtomolar inhibitory activity against thrombin. The crystallographic structure of a complex of XChimera with thrombin shows that it displays a trivalent binding mode in which it simultaneously blocks three functional sites of the protease, the active site and exosites I and II. This trivalent chimera exhibited ultrapotent anticoagulant activity in a suite of in vitro clotting assays and was also shown to possess potent in vivo antithrombotic activity in a murine model of thrombosis.