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.

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