Engineering ultrapotent trivalent anticoagulants through hybridisation of salivary peptides from multiple haematophagous organisms
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 <i>in vitro</i> clotting assays and was also shown to possess potent <i>in vivo</i> antithrombotic activity in a murine model of thrombosis.
Authors
- Daniel Ford (ORCID: https://orcid.org/0000-0002-4419-1553)
- Jorge Ripoll‐Rozada (ORCID: https://orcid.org/0000-0001-7235-317X)
- Angus S. Mackay (ORCID: https://orcid.org/0000-0002-0794-1415)
- Simone M. Schoenwaelder (ORCID: https://orcid.org/0000-0003-0465-5840)
- Joshua W. C. Maxwell (ORCID: https://orcid.org/0000-0003-1481-370X)
- Imala Alwis (ORCID: https://orcid.org/0000-0002-5459-9093)
- Shaun P. Jackson (ORCID: https://orcid.org/0000-0002-4750-1991)
- Richard J. Payne (ORCID: https://orcid.org/0000-0002-3618-9226)
- Rhyll E. Smythe (ORCID: https://orcid.org/0009-0002-3652-6197)
- Joana A. Santos (ORCID: https://orcid.org/0000-0001-8294-3405)
- Pedro José Barbosa Pereira (ORCID: https://orcid.org/0000-0003-0969-5438)
- Joanna Liu (ORCID: https://orcid.org/0009-0004-2554-4466)
- Zack Zuccolotto
- Cameron B J Trought (ORCID: https://orcid.org/0000-0003-1062-0214)
Institutions
- The University of Sydney (AU)
- Universidad de Cantabria (ES)
- Australian Research Council (AU)
- Consejo Superior de Investigaciones Científicas (ES)
- Charles Sturt University (AU)
- Universidade do Porto (PT)
- The Heart Research Institute (AU)
- Instituto de Biomedicina y Biotecnología de Cantabria (ES)
- ARC Centre of Excellence for Innovations in Peptide and Protein Science (AU)
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto (PT)
Publication Details
- Journal
- Chemical Science
- Published
- 2025-01-01
- DOI
- https://doi.org/10.1039/d5sc04734j
- Primary Topic
- Enzyme Production and Characterization
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- European Commission
- Medical Research Council
- National Health and Medical Research Council
- Fundação para a Ciência e a Tecnologia