Efficient Small-Molecule Reversal Agents for Anticoagulant Fondaparinux

Fondaparinux is a highly anionic synthetic heparinoid pentasaccharide used as an anticoagulant for specific clinical conditions and surgeries. As a non-natural small-molecule drug, it presents pharmacokinetic and pharmacodynamic advantages, as well as high stability and low immunogenicity, when compared with different forms of heparin. However, its broader usage is hampered by different factors like price, existence of alternative anticoagulants, or, specifically in this case, the lack of an effective antidote that is highly recommendable for avoiding uncontrolled bleeding. In this work, we describe two synthetic small molecules derived from spermine (3AC and 3FF) that efficiently revert the anticoagulant activity of fondaparinux. In an <i>in vitro</i> enzymatic assay related to blood coagulation, the spermine derivatives show potent activity as fondaparinux antidotes, with higher activity than ciraparantag (a small molecule in the clinical phase as an anticoagulant antidote) and much higher activity than protamine, the only approved antidote for unfractioned heparin but inefficient against fondaparinux. Remarkably, naked-eye <i>ex vivo</i> tests demonstrated their efficacy in freshly extracted mice blood. Mechanistic studies show that both small molecules strongly bind fondaparinux in buffered water, as detected by fluorescence and NMR spectroscopy and confirmed by molecular dynamics simulations. Thus, these spermine derivatives are promising reversal agents against heparinoid anticoagulants with a wide range of molecular weights, overcoming the drawbacks of those antidotes based on biomacromolecules.