Heparin (HP) and low molecular weight heparin (LWMH) are widely used clinical anticoagulant drugs. Their anticoagulant binding site to antithrombin III (AT-III) is well studied. A pentasaccharide sequence represents responsible for most of its anticoagulant activity. But the structure-activity relationship for heparin/LWMH oligosaccharides and other heparin-binding proteins remains incompletely understood. Here, we present a pull-down assay using a filter-entrapment enrichment that can obtain high protein binding HP/LWMH oligosaccharides. HP/LWMH was added to free heparin-binding proteins in a solution of binding buffer in a 50 kDa molecular weight cut-off centrifugal tube. Low-affinity HP/LMWH was washed through the centrifugal membrane and high-affinity HP/LWMH were eluted with a high concentration of sodium chloride. The proteins were retained by the membrane and can be reused after re-equilibration with binding buffer. Arixtra (Fondaparinux Sodium) is an ultra-low anticoagulant drug, which is a pentasaccharide that can be directly detected by Orbitrap MS was first tested with antithrombin III (AT-III) to confirm this method is working well. The proteins are required in the microgram level, and the protein remained activated after at least 10 cycles. Next, we tried HP/LWMH with antithrombin, thrombin and platelet factor 4 (PF4), and compared their structural motifs by disaccharides analysis, anticoagulant activity, nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) analysis and CE-MS for oligosaccharides. The results showed that there were many differences between low-binding and high-binding oligosaccharides. This method is high-throughput, efficient, reproducible and can be introduced to multiple proteins, thus results in a generation of a purified specific protein binding HP/LMWH oligosaccharides for structural characterization.

Structural motifs of heparin and low molecular weight heparin binding to anticoagulant proteins by pull-down assay

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.