A fluorescent probe assay (Heparin Red) for direct detection of heparins in human plasma.

ABSTRACT

Heparins are widely used anticoagulant drugs. The current monitoring practice for heparin in plasma, such as the chromogenic anti-factor Xa assay, relies on heparin-triggered activation of antithrombin, an inhibitor of coagulation proteases. Such assays are not applicable to the detection of non-anticoagulant heparins, an emerging class of drug candidates for therapeutic applications unrelated to anticlotting activity. This study describes the application of a commercially available fluorescent probe assay (Heparin Red) for the direct and sensitive detection of the "chemical" heparin in plasma, independent of any anticoagulant activity. The quantification range is about 0-5 µg/mL for both unfractionated heparin (corresponding to 0-1 IU/mL) and the low molecular weight heparin enoxaparin. The Heparin Red assay is of particular value for the quantification of non-anticoagulant heparins, as exemplified by the low molecular weight heparin derivative tafoxiparin and a N-desulfated-N-reacetylated heparin. Heparin octa- and decasaccharides are also detected. Graphical abstract Heparin quantification in plasma by mixing the sample with the Heparin Red reagent and fluorescence readout.