An assay to measure levels of factor Xa inhibitors in blood and plasma
Methodology paper published in Journal of Thrombosis and Haemostasis (2019)
Abstract
UNLABELLED: Essentials Direct oral anticoagulants (DOAC) are used for stroke and venous thromboembolism prevention. We report a new assay that measures anti-factor Xa DOAC levels in plasma and whole blood. Rivaroxaban and apixaban can be accurately quantified below trough levels. The ease and accuracy of the assay demonstrate its potential for point-of-care applications. BACKGROUND: Rivaroxaban and apixaban are the most commonly used anti-factor (F) Xa direct oral anticoagulants (DOAC), with indications for prevention of stroke in nonvalvular atrial fibrillation as well as treatment and prevention of venous thromboembolism. However, lacking is accessibility to a detection method that is able to quantify low levels of anti-FXa DOACs. OBJECTIVE: We report a new assay that measures anti-FXa DOAC levels in plasma and whole blood. METHODS: This is achieved by the use of a prothrombin derivative that is labeled with a fluorescent probe (Flu-II), which then acts as the macromolecular substrate to measure residual FXa activity. The Flu-II cleavage is then initiated by the addition of a solution containing FXa, FVa, and phospholipid vesicles composed of 75% PC and 25% PS (PCPS) vesicles with calcium, in the presence of hirudin to prevent feedback activity by the native thrombin generated. The Flu-II cleavage is monitored by fluorescence in real time where the initial rate of fluorescence change is inversely proportional to DOAC levels. RESULTS: In plasma systems, the assay demonstrates dose-response between 0 and 5 nmol/L rivaroxaban and between 0 and 10 nmol/L apixaban. Corn trypsin inhibitor did not affect this assay. With individual plasma samples, the assay showed excellent consistency and reproducibility. From 2 μL of whole blood, the assay showed dose-response between 0 and 2 nmol/L of DOACs in the final mixture of 100 μL, thus representing up to 100 nmol/L in circulating blood. CONCLUSION: The assay is ideal for rapidly and accurately measuring DOAC levels in plasma and blood, demonstrating its potential for point-of-care applications.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Reports a new fluorescent prothrombin-derivative assay using hirudin to prevent thrombin feedback, enabling rapid quantification of direct factor Xa inhibitors (rivaroxaban, apixaban) in plasma and whole blood with point-of-care potential.
Por qué esto importa para la hirudoterapia
This study reports a new fluorescence-based assay for measuring anti-factor Xa direct oral anticoagulant (DOAC) levels—rivaroxaban and apixaban—in plasma and whole blood, demonstrating dose-response down to low nanomolar concentrations from as little as 2 µL of blood. Hirudin appears in the methodology solely as a reagent added to the cleavage reaction to prevent feedback activity by native thrombin generated during the assay; it is not the subject of investigation. Consequently, the article has no direct relevance to hirudotherapy, the leech secretome, or ASH's core domain. The connection is limited to hirudin's routine laboratory use as an anticoagulant adjunct in a DOAC-monitoring assay, and no findings about hirudin itself, leech biology, or therapeutic leech application are reported.
Citación
An assay to measure levels of factor Xa inhibitors in blood and plasma.
Kim PY et al. · Journal of Thrombosis and Haemostasis, 2019
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: June 18, 2026