Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario, Canada.
Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada.
J Thromb Haemost. 2019 Jul;17(7):1153-1159. doi: 10.1111/jth.14451. Epub 2019 May 10.
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.
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.
We report a new assay that measures anti-FXa DOAC levels in plasma and whole blood.
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.
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.
The assay is ideal for rapidly and accurately measuring DOAC levels in plasma and blood, demonstrating its potential for point-of-care applications.
我们报告了一种新的测定法，用于测定血浆和全血中的抗 FXa 直接口服抗凝剂（DOAC）水平。
采用一种被荧光探针（Flu-II）标记的凝血酶原衍生物，作为测量剩余 FXa 活性的大分子量底物，从而实现这一点。然后，通过加入含有 FXa、FVa 和由 75% PC 和 25% PS（PCPS）组成的磷脂囊泡以及钙离子的溶液，在肝素存在的情况下，启动 Flu-II 裂解，以防止由内源性凝血酶产生的反馈活性。通过实时荧光监测 Flu-II 裂解，初始荧光变化速率与 DOAC 水平成反比。
在血浆系统中，该测定法在 0 至 5 nmol/L 利伐沙班和 0 至 10 nmol/L 阿哌沙班之间显示出剂量反应。玉米胰蛋白酶抑制剂不会影响该测定法。对于个体血浆样本，该测定法显示出极好的一致性和重现性。从 2 μL 的全血中，该测定法在最终 100 μL 混合物中显示出 0 至 2 nmol/L 的 DOAC 剂量反应，因此代表循环血液中的 100 nmol/L 左右。
该测定法非常适合快速准确地测量血浆和血液中的 DOAC 水平，展示了其在即时护理应用中的潜力。
