Novel Point-Of-Use FXa Assay For Monitoring Anticoagulant Therapy By Oral Factor Xa Inhibitors and Heparin

Introduction A highly specific, rapid, robust and sensitive assay for Factor Xa (FXa) activity will greatly improve monitoring and optimization of anticoagulant therapy by direct FXa inhibitors and by low molecular weight (LMW) heparins. We have developed a new point-of-use diagnostic system that will provide results in less than 30 minutes, enabling rapid medical decisions based on quantitation of FXa activity and anticoagulant drugs in patients’ blood. Anticoagulant therapy with unfractionated or LMW heparins needs aggressive monitoring in pregnant women, patients with renal insufficiency, and neonates, in particular in cases of treatment-related side effects, including heparin-induced thrombocytopenia and bleeding tendency (Chest, 2008). New oral anticoagulants rivaroxaban (Xarelto®) and apixaban (Eliquis®) have been effective in reducing risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and in preventing and treating deep vein thrombosis. Both drugs directly inhibit FXa and have significant therapeutic potential as an alternative to warfarin. Unlike warfarin, these drugs do not require constant monitoring and dose adjustment to maintain anticoagulation within the therapeutic interval. However, excessive anticoagulation and consequent hemorrhage risks have been sufficiently common problems to warrant clinical development of specific antidotes for FXa inhibitors. Currently available anti-Xa heparin assays may not be appropriate for rivaroxaban and apixaban measurements. Additionally, recently published data indicate that new oral anticoagulants interfere with the measurement of common test parameters, expanding the need for more specific diagnostic tests (Clinical Chemistry, February 2013). This novel rapid and specific FXa assay will allow monitoring LMW heparins and the new FXa inhibitors, identify heparin-refractory patients as well as those requiring antidotes to oral Factor Xa inhibitors, in addition to directly measuring FXa activity in patients with inherited deficiency of Factor X, congenital antithrombin deficiency, and acquired FXa deficiencies. Method A diagnostic assay to measure FXa activity in human plasma using electrochemiluminescence (ECL) technology was developed at Wellstat Diagnostics, LLC. The assay utilizes a synthetic peptide containing an FXa substrate sequence that is recognized by specific antibodies (labeled with an ECL-active ruthenium chelate) only after specific cleavage by FXa protease. The resulting ECL signal is directly proportional to FXa activity within the physiological range. The assay has been adapted to also provide quantitative measurement of activity of anticoagulant drugs that inhibit FXa. In the modified format the resulting ECL signal is inversely proportional to the concentration of rivaroxaban, apixaban or heparin in plasma. Results The ECL-based assay measures activity of anticoagulant drugs on FXa in human plasma with high intra- and inter-assay reproducibility, accuracy and specificity. Using FX-deficient plasma spiked with Factor X in the range 1-100 ng/mL, we have demonstrated linear increases of ECL signal directly proportional to the concentration of spiked Factor X activated by a specific Russell Viper Venom activator. Serial dilutions of anticoagulant drugs rivaroxaban, apixaban and enoxaparin (LMW heparin) in normal donor plasma showed exponential decreases of ECL signal in the range of concentrations relevant to the therapeutic doses of all three drugs. The dynamic range of the drug detection was 15 - 500 ng/mL for rivaroxaban and apixaban, and 0.2 - 2 IU/mL of enoxaparin. The assay was specific to activity of FXa, while insensitive to variable concentrations of other clotting factors in human plasma, as well as to the presence of other drugs in plasma that don’t directly inhibit FXa. Conclusions The point-of-use FXa assay is shown to be highly specific, robust, rapid and reproducible for measurements of anticoagulant activity of apixaban, rivaroxaban and enoxaparin in human plasma. Use of this point-of-use system for monitoring anticoagulant therapy may be further extended to measure other blood clotting factors (for example, Factor IXa, VIIa, FVIII), for monitoring activity of drugs acting on these factors and for rapid diagnosis of coagulation disorders. Disclosures: No relevant conflicts of interest to declare.