Residual fXa or IIa activity was measured in a plate reader by monitoring the initial rate of substrate cleavage at 405 nm. At different time points, a small aliquot of the reaction mixture was taken and quenched into a 96-well assay plate containing protamine (50 µg/mL) and a chromogenic substrate (100 µM) for fXa (Spectrozyme-Xa) or IIa (S2238). The reaction mixture contained fXa or IIa (20 nM), ATIII (200 nM), different amounts of unfractionated heparin and AnXa.

Methods: Inhibition of human fXa or thrombin (IIa) by ATIII in the presence of heparin was studied in an enzymatic assay in buffered solution. Here we report new in vitro data demonstrating that AnXa can also effectively reverse anticoagulation of unfractionated heparin. We previously reported nonclinical and Phase 2 clinical data in healthy subjects with enoxaparin, a low molecular weight heparin (LMWH). Andexanet alfa (AnXa) is a recombinant modified human fXa under development for the reversal of both direct and antithrombin III (ATIII)-dependent indirect fXa inhibitors. Although protamine sulfate can be used to neutralize heparin, its administration can cause severe hypotensive and anaphylactic reactions. Bleeding is the major complication that may result from these therapies. Background: Unfractionated heparin is used in the management of venous thromboembolism, stroke prevention in atrial fibrillation as well as in cardiac surgeries (e.g., valve replacement and other procedures requiring cardiopulmonary bypass).

The universal antidote