![]() ![]() These results indicate that AnXa was able to compete with not only fXa, but also IIa, for binding to heparinized-ATIII and reverse both the anti-fXa and anti-IIa activities of heparin. ![]() In human plasma, AnXa reversed the anti-IIa activity of heparin (0.8 IU/mL) with an estimated EC 50 comparable to anti-fXa reversal (anti-fXa:126 nM anti-IIa: 131nM). This suggests that interaction of AnXa with the primary binding site on the surface of heparin-activated ATIII (i.e., the reactive center loop (RCL)) might play a major role in the anti-IIa reversal as binding to the RCL was the minimal requirement for the inhibition of both fXa and IIa by ATIII. rivaroxaban) in the same enzymatic assay. The reversal effect of AnXa on IIa inhibition could be blocked by addition of a small molecule direct fXa inhibitor (e.g. ![]() AnXa dose-dependently reversed the IIa inhibition to its basal level seen without heparin. In the enzymatic assay with IIa and ATIII, heparin accelerated the reaction of IIa inhibition by ~1000-fold. Interestingly, AnXa was also able to reverse the heparin anti-IIa activity in the same assays. Results: AnXa dose-dependently reversed the anti-fXa activity of heparin in the enzymatic assay and in human plasma. Di-arginine piperazine (PER977), a positively charged small molecule, was also tested for comparison. Protamine sulfate was included as a control. Reversal of heparin was compared to enoxaparin. The reversal effect of AnXa on heparin anticoagulation and clot formation was further characterized by thromboelastography (TEG) in human plasma or whole blood. ![]() Reversal of heparin-induced clotting prolongation of human plasma was measured by a 96-well format turbidity assay using an aPTT reagent and Ca 2+. Reversal of heparin anti-fXa and anti-IIa activities by AnXa in human plasma was evaluated using modified anti-fXa and anti-IIa chromogenic assays. 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). ![]()
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