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). Bleeding is the major complication that may result from these therapies. Although protamine sulfate can be used to neutralize heparin, its administration can cause severe hypotensive and anaphylactic reactions. 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. We previously reported nonclinical and Phase 2 clinical data in healthy subjects with enoxaparin, a low molecular weight heparin (LMWH). Here we report new in vitro data demonstrating that AnXa can also effectively reverse anticoagulation of unfractionated heparin.
Methods: Inhibition of human fXa or thrombin (IIa) by ATIII in the presence of heparin was studied in an enzymatic assay in buffered solution. The reaction mixture contained fXa or IIa (20 nM), ATIII (200 nM), different amounts of unfractionated heparin and AnXa. 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). Residual fXa or IIa activity was measured in a plate reader by monitoring the initial rate of substrate cleavage at 405 nm. 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. Reversal of heparin-induced clotting prolongation of human plasma was measured by a 96-well format turbidity assay using an aPTT reagent and Ca2+. 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 was compared to enoxaparin. Protamine sulfate was included as a control. Di-arginine piperazine (PER977), a positively charged small molecule, was also tested for comparison.
Results: AnXa dose-dependently reversed the anti-fXa activity of heparin in the enzymatic assay and in human plasma. Interestingly, AnXa was also able to reverse the heparin anti-IIa activity in the same assays. In the enzymatic assay with IIa and ATIII, heparin accelerated the reaction of IIa inhibition by ~1000-fold. AnXa dose-dependently reversed the IIa inhibition to its basal level seen without heparin. The reversal effect of AnXa on IIa inhibition could be blocked by addition of a small molecule direct fXa inhibitor (e.g. rivaroxaban) in the same enzymatic assay. 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. In human plasma, AnXa reversed the anti-IIa activity of heparin (0.8 IU/mL) with an estimated EC50 comparable to anti-fXa reversal (anti-fXa:126 nM; anti-IIa: 131nM). 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 addition, AnXa dose-dependently and completely reversed prolongation of clotting by heparin (0.8 IU/mL) in human plasma as measured by the turbidity assay as well as parameters of heparin (1.0 IU/mL) anticoagulation as measured by TEG in either plasma or whole blood. As expected, protamine sulfate (5-10 µg/mL) neutralized heparin (1.0 IU/mL) anticoagulation in plasma in the TEG assay. At higher concentrations, protamine prevented clot formation, consistent with protamine's known anticoagulant effect when overdosed. No reversal activity was observed by PER977 in the same TEG assays using either plasma or whole blood.
Conclusions: The current and previously reported data demonstrate that AnXa could be a universal fXa inhibitor antidote to reverse a broad range of newer direct oral fXa inhibitors, ATIII-dependent indirect inhibitors, such as fondaparinux and enoxaparin, as well as unfractionated heparin. The mechanism for reversal of ATIII-dependent inhibitors by AnXa is distinct from other positively charged molecules that bind directly to heparin.
Lu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:Sea Lane Biotechnologies: Consultancy; 3-V Biosciences: Equity Ownership; Portola Pharmaceuticals, Inc.: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment.
Asterisk with author names denotes non-ASH members.