Abstract
Individuals anticoagulated with warfarin or heparin are typically treated with specific antidotes such as vitamin K or protamine, respectively, if they bleed or require surgery. In contrast, specific and effective antidotes are not available for the reversal of the anticoagulant effects of the low molecular weight heparins (LMWH) or the new oral anticoagulants targeting factor Xa (fXa) which are predictably only marginally affected by standard treatments using rfVIIa or fresh frozen plasma. The present study was designed to test the hypothesis that plasma-derived or recombinant fXa, modified to lack catalytic and membrane binding activities, could neutralize the anticoagulant activities of small molecule fXa inhibitors and LMWH. Plasma derived antidote (pd-Antidote) was prepared by chemical modification of the active site serine of fXa followed by chymotryptic removal of the Gla domain. Preliminary experiments showed that pd-Antidote dose dependently reversed the activity of rivaroxaban, apixaban or betrixaban, three small molecule fXa inhibitors currently in clinical trials. In a fXa amidolytic assay with 3nM enzyme, half maximal reversal of inhibitory activities (EC50) was attained at the following pd-Antidote concentrations: rivaroxaban =49 nM, apixaban = 122 nM, betrixaban = 41 nM. The pd-Antidote had no effect on the activity of fXa in the absence of inhibitors. Thus, active site inactivated pd-Antidote retained the ability to bind small molecule inhibitors of fXa. The activity of pd-Antidote was not limited to reversal of purified fXa catalytic activity. In a tissue factor-initiated thrombin generation assay in plasma, while pd-Antidote did not interfere with the normal function of prothrombinase complexes, it dose-dependently and completely reversed the inhibition produced by the small molecule fXa inhibitors. Pd-Antidote also demonstrated reversal of the in-vitro anticoagulant activity of the LMWH, enoxaparin. Addition of pd-Antidote (515nM) produced a 37% reduction of the clotting activity of enoxaparin (1.25Units/ml). In an activated partial thromboplastin time assay (aPTT), pd-Antidote also dose-dependently reversed the inhibitory effects of the fXa inhibitors. For example, aPTT prolongation by betrixaban (400nM) was reversed with an estimated EC50=650nM. Baseline aPTT was not altered upon addition of pd-Antidote up to a concentration of 2.5 μM, the highest concentration studied. Recombinant antidote (r-Antidote) was expressed in mammalian cells (Chinese hamster ovary) using a construct for human fXa with the S195A mutation and lacking the Gla-domain. The EC50’s for purified r-Antidote in reversing 7.5 nM fXa inhibitors in the fXa catalytic activity assay were: rivaroxaban =17 nM, apixaban = 45 nM, betrixaban = 15 nM. As expected, there was no inhibition of fXa activity by r-Antidote alone. Purified r-Antidote also reversed anticoagulant activity in plasma clotting assays. Prothrombin time (PT) extensions achieved by supratherapeutic concentrations of rivaroxaban (1 μM) or apixaban (1 μM) were completely normalized upon addition of r-Antidote (1.5 μM). Ex vivo clotting prolongation by an excess of betrixaban (300 nM) was essentially reversed (88% correction) by pre-incubation with r-Antidote (570 nM) prior to addition of aPTT reagents. The concentration of r-Antidote required for corrective activity was related to the inhibitory potency of the oral fXa inhibitors. PT or aPTT baselines did not change upon addition of r-Antidote alone at 1.9 μM, the highest concentration tested. We also examined the ability of both pd-Antidote and r-Antidote to alleviate inhibitory activities produced upon oral dosing of fXa inhibitors in mice. Perturbation of whole blood PT/INR in dosed animals was followed as a marker of anticoagulation. The effect following dosing of a fXa inhibitor could be reversed by a single intravenous injection of antidote. INR measurements in blood samples of treated animals showed a >50% reduction of observed inhibitory activity compared to control animals. Our results suggest that these plasma derived or recombinant proteins have the potential to act as universal antidotes for reversal of anticoagulation of all current fXa inhibitors, both small molecule and antithrombin dependent, in patients with bleeding related medical emergencies or those requiring cessation of anticoagulation prior to surgery.
The role of endothelium in factor Xa regulation: the effect of plasma proteinase inhibitors and hirudin