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The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.

Rivaroxaban (BAY 59-7939) is a novel, oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The antithrombotic activity of rivaroxaban has been demonstrated in several venous and arterial thrombosis models in rats, at doses that did not increase bleeding times. However, as with all anticoagulants, high doses of rivaroxaban cause prolongation of bleeding time. The aim of this study was to assess the ability of recombinant Factor VIIa (rFVIIa, NovoSeven®) to neutralize the anticoagulant effects of high-dose rivaroxaban. Animals were divided into four study groups receiving the following treatment and the appropriate placebo: rivaroxaban 2 mg/kg, rivaroxaban 2 mg/kg plus rFVIIa 100 or 400 μg/kg i.v, and rFVIIa 400 μg/kg i.v. Anesthetized rats were pretreated with rivaroxaban (or placebo); rFVIIa (or placebo) was then injected intravenously 1 minute after induction of bleeding. Mesenteric bleeding times were measured after cutting small branched arteries using microsurgery scissors, while the intestinal surface was superfused with 0.9% NaCl solution. Bleeding times were obtained from three control vessel incisions before treatment, and one after intravenous administration of rivaroxaban and/or rFVIIa (n=10). Prothrombin time (PT), thrombin generation (TG), and inhibition of FXa activity (activated by Russell’s viper venom) were measured in blood from animals that had received study medication (rivaroxaban 2 mg/kg and/or rFVIIa 400 μg/kg) or placebo, but had not been used for bleeding time determination (n=6). Administration of rFVIIa (400 μg/kg) reduced mesenteric bleeding time to 0.7-times baseline (Table). High-dose rivaroxaban increased bleeding time to 3.3-times baseline. The administration of rFVIIa to animals which had received rivaroxaban significantly reduced the bleeding times to 2.4- and 1.7-times baseline at doses of 100 μg/kg and 400 μg/kg, respectively. Administration of rFVIIa (400 μg/kg) reduced PT and the lag time of TG, but had no effect on endogenous thrombin potential (ETP: total amount of thrombin activity) in control animals. Rivaroxaban-induced prolongation of PT and lag time of TG were partially reversed by rFVIIa, as was the reduction in ETP. Rivaroxaban-induced inhibition of FXa activity was not affected by rFVIIa. These results demonstrate that rFVIIa partially reverses the anticoagulation effect (bleeding time, prolongation of PT, ETP, and lag time of TG) of rivaroxaban without affecting inhibition of FXa activity. Thus, these data indicate that rFVIIa has the potential to be used as an antidote to the oral, direct FXa inhibitor, rivaroxaban, if this was necessary. | Group | Change in bleeding time (x-fold) | PT (sec) | Thrombin generation (ETP) (nM x min) | Thrombin generation lag time (min) | Inhibition of FXa activity (%) | |:--------------------------------------:| -------------------------------- | -------- | ------------------------------------ | ---------------------------------- | ------------------------------ | | ETP: endogenous thrombin potential | | Placebo | 1 (baseline) | 15±0.4 | 516±16 | 1.2 ±0.04 | 0±3 | | rFVIIa 400 μg/kg | 0.7±0.1 | 7±0.2 | 507±19 | 0.7±0.01 | 1±4 | | Rivaroxaban 2 mg/kg | 3.3±0.5 | 97±5.6 | 363±14 | 2.4±0.2 | 77±4 | | Rivaroxaban 2 mg/kg + rFVIIa 400 μg/kg | 1.7±0.2 | 54±3.5 | 428±13 | 1.9±0.1 | 81±2