Taketoshi Furugohri

Impact of antithrombin deficiency on efficacy of edoxaban and antithrombin-dependent anticoagulants, fondaparinux, enoxaparin, and heparin

INTRODUCTION Oral factor Xa (FXa) inhibitors are a novel class of anticoagulants that, unlike heparins, are expected to demonstrate antithrombotic effects independent of plasma antithrombin (AT) concentrations. We utilized heterozygous AT-deficient (AT+/-) mice to determine the impact of AT deficiency on anticoagulant and antithrombotic effects of edoxaban, a direct FXa inhibitor, and compared with heparins (fondaparinux, enoxaparin, and unfractionated heparin [UHF]). MATERIALS AND METHODS The effects of edoxaban and heparins on in vitro prothrombin time and activated partial thromboplastin time were measured in plasma obtained from wild type (AT+/+) and AT+/- male mice. To assess the antithrombotic effects of these anticoagulants in vivo, venous thrombosis was induced in the inferior vena cava by FeCl3 treatment. Potency ratios of antithrombotic effects in AT+/- compared with AT+/+ mice were analyzed by a parallel line assay. RESULTS In vitro studies demonstrated that the clotting-time prolongation effects of edoxaban were not affected by heterozygous AT deficiency whereas those of AT-dependent anticoagulants were attenuated. In AT+/- mice, the antithrombotic effects of AT-dependent anticoagulants were less potent than those in AT+/+ mice. In contrast, edoxaban was equipotent in preventing thrombus formation in both wild-type and AT-deficient mice. The attenuated antithrombotic effects of fondaparinux, enoxaparin, and UFH in AT-deficient mice were restored by AT supplementation. Edoxaban exerts a comparable antithrombotic effect even in mice with low plasma AT antigen and activity to that in wild-type mice. CONCLUSION Edoxaban may potentially be prioritized over AT-dependent anticoagulants in patients with lower plasma AT concentration.

The effects of warfarin and edoxaban, an oral direct factor Xa inhibitor, on gammacarboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats

INTRODUCTION Osteocalcin plays a role in bone homeostasis. The vitamin K cycle is essential for the gamma-carboxylation of glutamic acid residues in osteocalcin. Some evidence suggests that long-term warfarin therapy, which inhibits the vitamin K cycle and prevents gamma-carboxylation, is associated with increased bone-fracture risk. The aim of this study was to determine the effects of warfarin and edoxaban, a direct factor Xa inhibitor, on the serum concentration of total, gamma-carboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats. MATERIALS AND METHODS Rats received orally administered warfarin or edoxaban, and 24h later serum and plasma were prepared. Osteocalcin level in serum was measured with ELISA. A Gla-osteocalcin was precipitated by the addition of hydroxyapatite, and the resulting supernatant was used for measuring uc-osteocalcin. Prothrombin time (PT) of plasma was also measured. RESULTS Warfarin at 1mg/kg (a dose which prolonged PT 2.62-fold) markedly increased the serum level of uc-osteocalcin and slightly increased the total osteocalcin level compared with control in rats. Serum Gla-osteocalcin significantly decreased by warfarin. Edoxaban at 1mg/kg (an antithrombotic dose) and 54mg/kg (a dose which prolonged PT 2.25-fold) had no effects on total, uc-, and Gla-osteocalcin levels. CONCLUSIONS This study demonstrates that warfarin impaired the carboxylation of osteocalcin in rats. In contrast, edoxaban at or higher doses than needed for an antithrombotic effect sustained the circulating Gla-osteocalcin level. These findings suggest that edoxaban has no effects on the production of Gla-osteocalcin and thus, may have a lower risk of adverse effects on bone health.

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin-antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin-antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin-antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15-234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation.

A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression.

Paradoxical enhancement of the intrinsic pathway-induced thrombin generation in human plasma by melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, or heparin

INTRODUCTION The blood coagulation cascade consists of two pathways, the tissue factor (TF)-dependent extrinsic pathway and the contact factor-dependent intrinsic pathway. We have previously shown that a direct thrombin inhibitor, melagatran, paradoxically increased TF-induced thrombin generation (TG) in thrombomodulin (TM)-containing human plasma in vitro. However, the effect of melagatran on the intrinsic pathway-induced TG remains to be investigated. We investigated whether melagatran enhances the intrinsic pathway-induced TG. METHODS AND RESULTS TG was induced by kaolin in human plasma and assayed by the calibrated automated thrombography method. Melagatran at 150 and 300 nM significantly increased the peak level (2.40-fold) and endogenous thrombin potential of TG in normal plasma in the presence of 5 nM TM. In the absence of TM or in protein C (PC)-deficient plasma, the paradoxical enhancement of TG by melagatran disappeared. A direct FXa inhibitor, edoxaban, and an antithrombin-dependent anticoagulant, unfractionated heparin (UFH), did not increase, but simply decreased TG under each condition in a concentration dependent manner. CONCLUSION Melagatran enhanced the intrinsic pathway-induced TG as well as the extrinsic pathway-induced TG in human plasma under the condition where PC system is active. In contrast, edoxaban and UFH showed concentration-dependent decrease of TG, but no enhancement. These results indicated that edoxaban and UFH may have a low risk of the paradoxical enhancement of TG by both the extrinsic and intrinsic pathway activation.

Tranexamic Acid Failed to Reverse the Anticoagulant Effect and Bleeding by an Oral Direct Factor Xa Inhibitor Edoxaban

Background/Aims: Agents to reverse the anticoagulant effect of edoxaban, an oral direct factor Xa inhibitor, would be desirable in emergency situations. The aim of this study is to determine the effect of tranexamic acid, an antifibrinolytic agent, on the anticoagulant activity and bleeding by edoxaban in rats. Methods: A supratherapeutic dose of edoxaban (3 mg/kg) was intravenously administered to rats. Three minutes after dosing, tranexamic acid (100 mg/kg) was given intravenously. Bleeding was induced by making an incision with a blade on the planta 8 min after edoxaban injection and bleeding time was measured. Prothrombin time (PT) and clot lysis were examined. Results: A supratherapeutic dose of edoxaban significantly prolonged PT and bleeding time. Tranexamic acid did not affect PT or bleeding time prolonged by edoxaban, although tranexamic acid significantly inhibited clot lysis in rat plasma. Conclusion: An antifibrinolytic agent tranexamic acid failed to reverse the anticoagulant effect and bleeding by edoxaban in rats.

Role of bradykinin-no pathway in prevention of cardiac hypertrophy by ace inhibitor

Ishigai, Yutaka, Tomohiro Mori, Toshiko Ikeda,Akiko Fukuzawa, and Toshiro Shibano. Role of bradykinin-NO pathway in prevention of cardiac hypertrophy by ACE inhibitor in rat cardiomyocytes. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H2659–H2663, 1997.—To examine whether the bradykinin-nitric oxide (NO) pathway directly participates in the antihypertrophic property of angiotensin-converting enzyme (ACE) inhibitors in congestive heart failure, the effects of bradykinin were studied in rat cultured heart cells. Bradykinin (0.1, 1 nM) prevented the phenylephrine-induced increase in protein/DNA content, an index of hypertrophy of heart cells, and amplified the nitrite/nitrate content in the medium. Perindoprilat (1 μM), an ACE inhibitor, also restrained the progression of cardiac hypertrophy and augmented NO release. These effects of perindoprilat were abolished by HOE-140 (kinin B2 antagonist), Nv-nitro-Larginine (NO synthase inhibitor), and methylene blue (guanylate cyclase inhibitor). Furthermore, there was a significant correlation between protein/DNA content and nitrite/nitrate content. These results indicate that bradykinin inhibits the progression of cardiac hypertrophy due to the increase in NO release and that perindoprilat produces beneficial effects on cardiac hypertrophy by stimulating the bradykinin-NO pathway.