Search

Previously, we reported that a direct thrombin inhibitor melagatran paradoxically increased thrombin generation in human plasma in the presence of thrombomodulin. The aim of this study is to test the hypothesis that melagatran may exert a deleterious effect on tissue-type plasminogen activator (t-PA)-induced fibrinolysis via enhancement of thrombin generation and subsequent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and factor XIII (FXIII). Clot formation in human plasma containing t-PA and thrombomodulin was induced by tissue factor. The absorbance at 405 nm was measured to obtain clot lysis time. Effects of melagatran and a factor Xa inhibitor edoxaban on clot lysis time were determined. In the presence of thrombomodulin, melagatran significantly prolonged clot lysis time, but edoxaban shortened it. In the absence of thrombomodulin, melagatran did not inhibit fibrinolysis. Prolongation of clot lysis time by melagatran was reversed by activated protein C (which suppressed thrombin generation increased by melagatran) and a TAFIa inhibitor. Melagatran significantly suppressed plasmin generation, while edoxaban significantly increased it. However, both melagatran and edoxaban suppressed FXIII activation. In the clot formed in the presence of melagatran and edoxaban, the fibrin fibre was thin compared with control, showing no clear difference in the clot structures between melagatran and edoxaban. These results indicated that melagatran, not edoxaban, prolonged clot lysis time through the paradoxical enhancement of thrombin generation, and subsequent TAFI activation and inhibition of plasmin generation. Neither FXIII activation nor change in fibrin clot structure contributed to the inhibition of fibrinolysis by melagatran.

Fibrinolysis is regulated by the thrombin/thrombin-activatable fibrinolysis inhibitor (TAFI) system. Thus, anticoagulants and inhibitors of TAFI are expected to accelerate fibrinolysis. The combined effects of an anticoagulant and a TAFIa inhibitor on fibrinolysis remain unknown. The aim of this study was to evaluate the combined effect of edoxaban, an oral direct factor Xa (FXa) inhibitor, and a TAFIa inhibitor, potato tuber carboxypeptidase inhibitor (PCI) on tissue-type plasminogen activator (t-PA)-induced clot lysis in human plasma in vitro. Pooled human plasma (containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban and/or PCI. Clot formation was induced by 2.5 pM tissue factor and 4 µM phospholipids and clot lysis time was examined. Plasma plasmin-α2 antiplasmin complex (PAP) concentration was measured as a marker of plasmin generation. Edoxaban or PCI alone significantly shortened the t-PA-induced clot lysis time and plasma PAP concentration. The combination of these compounds significantly accelerated the clot lysis compared with the inhibitors alone. Addition of PCI (0.3, 1, and 3 μg/mL) to 75 ng/mL edoxaban increased plasma PAP concentration compared with edoxaban alone; however, compared with PCI alone only the combination of 0.3 μg/mL PCI + 75 ng/mL edoxaban showed the significant increase in PAP concentration. Concomitant use of an oral direct FXa inhibitor, edoxaban, and a TAFIa inhibitor, PCI, significantly accelerate fibrinolysis via enhancement of plasmin generation. These results suggest that the combination of edoxaban and a TAFIa inhibitor might be beneficial for the treatment of thromboembolic diseases.

Juvenile myelomonocytic leukemia (JMML) is a pediatric hematological malignancy with a poor prognosis. Although several case series have been published describing hematological and molecular responses to azacitidine (AZA) treatment in patients with JMML, the efficacy and safety profile of AZA is not well investigated, especially in Asian children and children undergoing hematopoietic stem cell transplantation (HSCT). We retrospectively analyzed 5 patients who received a total of 12 cycles (median 2 cycles) of AZA treatment in Japan. All five patients were boys and their ages at the time of treatment were 21, 23, 24, 26, and 46 months, respectively. All five patients tolerated AZA treatment, including four patients who received AZA after HSCT. Therapeutic toxicity with AZA was mostly limited to hematological toxicity. The only serious non-hematological adverse event was hyperbilirubinemia (grades III-IV) observed in a patient who received AZA after a second HSCT. Two out of five patients treated with AZA achieved a partial response (PR), while three patients treated for post-transplant relapse did not have an objective response. Future prospective studies should be conducted to develop combination therapies with AZA and other molecular targeted drugs for high-risk patients.

Background Anticoagulation therapy, particularly subcutaneous heparin therapy, is recommended for cancer-associated thrombosis. However, not starting or discontinuing anticoagulation was not rare. The aim of the present study was to examine the practical issues related to anticoagulation therapy and effects of subcutaneous heparin therapy for cancer-associated stroke. Methods Patients with cancer-associated stroke in our stroke center between October 2014 and August 2017 who were diagnosed as having acute ischemic stroke based on diffusion-weighted imaging were retrospectively enrolled. Baseline clinical characteristics, heparin injection, reasons for no subcutaneous heparin therapy, and clinical outcomes were collected. Results A total of 59 patients with cancer-associated stroke (75 ± 10 years old, male 42%) were enrolled. Lung cancer was the most frequently observed cancer (n = 17, 29%), followed by gastric cancer (n = 8, 14%) and pancreatic cancer (n = 8, 14%). Of the 19 patients (32%) who underwent subcutaneous heparin therapy, it was discontinued in 9 (47%), mainly because of patients’ medical conditions (deterioration of cancer or hemorrhagic complication). Ten patients with long-term subcutaneous heparin therapy did not have stroke recurrence. In contrast, among nine patients who discontinued subcutaneous heparin therapy, three (33%) had recurrence of ischemic stroke. Of the 40 patients without subcutaneous heparin therapy, the main reasons for no subcutaneous heparin therapy were the patients’ medical conditions (n = 22, 55%). Conclusions Although subcutaneous heparin therapy was given to only one third of cancer-associated stroke patients, long-term subcutaneous heparin therapy might prevent recurrence of cancer-associated stroke.

Background/Aims: Stent thrombosis is a serious complication after percutaneous coronary intervention and femoropopliteal endovascular intervention. The aim of this study was to determine the antithrombotic effects of a direct factor Xa inhibitor edoxaban alone or in combination with antiplatelet agents in a rat model of stent thrombosis. Methods: Stainless steel stents (4 stents per rat) were placed in an extracorporeal arteriovenous shunt. The shunt was inserted into the carotid artery and the jugular vein in each rat to circulate blood. Stent thrombosis was induced by exposing the stents to arterial blood for 30 min. Protein content of the thrombus was measured. Edoxaban and antiplatelet agents (aspirin, clopidogrel, and ticagrelor) were orally administered before the thrombus induction. Results: Edoxaban (0.3–3 mg/kg), clopidogrel (1–10 mg/kg), aspirin (10–100 mg/kg), and ticagrelor (0.3–3 mg/kg) exerted significant and dose-dependent antithrombotic effects in a rat stent thrombosis model. The effect of edoxaban was comparable to that of antiplatelet agents. The combination of submaximal doses of edoxaban and clopidogrel or aspirin significantly potentiated the antithrombotic effects compared with antiplatelet agents alone. Conclusion: These results suggest that edoxaban alone and in combination with clopidogrel or aspirin are promising antithrombotic therapies for the prevention of stent thrombosis.

We report a pediatric case aged 10 years with Granulicatella adiacens-associated chronic mandibular osteomyelitis. The causative pathogen was uncertain because polymicrobial species were detected from the bacterial culture in bone marrow fluid. In contrast, G. adiacens was predominantly identified in the clone library analysis of the bacterial 16S rRNA gene sequence. Vancomycin to which G. adiacens was reported to be susceptible was not administrated sufficiently to this patient because of its adverse event, whereas linezolid and ciprofloxacin was alternatively effective for the treatment of chronic mandibular osteomyelitis.

Here we report a case of a 12-year-old girl who was referred to our department because of marked short stature of more than -5 SD below the median. Although her growth failure began suddenly at 6 years of age, she never had an examination because she had no other symptoms. Brain MRI examination suggested a tumor in the suprasellar region, and endocrine examination revealed combined pituitery hormone deficiency due to the tumor. Before surgery, the supplementation with hydrocortisone and levothyroxine was initiated. The pathological diagnosis of the surgically removed tumor was xanthogranuloma. The pattern of her growth curve showed a growth failure with sudden onset, which is a typical pattern of short stature secondary to pituitary disfunction including growth hormone deficiency associated with brain tumors. This case suggests that growth failure could be the only symptom in pediatric cases with brain tumors. Improved awareness regarding the association of growth failure with brain tumors is needed for earlier diagnosis and treatment. Furthermore, the growth curves should be carefully evaluated in regular health examinations at school.

A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.

In addition to platelet aggregation, coagulation activation is considered to be involved in arterial thrombosis. In this study, we determined antithrombotic effects of edoxaban, an oral factor Xa (FXa) inhibitor, as both a monotherapy and in combination with antiplatelet agents in a rat model of arterial thrombosis. We further examined its effects on a procoagulant biomarker and bleeding. Arterial thrombosis was induced by topical application of 15% ferric chloride to rat abdominal aortas. Bleeding time was measured by a tail incision method. Edoxaban, clopidogrel, and aspirin were orally administered 30min, 4h, and 2h before thrombus or bleeding induction. As a biomarker of coagulation activation, plasma thrombin-antithrombin complex (TAT) was measured. Edoxaban dose-dependently prevented arterial thrombosis in a manner comparable to clopidogrel and aspirin. The combination of edoxaban plus clopidogrel or edoxaban plus aspirin significantly potentiated the antithrombotic effects compared with these drugs alone. The combination of edoxaban and clopidogrel was more potent than clopidogrel and aspirin. Plasma TAT concentration was elevated after thrombus induction and suppressed by edoxaban and clopidogrel, but not by aspirin, suggesting P2Y12 receptor-mediated platelet procoagulant activity. Bleeding time was prolonged by the coadministration of edoxaban and clopidogrel, but not by edoxaban and aspirin. In conclusion, the present study demonstrates that the monotherapy with edoxaban and combination therapy with edoxaban plus clopidogrel or edoxaban plus aspirin are promising options for the prevention of arterial thrombosis as effective as the standard antiplatelet agents; however, a combination of edoxaban and clopidogrel increased the risk of bleeding.

Acquired hemophilia A (AHA), a bleeding disorder caused by autoantibodies against FVIII, has the potential for life-threatening bleeding. The annual onset rate is said to be one in 4 million people, but diagnosis examples increase in adults because a disorder concept penetrated. AHA is quite rare in children, with an incidence rate of 0.045 per 1 million, but early detection is crucial because serious bleeding can happen, as in adults. We report a pediatric case who received an early diagnosis of AHA by an activated partial thromboplastin time (APTT) cross-mixing test. The 12-year-old girl had neither a past history nor a family history of bleeding episodes. She presented with intramuscular bleeding and epistaxis without trauma or medication. At diagnosis, her blood test showed prolonged APTT. Other hemostatic tests, such as the platelet count, prothrombin time and fibrinogen concentration, were within the normal range. We administered an APTT cross-mixing test that detected an inhibitor pattern and inhibitory antibodies against factors VIII. As a result, we administered prednisolone and the inhibitor disappeared after 1.5 months. In conclusion, AHA is a bleeding disorder which should be considered even in children due to the potential for life-threatening bleeding. Furthermore, the APTT cross-mixing test is useful for screening coagulation factor deficiencies and inhibitors.

Vascular injury activates the coagulation cascade. Some studies report that coagulation factor Xa and thrombin are implicated in proliferation of vascular smooth muscle cells and neointimal hyperplasia after vascular injury. The aim of this study was to determine the effect of an oral direct factor Xa inhibitor, edoxaban, on neointimal hyperplasia following the carotid artery injury in apolipoprotein E (ApoE)-deficient mice. Vascular injury was induced by the application of 10% ferric chloride to the carotid artery for 3 min in ApoE-deficient mice. After vascular injury, all animals were fed with high-cholesterol chow for 6 weeks. Edoxaban at 15 mg/kg was orally administered to the mice 1 h before (n = 10) or 1 h after (n = 9) ferric chloride injury, and thereafter 10 mg/kg edoxaban was orally administered b.i.d. for 6 weeks. Thrombus formation and neointimal hyperplasia were evaluated. Treatment with 15 mg/kg edoxaban before vascular injury almost completely inhibited thrombus formation, and following chronic administration of edoxaban significantly suppressed neointimal hyperplasia. In the mice treated with edoxaban after vascular injury, there was wide interindividual variability. In some mice (four out of nine) the neointimal hyperplasia was inhibited like in edoxaban-pretreated mice, but there was no statistical difference compared with control. This study demonstrated that inhibition of the coagulation and thrombosis by edoxaban ameliorated neointimal hyperplasia caused by vascular injury and high-cholesterol diets in ApoE-deficient mice. This suggests that factor Xa has a crucial role in the formation of neointima following vascular injury.The abstract should be followed by 3-4 bullet points that highlight major findings. The final bullet point should emphasize future directions for research.

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.

Edoxaban is an oral and direct activated factor X inhibitor. In this study, the acute treatment effect of edoxaban on venous thrombosis is investigated in rats by single and multiple administrations, and compared to the conventional parenteral anticoagulants, enoxaparin and fondaparinux. Venous thrombus was induced in the inferior vena cava by partial stenosis plus topical application of 10% ferric chloride for 5min. After 1-h thrombus maturation, oral edoxaban and subcutaneous enoxaparin and fondaparinux were given. In the single administration experiment, thrombus weight was measured 1 or 4h after thrombus induction. In the multiple administration experiments, edoxaban was orally administered once daily (QD) and twice daily (BID) for 3 days. In the single administration experiment, oral administration of edoxaban (3.0 and 10mg/kg) 1h after thrombus formation significantly regressed the venous thrombus compared to the thrombus at 1h after thrombus formation. Similarly the significant venous thrombus regression was observed with enoxaparin (10mg/kg) and fondaparinux (0.30-3.0mg/kg). In the multiple administration experiment, both QD and BID administration of edoxaban at daily doses of 5 and 10mg/kg exerted significant treatment effects. QD administration of edoxaban including lower doses (1-10mg/kg) significantly reduced thrombus weight. Edoxaban administered QD and BID was effective in the treatment of venous thrombosis, and the treatment effect of edoxaban was comparable to the conventional parenteral anticoagulants. These data demonstrate the potential of edoxaban as an oral anticoagulant in the acute treatment of venous thromboembolism.

In this study, oxytocin receptors (OTRs) in the islets of Langerhans were detected using real-time RT-PCR and immunohistochemical technique. Indeed, OTR mRNA was expressed in the rat pancreas. Double immunohistochemical staining for OTR and either glucagon or insulin demonstrated their co-localization in A-cells or B-cells, respectively. OTR-immunoreactivity in A-cells was stronger than that of B-cells. All A-cells and 94.8% of B-cells were OTR-immunoreactive. We reveal the statistically significant relations of OTR with A-cells and B-cells in the islets of Langerhans. This is the first demonstration of the OTR localization in the islets of Langerhans immunohistochemically. It suggests that oxytocin (OT) is involved in the release of insulin and glucagon.

SummaryEdoxaban, an oral, direct factor Xa inhibitor, has a similar or low incidence of bleeding events compared with other anticoagulants in clinical trials. Therefore, agents to reverse the anticoagulant effects of edoxaban could be desirable in emergency situations. In this study, the reversal effects of haemostatic agents were determined on prothrombin time (PT) prolongation in vitro and bleeding time prolongation in vivo by edoxaban. PT using human plasma was measured in the presence of edoxaban at therapeutic and excess concentrations with the haemostatic agents, prothrombin complex concentrate (PPSB-HT), activated prothrombin complex concentrate (Feiba), and recombinant factor VIIa (rFVIIa). In rats, rFVIIa and Feiba was given during intensive anticoagulation with edoxaban. The haemostatic effect was evaluated in a model of planta template bleeding and a potential prothrombotic effect was evaluated in a venous thrombosis model. PPSB-HT, Feiba, and rFVIIa concentration-dependently shortened PT prolonged by edoxaban. Among these, rFVIIa and Feiba showed potent activities in reversing the PT prolongation by edoxaban. rFVIIa (1 and 3 mg/kg, i.v.) and Feiba (100 U/kg, i.v.) significantly reversed edoxaban (1 mg/kg/h)-induced prolongation of bleeding time in rats. In a rat venous thrombosis model, no potentiation of thrombus formation was observed when the highest dose (3 mg/kg) of rFVIIa was added to edoxaban (0.3 and 1 mg/kg/h) compared with the control. The present study indicated that rFVIIa, Feiba, and PPSB-HT have the potential to be reversal agents for edoxaban.

SummaryEdoxaban is an oral, direct factor Xa (FXa) inhibitor under late-phase clinical development. This study compared the antithrombotic efficacy of edoxaban with that of an indirect FXa inhibitor, fondaparinux, in in vivo venous and arterial thrombosis models and in ex vivo perfusion chamber thrombosis model under low and high shear rates in rats. Venous and arterial thrombi were induced by platinum wire insertion into the inferior vena cava and by application of FeCl3 to the carotid artery, respectively. The perfusion chamber thrombus was formed by blood perfusion into a collagen-coated capillary at 150 s-1 (low shear rate) and 1,600 s-1 (high shear rate). Effective doses of edoxaban that reduced thrombus formation by 50% (ED50) in venous and arterial thrombosis models were 0.076 and 0.093 mg/kg/h, respectively. In contrast, ED50 of fondaparinux in the arterial thrombosis model (>10 mg/kg/h) was markedly higher compared to ED50 in the venous thrombosis model (0.021 mg/kg/h). In the perfusion chamber thrombosis model, the ratio of ED50 under high shear rate (1.13 mg/kg/h) to that under low shear rate (0.63 mg/kg/h) for edoxaban was 1.9, whereas that for fondaparinux was more than 66. While the efficacy of fondaparinux markedly decreased in arterial thrombosis and in a high-shear state, edoxaban exerted consistent antithrombotic effects regardless of flow conditions. These results suggest that shear rate is a key factor in different antithrombotic effects between edoxaban and fondaparinux.

Introduction: Direct oral anticoagulants are as effective as vitamin K antagonists for the treatment of venous thromboembolism and are associated with less bleeding. We have reported that a direct oral factor Xa inhibitor edoxaban exerts a thrombus resolution effect in a rat model of venous thrombosis and enhances tissue plasminogen activator (t-PA)-induced clot lysis in human plasma. However, the mechanism underlying the thrombus resolution effect and fibrinolysis enhancement by edoxaban remains to be determined. Purposes: To evaluate the effect of edoxaban on plasmin generation during clot lysis in human plasma in vitro. To determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in the enhancement of fibrinolysis by edoxaban. Methods: Pooled human normal plasma or TAFI-deficient plasma (both containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban, an activated TAFI (TAFIa) inhibitor (a fibrinolysis enhancer) potato tuber carboxypeptidase inhibitor (PCI), or vehicle. Clot was induced by adding 2.5 pM tissue factor and 4 µM phospholipids. To monitor the clot formation and lysis, the absorbance of plasma at 405 nm was measured every 30 sec. Clot lysis time was defined as the interval between the time of the midpoint of the clear to maximum turbidity transition and the midpoint of the maximum turbidity to clear transition. Plasmin-α2 antiplasmin complex (PAP) concentration was measured by ELISA as an indicator of plasmin generation. Results: In normal plasma, PCI accelerated clot lysis and increased plasma PAP concentration. There was a correlation between plasma PAP concentration and percent of clot lysis, indicating that plasma PAP concentration is an appropriate marker of fibrinolysis. Edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) significantly shortened clot lysis time and elevated plasma PAP concentration. The additive combined effect of edoxaban and PCI was observed. In TAFI-deficient plasma, clot lysis time was shortened and plasma PAP concentration increased compared with normal plasma. In these samples, the effects of edoxaban and PCI on clot lysis and plasma PAP concentration were markedly diminished as compared with normal plasma. Conclusions: Edoxaban at clinically relevant concentrations enhanced t-PA-induced clot lysis with increasing plasma PAP concentration in human plasma. The effects of edoxaban on clot lysis and plasmin generation were diminished in TAFI-deficient plasma. A TAFIa inhibitor PCI exerted similar effects. These data suggest that edoxaban enhanced fibrinolysis via inhibition of TAFI activation and enhancement of plasmin generation. Disclosures Morishima: Daiichi Sankyo Co., Ltd.: Employment. Honda:Daiichi Sankyo Co., Ltd.: Employment. Furugohri:Daiichi Sankyo Co., Ltd.: Employment.

Introduction Tissue factor-induced platelet aggregation and factor Xa (FXa) activity bound to clot contribute to the formation and growth of thrombus. The effects of edoxaban, a direct FXa inhibitor, on these responses were determined and compared with that of fondaparinux, an antithrombin-dependent (indirect) FXa inhibitor. Material and methods Human platelet aggregation was induced by human tissue factor (Dade Innovin or RecombiPlasTin) in platelet-rich plasma spiked with edoxaban or fondaparinux. Clot formed from human whole blood was incubated with 0.9 μM prothrombin in the absence or presence of FXa inhibitors. As the index of FXa activity, the amount of prothrombin fragment F1 + 2 was measured with an ELISA. Free FXa activity was measured using human FXa and its chromogenic substrate S-2222. Results Edoxaban inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner with the IC50 values of 150 and 110 nM for Dade Innovin and RecombiPlasTin-induced platelet aggregation, respectively. At 1 μM, edoxaban completely inhibited the aggregation. Fondaparinux inhibited RecombiPlasTin-induced aggregation with the IC50 of 9.3 μM, but did not show complete inhibition up to 30 μM and had no effect on Dade Innovin-induced aggregation. Edoxaban inhibited both free and clot-bound FXa with the IC50 of 2.3 and 8.2 nM, respectively. Fondaparinux inhibited free FXa (IC50 5.4 nM), but 40-times higher concentration were required to inhibit clot-bound FXa (IC50 217 nM). Conclusions Edoxaban, a direct FXa inhibitor, was a more potent inhibitor of tissue factor-induced platelet aggregation and clot-bound FXa than fondaparinux, an indirect FXa inhibitor.

Introduction Patients with atrial fibrillation undergoing percutaneous coronary intervention may require combination therapy with anticoagulants and antiplatelet agents. The objectives of this study were to establish an assay which can evaluate the effects of both anticoagulants and P2Y 12 receptor antagonists and determine the effects of edoxaban, a direct factor Xa inhibitor, and P2Y 12 receptor antagonists (clopidogrel and ticagrelor) alone and when combined. Material and methods Human platelet-rich plasma (PRP) from healthy subjects was stimulated with adenosine diphosphate (ADP) plus tissue factor. Thrombin generation was measured by means of calibrated automated thrombography. Results Combination of 10μM ADP and low concentration (0.25 pM) tissue factor induced reproducible thrombin generation in human PRP. Edoxaban (40 and 80ng/mL), active metabolite of clopidogrel (AM-clopidogrel, 10 and 20μg/mL), and ticagrelor (3μg/mL) alone inhibited ADP plus tissue factor-induced thrombin generation. Edoxaban suppressed all 5 parameters (lag time, peak, time to peak, endogenous thrombin potential, and maximum rate), whereas AM-clopidogrel and ticagrelor inhibited 4 and 3 parameters, respectively. Concomitant treatment with edoxaban and AM-clopidogrel or ticagrelor produced an additive inhibition of thrombin generation compared to the single treatments. Conclusions The thrombin generation assay induced by ADP plus tissue factor can detect the activities of both edoxaban and P2Y 12 receptor antagonists. Combination of edoxaban and a P2Y 12 receptor antagonist shows additive inhibition. These results suggest that ADP plus tissue factor-induced thrombin generation may be a useful measurement to assess the combination effects of anticoagulants and P2Y 12 receptor antagonists in a single assay.

Background Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants. Objectives To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage. Methods Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail. Results In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose–response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin. Conclusions These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.

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.

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 24 h 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 1 mg/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 1 mg/kg (an antithrombotic dose) and 54 mg/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.

Introduction Factor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage. Methods Rats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4 days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60 min. Tail template bleeding time was measured after making an incision on the tail. Results Edoxaban at 0.3, 1 and 3 mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED 50 ) was 1.9 mg/kg. At supra-therapeutic doses (10 and 20 mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED 50 values of warfarin and enoxaparin were 0.12 mg/kg and 500 IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16 mg/kg and 1700 IU/kg, respectively. The safety margin (ratio of BT2 to ED 50 ) of edoxaban (> 10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4). Conclusions Edoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.

We compared the antithrombotic properties of a factor Xa inhibitor (DX-9065a) with those of a thrombin inhibitor (melagatran) in a rat disseminated intravascular coagulation model and a rat venous thrombosis model. Rat disseminated intravascular coagulation and venous thrombosis models were produced by injection of tissue factor and platinum wire placement, respectively. DX-9065a exerted antithrombotic effects dose dependently in both models. Melagatran was also effective in the venous thrombosis model, whereas it showed an aggravation in the disseminated intravascular coagulation model at low but not high doses. In the in vitro study, DX-9065a decreased the C max of the thrombin generation curve in plasma irrespective of whether protein C was present or not. However, melagatran increased the C max at low concentrations when protein C was present. This increase was not detected in protein C-deficient plasma. These results suggest that, unlike DX-9065a, melagatran in low doses aggravates disseminated intravascular coagulation by increasing thrombin generation, which may be partly due to suppression of negative feedback by activated protein C.

Abstract 3319 Background: Hemorrhage is an unwanted effect of any anticoagulant. In animal studies, edoxaban, a novel direct factor Xa (FXa) inhibitor, does not cause significant bleeding at therapeutic doses; however at higher than 10-times the doses the compound prolongs bleeding time. In clinical studies, edoxaban causes bleeding complications comparable to standard anticoagulants, warfarin and enoxaparin in patients with atrial fibrillation and patients undergoing orthopedic surgery. In case of emergency, antidotes to reverse the anticoagulant effect of edoxaban would be helpful. Objective: To determine the effects of three hemostatic agents: anti-inhibitor coagulant complex, prothrombin complex concentrate, and recombinant factor VIIa (rFVIIa) on prothrombin time (PT) prolonged by edoxaban using pooled human plasma in vitro. Materials & Methods: Anti-inhibitor coagulant complex (Feiba), prothrombin complex concentrate (PPSB-HT), and rFVIIa were purchased from Baxter, Nihon Pharmaceutical, and Novo Nordisk Pharma. Pooled human normal plasma was obtained from George King Bio-Medical. PT was measured with a microcoagulometer (Amelung KC-10A micro) as follows. Five μL of anti-inhibitor coagulant complex, prothrombin complex concentrate, and rFVIIa solutions or vehicle (saline) and 45 μL of plasma spiked with edoxaban or 5% DMSO-saline solution were added to a cuvette and pre-incubated at 37°C for 1 min. Coagulation was started by the addition of 100 μL of HemosIL PT-fibrinogen HS PLUS (Instrumentation Laboratory) to the mixture. The clotting time was measured. Results: Mean PT of the control plasma in three experiments was 17.8 − 18.4 sec. Edoxaban at concentrations of 150 and 300 ng/mL significantly prolonged PT. Addition of anti-inhibitor coagulant complex (0.15, 0.5 and 1.5 U/mL) significantly and concentration-dependently shortened the prolonged PT caused by edoxaban. At the maximum concentration (1.5 U/mL), anti-inhibitor coagulant complex reversed PT to 19.5 and 25.0 sec from 31.5 and 43.7 sec in the presence of 150 and 300 ng/mL edoxaban, respectively. Prothrombin complex concentrate (0.15, 0.5 and 1.5 U/mL) and rFVIIa (100, 300 and 1000 ng/mL) also significantly and concentration-dependently reversed anticoagulant effect of edoxaban. At the maximum concentration of prothrombin complex concentrate (1.5 U/mL), PT was shortened to 23.7 and 31.6 sec from 30.7 and 42.5 sec in the presence of 150 and 300 ng/mL edoxaban. rFVIIa at the maximum concentration (1000 ng/mL) reversed PT to 17.3 and 24.2 sec from 30.7 and 43.1 sec in the presence of 150 and 300 ng/mL edoxaban. Anti-inhibitor coagulant complex, prothrombin complex concentrate and rFVIIa each alone significantly shortened PT in a concentration-dependent manner. In the presence of anti-inhibitor coagulant complex (1.5 U/mL), prothrombin complex concentrate (1.5 U/mL), and rFVIIa (1000 ng/mL), PT was 13.1, 15.1, and 11.6 sec, respectively. Conclusions: The present study demonstrates that anti-inhibitor coagulant complex, prothrombin complex concentrate, and rFVIIa effectively reversed the anticoagulant effect of edoxaban in pooled human plasma in vitro. Therefore, it is suggested that these hemostatic agents have the potential to be antidotes to edoxaban in cases of hemorrhage. Disclosures: Morishima: Daiichi Sankyo Co., Ltd.: Employment. Off Label Use: Drugs: anti-inhibitor coagulant complex, prothrombin complex concentrate, and recombinant factor VIIa Purpose: Reversal of the anticoagulant effect of edoxaban, a factor Xa inhibitor. Honda:Daiichi Sankyo Co., Ltd.: Employment. Shibano:Daiichi Sankyo Co., Ltd.: Employment.

16,16-Dimethyl prostaglandin E2 was examined for its ability to inhibit complement-mediated in vitro hepatocytolysis by an antigen-antibody reaction. In the presence of fresh rat serum as a source of complement, 5-min culture of isolated rat hepatocytes with a monoclonal antibody against a rat liver-specific membranous antigen resulted in a marked, significant elevation in lactate dehydrogenase leakage into the culture medium. However, with heat-inactivated rat serum, such a reaction did not occur, indicating that the hepatocytolysis induced by the antibody was attributable to the membrane damaging action of complement activated by an antigen-antibody reaction. Pretreatment of the hepatocyte with 16,16-dimethyl prostaglandin E2 significantly suppressed the cytolytic reaction induced by the antibody in a concentration-dependent manner. These results show that 16,16-dimethyl prostaglandin E2 is capable of protecting isolated rat hepatocytes against the membrane-damaging insult of activated complement.

16,16-Dimethyl prostaglandin E2, a known cytoprotective agent, was examined for its ability to protect the liver against complement-mediated necrosis induced by an intravenous injection of a monoclonal antibody against a rat liver—specific antigen in rats. The hepatic injury induced by the antibody was characterized by (a) rapid development of numerous massive hemorrhagic foci of necrotic liver cells, (b) marked increases in serum liver enzyme activities and (c) pronounced reduction in the CH50 level, presumably as a result of complement consumption in the liver. Pretreatment with 16,16-dimethyl prostaglandin E2 at intraperitoneal doses of 20 and 100 μg/kg suppressed the hepatic injury, as evidenced by markedly mitigated liver-cell necrosis and much smaller increases in the serum-enzyme activities compared with the values in diseased control animals. The prostaglandin analogue failed to prevent serum complement consumption in response to the antibody injection or affect the CH50 level at the preinjury stage, indicating that neither complement inactivation nor interference with the antigen-antibody reaction was involved in the hepatic protection. The hepatoprotective doses of 16,16-dimethyl prostaglandin E2 produced a significant increase in liver cyclic AMP content in a dose-related manner. In addition, intravenous dibutyryl cyclic AMP at 3 and 10 mg/kg dose-dependently prevented histological and biochemical changes in the hepatic damage without altering the rate of reduction in serum complement activity. Like 16,16-dimethyl prostaglandin E2, dibutyryl cyclic AMP did not affect the preinjury CH50 level. These results suggest that increased hepatocellular cyclic AMP is responsible, at least in part, for the hepatic protection afforded by 16,16-dimethyl prostaglandin E2 against the membrane-damaging insult of activated complement, possibly through the stabilization of the plasma membrane.

Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

DU-176b is a novel potent, orally active and selective direct inhibitor of factor Xa (FXa). Direct FXa inhibitors have been reported to exert little effect on bleeding time at antithrombotic doses in animal studies. The aim of the present study was to compare the antithrombotic and hemorrhagic effects of DU-176b with unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin) and warfarin in rat models of thrombosis and hemorrhage. Rats were treated with DU-176b, UFH and LMWH by continuous intravenous infusion for 2 – 2.5 h, and with warfarin orally once daily for 4 days before thrombosis or hemorrhage. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava and left for 60 min. Tail template bleeding time was measured after an incision on the tail. DU-176b dose-dependently inhibited thrombus formation in the venous thrombosis model. The dose required for 50% inhibition (ED50) was 0.076 mg/kg/h. In contrast, the dose of DU-176b to double template bleeding time (BT2) was 0.75 mg/kg/h, indicating 10-fold dissociation of the doses of antithrombotic and hemorrhagic effects. UFH, LMWH and warfarin also prevented thrombus formation (ED50 = 56 U/kg/h, 66 U/kg/h and 0.16 mg/kg/day, respectively), but prolonged bleeding time at slightly higher doses (BT2 = 73 U/kg/h, 135 U/kg/h and 0.21 mg/kg/day, respectively) than the effective doses. The dissociation of the doses for these compounds was only 1.3, 2.0 and 1.3-fold, respectively. Moreover, the slope of dose-antithrombotic response curve of DU-176b was significantly slighter than those of UFH, LMWH and warfarin, indicating that the therapeutic dose range of DU-176b would be wider than those of the other anticoagulants. These results suggest that direct and selective inhibition of FXa by DU-176b is preferable for the treatment of thrombotic diseases in the aspect of lack of compromising primary hemostasis.

Factor Xa (FXa) is an attractive target for the treatment of thrombosis due to its crucial role in the blood coagulation cascade. Fondaparinux, a selective FXa inhibitor, has been approved for clinical use to prevent deep vein thrombosis after orthopedic surgery; however, it requires antithrombin (AT) to exert its antithrombotic effect. It is reported that AT dependent anticoagulants such as heparin are less effective to suppress platelet-rich arterial-type thrombus due to its inaccessibility to thrombus-bound FXa/thrombin. We have developed a potent direct (i.e. AT independent) FXa inhibitor, DU-176b. The objective of this study is to compare the antithrombotic properties of a direct selective FXa inhibitor, DU-176b, with an AT dependent selective FXa inhibitor, fondaparinux. We evaluated the antithrombotic effects of DU-176b and fondaparinux in rat models of arterial and venous thrombosis. The arterial and venous thrombosis was induced by topical application of ferric chloride to the carotid artery and by insertion of a platinum wire into the inferior vena cava, respectively. DU-176b (0.05 – 1.25 mg/kg/h) and fondaparinux (1 – 10 mg/kg/h for arterial thrombosis and 0.03 – 1 mg/kg/h for venous thrombosis) were intravenously administered as continuous infusions. DU-176b prevented both arterial and venous thrombosis in the same dose range. In contrast, the effective doses of fondaparinux markedly differed between these models. A higher dose of fondaparinux more than 100 times was required to inhibit arterial thrombosis compared with venous thrombosis. These results suggest that direct inhibition of FXa is a preferable strategy to AT dependent inhibition for the prevention of thrombus formation in the arteries.

Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.