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Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats
European Journal of Pharmacology 686 (2012) 74–80
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Cardiovascular Pharmacology
Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats Taketoshi Furugohri ⁎, Toshio Fukuda, Naoki Tsuji, Akemi Kita, Yoshiyuki Morishima, Toshiro Shibano Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan
a r t i c l e
i n f o
Article history: Received 19 December 2011 Received in revised form 26 March 2012 Accepted 5 April 2012 Available online 21 April 2012 Keywords: Edoxaban Factor Xa inhibitor Tissue factor Hypercoagulation Thrombin inhibitor
a b s t r a c t 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 10 min after tissue factor injection. Platelet numbers, thrombin–antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1 mg/kg, i.v.) suppressed platelet consumption and thrombin–antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1 mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. In addition, although melagatran (3 mg/kg, i.v.) improved coagulation status when tissue factor was given 5 min after the drug administration, and 2, 4 and 8 h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15–234 ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190 ng/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. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Factor Xa plays an important role in the blood coagulation cascade, serving as the juncture between the intrinsic and extrinsic system leading to the generation of thrombin, thus factor Xa is an attractive target for the prevention and treatment of thromboembolic diseases (Ansell, 2007). Thrombin also has a key role in the blood coagulation cascade (Weitz, 2007). Therefore, several oral direct factor Xa inhibitors and an oral direct thrombin inhibitor have been launched. But, there are some concerns about thrombogenesis by direct thrombin inhibitors. Compared to warfarin/placebo, the elevated risk of arterial cardiovascular events in patients treated with ximelagatran (FDA, 2004), a prodrug of direct thrombin inhibitor melagatran, and dabigatran (Connolly et al., 2009) were reported. We (Furugohri et al., 2005) and other group (Perzborn et al., 2008a) have previously demonstrated that a direct thrombin inhibitor, melagatran, induces a paradoxical activation of coagulation pathway in a rat model of tissue factor-induced hypercoagulation. Low-dose
⁎ Corresponding author. Tel.: + 81 3 3492 3131; fax: + 81 3 5436 8587. E-mail address: [email protected] (T. Furugohri). 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.04.031
oral administration of melagatran enhances platelet consumption and thrombin–antithrombin complex generation, suggesting that this paradoxical phenomenon may be implicated in coagulation activation observed with direct thrombin inhibitors in clinical trials. We presented the precise mechanism of this paradoxical activation of coagulation by the direct thrombin inhibitor in our in vitro studies (Furugohri et al., 2011; Morishima et al., 2006). In short, direct thrombin inhibitors increase the activation of coagulation by suppression of the thrombininduced negative-feedback system through the inhibition of protein C activation. The same result was reported by other group (Perzborn and Harwardt, 2008b). However, the precise mechanism underlying the paradoxical coagulation activation in vivo by melagatran remains to be fully elucidated. Thus, in this study, we determined the relationship between the plasma concentrations of melagatran and the coagulation status by changing treatment conditions such as doses of the compound and timing of coagulation induction after the administration of melagatran. In terms of the paradoxical coagulation activation by other anticoagulants, the effects of edoxaban (the free form of edoxaban tosilate hydrate: Japanese Accepted Name), a recently approved direct factor Xa inhibitor in Japan, on tissue factor-induced hypercoagulation are not being investigated, even though we have shown that a factor Xa
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inhibitor, DX-9065a, does not cause the aggravation of coagulation status induced by tissue factor. Moreover, it is not examined whether a different type of anticoagulant heparin, which exerts an antithrombin-dependent inhibition of thrombin and factor Xa, induces hypercoagulation in rats treated with tissue factor. Therefore, we directly compared the potential of edoxaban and heparin with that of melagatran to aggravate tissue factor-induced coagulation in rats. 2. Materials and methods 2.1. Reagents and drugs Edoxaban and melagatran were synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Heparin sodium was purchased from Novo Nordisk A/S (Copenhagen, Denmark). Tissue factor (Thromboplastin C Plus) and Enzygnost TAT micro were purchased from Siemens AG (Munich, Germany). Halothane was from Takeda Pharmaceutical (Osaka, Japan) and thiopental sodium was from Mitsubishi Tanabe Pharma (Osaka, Japan). 2.2. Animals Animal facilities, animal care and study programs were in accordance with the in-house guidelines of the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd. Eight or nine-week-old male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan) and maintained on an 8:00 am/8:00 pm light/dark schedule, temperature (23 ± 2 °C) and humidity (55 ± 20%). Rats were housed 5–6 per cage and food and water were available ad libitum. They were acclimated for 1 or 2 weeks. 2.3. Tissue factor-induced hypercoagulation model Rats (232–284 g) were anesthetized with thiopental (100 mg/kg, i.p.). Coagulation was induced by the injection of 2.8 U/kg tissue factor into the femoral vein. Blood samples were collected into plastic syringe containing citrate solution (9 volumes of blood to 1 volume of 3.13% sodium citrate solution) 10 min after tissue factor injection. Immediately after blood collection, platelet number was measured using an automatic hematology analyzer MEK-6358 (Nihon Kohden, Tokyo, Japan). Then, blood samples were centrifuged at 1500 ×g for 10 min at 4 °C and plasma samples were prepared. Plasma was stored at −70 °C until the measurement of the following parameters: thrombin–antithrombin complex and plasma concentrations of drugs. Thrombin-antithrombin complex concentrations were assayed according to the previous study with Enzygnost TAT micro (Morishima et al., 1997). 2.4. Drug treatment 2.4.1. Dose-dependent effects of a direct thrombin inhibitor, heparin and a factor Xa inhibitor on hypercoagulation Under the thiopental (100 mg/kg, i.p.) anesthesia, melagatran at doses of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1 mg/kg or heparin at doses of 0.1, 0.3, 1, 10 and 100 U/kg was injected into the jugular vein of rats. Hypercoagulation was induced 5 min after drug administration. Edoxaban (0.1, 0.5 and 2.5 mg/kg) was orally administered by gavage to fasted rats, and then the rats were anesthetized with thiopental. Hypercoagulation was induced 30 min after drug administration. 2.4.2. Time-dependent effects of direct thrombin inhibitor and factor Xa inhibitor after drug treatment on hypercoagulation The experiment protocol is shown in Fig. 1. Under the halothane (2–3%) anesthesia, melagatran (3 mg/kg) or edoxaban (0.3 mg/kg) was injected into the jugular vein of rats. After their administration, the rats were awoken except 5 min after dosing. Hypercoagulation was induced 5 min, 2, 4, 8 and 16 h after dosing under the thiopental anesthesia.
Fig. 1. The experimental protocol of time-dependent experiments.
2.5. Measurement of plasma concentration The plasma concentrations of drugs were measured according to the previous study (Furugohri et al., 2008; Zafar et al., 2007) by liquid chromatography-tandem mass spectrometry analysis. Lower limit of quantitation is shown Table 1–4. 2.6. Statistical analysis Analyses were performed using EXSAS ver.7.10 (ARM SYTEX, Osaka, Japan) based on SAS release 8.2 (SAS Institute Japan, Tokyo, Japan). Data are expressed as means ± S.E.M. unless otherwise noted and statistical significance was measured at the level of P b 0.05. Comparison of the platelet number and thrombin–antithrombin complex concentration between groups were analyzed by t-test or Dunnett multiple comparison method. Mortality rate of rats up to 60 min after tissue factor injection was analyzed by Fisher test. 3. Results 3.1. Dose-dependent effects of melagatran, heparin and edoxaban on hypercoagulation 3.1.1. Melagatran In the sham group, platelet number and thrombin–antithrombin complex concentration were 79.2 ± 2.1 × 10 4 cells/μl and 3.6 ± 0.4 ng/ ml, respectively (Fig. 2). Intravenous injection of tissue factor significantly reduced platelet number to 47.5 ± 2.3 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin–antithrombin complex to 341.9 ± 37.7 ng/ml (P b 0.001) (Fig. 2). Compared to the tissue factor-treated control group, a high dose of melagatran (1 mg/kg, i.v.) given 5 min before the hypercoagulation induction suppressed platelet consumption (68.0 ± 3.1 × 10 4 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (152.5 ± 6.0 ng/ml, P b 0.01). The plasma concentration of melagatran was 1190±182 ng/ml (Table 1). Lower doses of melagatran (0.01, 0.03 and 0.1 mg/kg, Table 1 Dose-dependent effect of melagatran on mortality in tissue factor-induced hypercoagulation rats and plasma melagatran concentrations. Treatment Control Melagatran
Dose (mg/kg, i.v.)
Mortality
Concentration (ng/ml)
0.001 0.003 0.01 0.03 0.1 0.3 1
0/6 0/6 3/6 4/6 4/6 3/6 0/6 0/6
1.3 ± 0.2 4.5 ± 1.2 22 ± 0 81 ± 6 234 ± 31 454 ± 31 1190 ± 182
Concentrations represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml.
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Table 2 Plasma edoxaban concentrations after orally administration in a tissue factor-induced hypercoagulation model. Treatment Edoxaban
Dose (mg/kg, p.o.)
Concentration (ng/ml)
0.1 0.5 2.5
b 5.0 16.2 ± 5.0 163 ± 41
Data represent the means ± S.D. Lower limit of quantitation is 5.0 ng/ml.
Table 4 Plasma edoxaban concentrations after i.v. administration in a tissue factor-induced hypercoagulation model. Treatment
Time after i.v.
Concentration (ng/ml)
Edoxaban 0.3 mg/kg, i.v.
5 min 2h 4h 8h 16 h
43 ± 4 4.2 ± 0.9 1.4 ± 0.3 b 1.0 b 1.0
Data represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml.
i.v.) significantly enhanced platelet consumption (9.1 ± 2.0, 6.4 ± 0.5, 10.3 ± 1.1 ×104 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (753.3 ± 61.4, 868.6± 49.4, 918.1 ± 13.5 ng/ml, P b 0.001). The range of mean plasma melagatran concentrations at which melagatran aggravated hypercoagulation was 22–234 ng/ml (Table 1). In this aggravating range, melagatran showed the tendency to increase mortality at 0.01 and 0.03 mg/kg (P = 0.06) (Table 1). Very low doses (0.001 and 0.003 mg/kg, i.v.) of melagatran did not affect coagulation. 3.1.2. Heparin In the sham group, platelet number and thrombin–antithrombin complex concentration were 74.1±3.0×104 cells/μl and 7.3±1.5 ng/ml, respectively (Fig. 3). Compared to the sham group intravenous injection of tissue factor significantly reduced platelet number to 40.6 ± 2.2 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin– antithrombin complex to 826.6 ± 89.4 ng/ml (P b 0.001). Heparin at 100 U/kg, i.v. inhibited platelet consumption (68.9 ± 1.6 × 10 4 cells/μl, P b 0.01) and thrombin–antithrombin complex generation (198.0 ± 23.8 ng/ml, P b 0.001) compared to those in the control group (Fig. 3). Lower doses of heparin (0.1, 0.3, 1 and 10 U/kg, i.v.) have no effects on platelet consumption and thrombin–antithrombin complex generation.
3.2.1. Melagatran Intravenous injection of tissue factor significantly reduced platelet number from 82.0 ± 1.9 × 10 4 cells/μl to 48.2 ± 2.7 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin–antithrombin complex from 5.0 ± 0.5 ng/ml to 384.7 ± 44.7 ng/ml (P b 0.001) compared to those in the sham group (Fig. 5). Melagatran inhibited platelet consumption (80.6±1.7×104 cells/μl, Pb 0.001) and thrombin– antithrombin complex generation (48.9±1.0 ng/ml, Pb 0.001) when hypercoagulation was induced 5 min after the drug administration (Fig. 5). These data indicated that melagatran effectively inhibited hypercoagulation at this time point with a mean plasma concentration of 3987 ng/ml. Melagatran, however, enhanced platelet consumption (5.7±0.4, 10.6±0.6, 23.1±4.3×104 cells/μl, Pb 0.001) and thrombin– antithrombin complex generation (836.1±21.5, 756.1±8.9, 603.0± 60.9 ng/ml, Pb 0.001) (Fig. 5), when coagulation was induced 2, 4 and 8 h after dosing. Significant numbers of rats died when coagulation was
3.1.3. Edoxaban Edoxaban (0.1, 0.5 and 2.5 mg/kg, p.o.) inhibited platelet consumption (47.8±2.8, 50.2±1.2, 56.9±2.4×104 cells/μl, Pb 0.05, Pb 0.01, Pb 0.001) and thrombin–antithrombin complex generation (173.1±22.0, 108.3± 17.2, 38.6±3.2 ng/ml, Pb 0.05, Pb 0.001, Pb 0.001) compared to those in the control group (Fig. 4). Mean plasma concentrations of edoxaban were b5.0, 16.2 and 163 ng/ml, respectively (Table 2). 3.2. Time-dependent effects of melagatran and edoxaban after drug treatment on hypercoagulation The time-dependent effects after the injection of melagatran (3 mg/kg, i.v.) or edoxaban (0.3 mg/kg, i.v.) on hypercoagulation were evaluated in a tissue factor-induced rat hypercoagulation model.
Table 3 Time-dependent effect after melagatran treatment on mortality in tissue factorinduced hypercoagulation rats and plasma melagatran concentrations. Treatment
Time after i.v.
Mortality
Concentration (ng/ml)
Control Melagatran 3 mg/kg, i.v.
5 min
0/8 0/8
3987 ± 338
2h 4h 8h 16 h
a
7/8 7/8a 3/7 0/8
126 ± 12 55 ± 15 15 ± 11 1.2 ± 1.2
The experiment protocol is shown in Fig. 1. Melagatran (3 mg/kg) was injected into the jugular vein of rats. Hypercoagulation was induced 5 min, 2, 4, 8 and 16 h after melagatran administration. Concentrations represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml. a Indicates statistically significant differences vs control values (P b 0.01).
Fig. 2. Effects of melagatran on tissue factor-induced hypercoagulation in rats. Melagatran at 1 mg/kg, i.v. suppressed platelet consumption (A) and thrombin– antithrombin complex generation (B) compared to the control group. Lower doses of melagatran (0.01–0.1 mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. Data represent means ± S.E.M. (n = 3–6). ### P b 0.001 vs. sham. ** P b 0.01, *** P b 0.001 vs. control.
T. Furugohri et al. / European Journal of Pharmacology 686 (2012) 74–80
Fig. 3. Effects of heparin on tissue factor-induced hypercoagulation in rats. Heparin at 100 U/kg, i.v. suppressed platelet consumption (A) and thrombin–antithrombin complex generation (B) compared to the control group. Lower doses of heparin (0.1– 10 U/kg, i.v.) have no effects on platelet consumption and thrombin–antithrombin complex generation.Data represent means ± S.E.M. (n = 5–6). ### P b 0.001 vs. sham. ** P b 0.01, *** P b 0.001 vs. control.
induced 2 and 4 h after dosing of melagatran (Table 3), probably due to respiratory distress provoked by pulmonary embolism. These findings demonstrated that melagatran aggravated hypercoagulation at these time points. After 16 h, the effects of melagatran towards inhibition or enhancement of coagulation disappeared. The range of plasma melagatran concentrations at which melagatran aggravated intravascular coagulation was 15–126 ng/ml (Table 3). 3.2.2. Edoxaban Edoxaban inhibited platelet consumption (84.3 ± 6.1 × 10 4 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (26.2 ± 1.5 ng/ml, P b 0.001) compared to those in the control group (platelet number: 59.1±3.5× 104 cells/μl, thrombin–antithrombin complex concentration: 315.6±45.7 ng/ml), when hypercoagulation was induced 5 min after dosing (Fig. 6). At this time, plasma concentration of edoxaban was 43 ± 4 ng/ml (Table 4). Edoxaban had no effects on platelet number and significantly inhibited thrombin–antithrombin complex generation 2–16 h after drug administration (plasma concentration of edoxaban: b1.0–4.2 ng/ml, Table 4). Unlike melagatran, edoxaban did not show any exacerbation of tissue factor-induced hypercoagulation at any time points. 3.3. Relationship between plasma melagatran concentration and platelet number or thrombin–antithrombin complex concentration To determine the relationship between plasma melagatran concentration and platelet number or thrombin–antithrombin complex
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Fig. 4. Effects of edoxaban on tissue factor-induced hypercoagulation in rats. Edoxaban dose-dependently inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) compared to the control group. Data repressent means± S.E.M. (n= 6). ### P b 0.001 vs. sham. * P b 0.05, ** P b 0.01, *** P b 0.001 vs. control.
concentration, platelet numbers and thrombin–antithrombin complex concentrations obtained from dose- and time-dependent studies were plotted against plasma melagatran concentrations (Fig. 7). Higher concentrations (>454 ng/ml) of melagatran inhibited platelet consumption and thrombin–antithrombin complex generation. Lower concentrations (15–234 ng/ml) of melagatran aggravated hypercoagulation regardless of the different treatment conditions. These results indicated that aggravation of hypercoagulability by melagatran depends on its plasma concentrations at the induction of hypercoagulation. On the other hand, edoxaban inhibited but not aggravated hypercoagulation, regardless of its plasma concentrations. 4. Discussion Hypercoagulability after cessation of anticoagulant treatment has been concerned. We have shown that low-dose administration of a direct thrombin inhibitor melagatran, but not a prototype direct factor Xa inhibitor (DX-9065a), significantly worsens the coagulation status induced by tissue factor injection. In this study, we clarified the relationship between the plasma concentrations of melagatran and aggravation or improvement of coagulation status. Furthermore, we directly compared the potential of a novel oral factor Xa inhibitor, edoxaban with a Ki value of 0.561 nM (Furugohri et al., 2008), and an antithrombin-dependent anticoagulant, heparin, with that of a direct thrombin inhibitor, melagatran, to aggravate tissue factor-induced coagulation in rats. Tissue factor is a critical factor responsible for the formation of arterial thrombi, which is the cause of myocardial infarction and ischemic stroke. Tissue factor is a membrane-bound protein that is
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Fig. 5. Time-dependent effects after melagatran treatment on hypercoagulation. Melagatran inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) when hypercoagulation was induced 5 min after the drug administration. However, melagatran enhanced platelet consumption and thrombin–antithrombin complex generation, when hypercoagulation was induced 2, 4 and 8 h after dosing.Data represent means ± S.E.M. (n= 5–8). ### P b 0.001 vs. sham. *** P b 0.001 vs. control.
Fig. 6. Time-dependent effects after edoxaban treatment on hypercoagulation. Edoxaban inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) just after the drug administration (5 min). Edoxaban did not exert any deleterious effects and had no effects on platelet number and significantly inhibited thrombin–antithrombin complex generation 2–16 h after drug administration.Data represent means± S.E.M. (n= 6). ## P b 0.01, ### P b 0.001 vs. sham. * P b 0.05, *** P b 0.001 vs. control.
not normally expressed on vascular cells which are in contact with blood. Under pathological circumstances, however, tissue factor expression is induced in cells such as monocytes and endothelial cells and tissue factor is localized in the lipid core within the atherosclerotic lesions of the coronary (Hathcock, 2004) and carotid (Jander et al., 2001) arteries. Tissue factor initiates the extrinsic pathway of coagulation and contribute to thrombus formation after the plaque rupture leading to the onset of coronary artery diseases and ischemic stroke. Therefore, we used tissue factor as a trigger of hypercoagulation in our model to mimic the initiation of thrombus formation at the site of atherosclerotic plaques (Asakura et al., 2002; Dickneite et al., 1995; Hara et al., 1995; Yamazaki et al., 1994). Our study clearly demonstrates that the effects of melagatran on coagulation depended on the plasma drug concentration. The coagulation status was improved at high melagatran concentrations (1190 ng/ml or more) and aggravated at lower concentrations (15–234 ng/ml) in both dose-dependent and time-dependent models. Therefore, we speculate that when the plasma concentrations of the direct thrombin inhibitor decline below therapeutic ranges, the paradoxical effect (enhancement of coagulation activity) might occur. On the other hand, a direct factor Xa inhibitor, edoxaban, and an antithrombin-dependent anticoagulant, heparin, did not exert any deleterious effects, regardless of its plasma concentration or at any time points under this examination conditions. This phenomenon in vivo is well matched with that observed in our in vitro studies (Furugohri et al., 2011), in which low concentrations (4–315 ng/ml) of melagatran enhances thrombin generation in human plasma whereas at high concentration (630 ng/ml) the compound
inhibits it. In consistent with the results of the present study, direct factor Xa inhibitors and antithrombin-dependent anticoagulants do not enhance thrombin generation in our in vitro studies. The elevated risks of arterial cardiovascular events in patients treated with direct thrombin inhibitors were reported. Compared to warfarin/ placebo in patients undergoing orthopaedic surgery, ximelagatran, a prodrug of direct thrombin inhibitor melagatran, significantly elevates the risk of arterial cardiovascular events (FDA, 2004). Moreover, although it is marginally, another direct thrombin inhibitor, dabigatran, also increases the rate of myocardial infarction compared to warfarin (0.74%/year vs. 0.53%/year) in the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) study (Connolly et al., 2009). To the contrary, the symptoms of myocardial infarction were not observed in clinical studies with direct factor Xa inhibitors: Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET-AF) study (Patel et al., 2011) and Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) study (Granger et al., 2011). Thus, hypercoagulation observed in our in vitro and in vivo studies might contribute to clinical cardiovascular events like myocardial infarction associated with anticoagulant therapies with direct thrombin inhibitors. We clearly pointed out the precise mechanism of enhancement of thrombin generation by the direct thrombin inhibitor in our in vitro study (Furugohri et al., 2011). In short, direct thrombin inhibitors increase thrombin generation by suppression of the thrombin-induced negative-feedback system through the inhibition of protein C
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enhance thrombin generation in our in vitro study. Edoxaban is a highly selective factor Xa inhibitor and has no effect on serine protease activity of activated protein C. Heparin is not a selective thrombin inhibitor, which has inhibitory effects on multiple activated coagulation factors including thrombin, factor Xa, factor IXa and protein C activation via antithrombin (Mattsson et al., 2001). These differences in mechanism of action between factor Xa inhibitors/heparin and a direct thrombin inhibitor may result in the different regulation of blood coagulation. Further studies may be required to clarify the distinctions between these anticoagulants in vivo. In conclusion, we have shown that at certain concentrations a direct thrombin inhibitor, melagatran, aggravates coagulation status initiated by the injection of tissue factor. The inhibition or activation of coagulation clearly depends on the plasma concentration of melagatran. This phenomenon may implicate the coagulation activation observed with direct thrombin inhibitors in the clinical trials. On the other hand, a factor Xa inhibitor, edoxaban, and heparin does not exert any deleterious effects on tissue factor-induced hypercoagulation. Therefore, direct factor Xa inhibitors are more useful than direct thrombin inhibitors in terms of lower possibility of the activation of coagulation pathway. References
Fig. 7. Relationship between plasma concentration of melagatran and platelet number (A) or thrombin–antithrombin complex (B) in a tissue factor-induced hypercoagulation model in rats. The data of platelet numbers and thrombin–antithrombin complex concentrations in dose- and time-dependent studies were plotted against plasma melagatran concentrations. Higher concentrations (>454 ng/ml) of melagatran inhibited platelet consumption and thrombin–antithrombin complex generation. Lower concentrations (15–234 ng/ml) of melagatran enhanced platelet consumption and thrombin– antithrombin complex generation and increased mortality. Circles are data from the dosedependent study. Squares are from the time-dependent study. Open and closed symbols indicate survival and dead rats, respectively.
activation. Protein C is activated by the thrombin-thrombomodulin complex and activated protein C acts as a negative regulator of the coagulation pathway (Dahlback and Villoutreix, 2003; Esmon, 2003). Activated protein C exerts a potent anticoagulant effect via proteolysis of factor Va and VIIIa (negative feedback). Melagatran inhibits the activation of protein C through the inhibition of thrombomodulinbound thrombin (Mattsson et al., 2001). Therefore, it is likely that the inhibition of the negative feedback loop by melagatran may enhance tissue factor-induced hypercoagulation in vivo. This idea is also supported by a finding that relative deficiency in protein C is one drive for the hypercoagulable state during initiation of treatment with vitamin K antagonists (Wittkowsky, 2005). The higher concentration of thrombin inhibitors could be enough to directly inhibit thrombin activity and suppressed thrombin generation through the inhibition of the positive-feedback activation of factor V, VII, VIII and XI, which are converted to active forms by thrombin. Since the positive feedback reactions by thrombin play an important role in thrombin generation (He et al., 2001), the inhibition of the reactions by the higher concentration of thrombin inhibitors may predominate over the thrombin generation enhanced by the inhibition of protein C activation. In contrast, we demonstrated that edoxaban and heparin does not
Ansell, J., 2007. Factor Xa or thrombin: is factor Xa a better target? J. Thromb. Haemost. 5 (Suppl. 1), 60–64. Asakura, H., Ichino, T., Yoshida, T., Suga, Y., Ontachi, Y., Mizutani, T., Kato, M., Ito, T., Yamazaki, M., Aoshima, K., Morishita, E., Saito, M., Miyamoto, K.I., Nakao, S., 2002. Beneficial effect of JTV-803, a new synthetic inhibitor of activated factor X, against both lipopolysaccharide-induced and tissue factor-induced disseminated intravascular coagulation in rat models. Blood Coagul. Fibrinolysis 13, 233–239. Connolly, S.J., Ezekowitz, M.D., Yusuf, S., Eikelboom, J., Oldgren, J., Parekh, A., Pogue, J., Reilly, P.A., Themeles, E., Varrone, J., Wang, S., Alings, M., Xavier, D., Zhu, J., Diaz, R., Lewis, B.S., Darius, H., Diener, H.C., Joyner, C.D., Wallentin, L., RE-LY Steering Committee and Investigators, 2009. Dabigatran versus warfarin in patients with atrial fibrillation. N. Engl. J. Med. 361, 1139–1151. Dahlback, B., Villoutreix, B.O., 2003. Molecular recognition in the protein C anticoagulant pathway. J. Thromb. Haemost. 1, 1525–1534. Dickneite, G., Seiffge, D., Diehl, K.H., Reers, M., Czech, J., Weinmann, E., Hoffmann, D., Stüber, W., 1995. Pharmacological characterization of a new 4-amidinophenylalanine thrombin-inhibitor (CRC 220). Thromb. Res. 77, 357–368. Esmon, C.T., 2003. The protein C pathway. Chest 124, 26S–32S. FDA, 2004. Integrated executive summary of FDA review for NDA 21–686 Exanta (Ximelagatran). Food and Drug Administration, Washington (DC). Furugohri, T., Shiozaki, Y., Muramatsu, S., Honda, Y., Matsumoto, T., Isobe, K., Sugiyama, N., 2005. Different antithrombotic properties of factor Xa inhibitor and thrombin inhibitor in rat thrombosis models. Eur. J. Pharmacol. 514, 35–42. Furugohri, T., Isobe, K., Honda, Y., Kamisato-Matsumoto, C., Sugiyama, N., Nagahara, T., Morishima, Y., Shibano, T., 2008. DU-176b, a potent and orally active factor Xa inhibitor: in vitro and in vivo pharmacological profiles. J. Thromb. Haemost. 6, 1542–1549. Furugohri, T., Sugiyama, N., Morishima, Y., Shibano, T., 2011. Antithrombin-independent thrombin inhibitors, but not direct factor Xa inhibitors, enhance thrombin generation in plasma through inhibition of thrombin-thrombomodulin-protein C system. Thromb. Haemost. 106, 1076–1083. Granger, C.B., Alexander, J.H., McMurray, J.J., Lopes, R.D., Hylek, E.M., Hanna, M., AlKhalidi, H.R., Ansell, J., Atar, D., Avezum, A., Bahit, M.C., Diaz, R., Easton, J.D., Ezekowitz, J.A., Flaker, G., Garcia, D., Geraldes, M., Gersh, B.J., Golitsyn, S., Goto, S., Hermosillo, A.G., Hohnloser, S.H., Horowitz, J., Mohan, P., Jansky, P., Lewis, B.S., Lopez-Sendon, J.L., Pais, P., Parkhomenko, A., Verheugt, F.W., Zhu, J., Wallentin, L., ARISTOTLE Committees and Investigators, 2011. Apixaban versus warfarin in patients with atrial fibrillation. N. Engl. J. Med. 365, 981–992. Hara, T., Yokoyama, A., Tanabe, K., Ishihara, H., Iwamoto, M., 1995. DX-9065a, an orally active, specific inhibitor of factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb. Haemost. 74, 635–639. Hathcock, J., 2004. Vascular biology-the role of tissue factor. Semin. Hematol. 41, 30–34. He, R., Xiong, S., He, X., Liu, F., Han, J., Li, J., He, S., 2001. The role of factor XI in a dilute thromboplastin assay of extrinsic coagulation pathway. Thromb. Haemost. 85, 1055–1059. Jander, S., Sitzer, M., Wendt, A., Schroeter, M., Buchkremer, M., Siebler, M., Müller, W., Sandmann, W., Stoll, G., 2001. Expression of tissue factor in high-grade carotid artery stenosis: association with plaque destabilization. Stroke 32, 850–854. Mattsson, C., Menschik-Lundin, A., Nylander, S., Gyzander, E., Deinum, J., 2001. Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modulated activation of protein C in vitro. Thromb. Res. 104, 475–486. Morishima, Y., Tanabe, K., Terada, Y., Hara, T., Kunitada, S., 1997. Antithrombotic and hemorrhagic effects of DX-9065a, a direct and selective factor Xa inhibitor: comparison with a direct thrombin inhibitor and antithrombin III-dependent anticoagulants. Thromb. Haemost. 78, 1366–1371.
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Morishima, Y., Furugohri, T., Shiozaki, Y., Sugiyama, N., Shibano, T., 2006. Antithrombinindependent thrombin inhibitors, but not factor Xa inhibitors, enhance thrombin generation in human plasma via inhibition of Thrombin-Thrombomodulin-Protein C System. Blood (ASH Annual Meeting Abstracts), 108, p. 914. Patel, M.R., Mahaffey, K.W., Garg, J., Pan, G., Singer, D.E., Hacke, W., Breithardt, G., Halperin, J.L., Hankey, G.J., Piccini, J.P., Becker, R.C., Nessel, C.C., Paolini, J.F., Berkowitz, S.D., Fox, K.A., Califf, R.M., ROCKET AF Investigators, 2011. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N. Engl. J. Med. 365, 883–891. Perzborn, E., Harwardt, M., 2008. Direct thrombin inhibitors, but not factor Xa inhibitors, enhance thrombin formation in human plasma by interfering with the Thrombin-Thrombomodulin-Protein C system. Blood (ASH Annual Meeting Abstracts), 112, pp. 198–199. Perzborn, E., Buetehorn, U., Fischer, E., Harwardt, M., Klages, M., Trabandt, A., 2008. Hypercoagulability in rats is increased by low doses of the direct thrombin
inhibitor melagatran, but is reduced by the direct factor Xa inhibitor rivaroxaban. Eur. Heart J. 29, 828–829 (abstract supplement). Weitz, J.I., 2007. Factor Xa or thrombin: Is thrombin a better target? J. Thromb. Haemost. 5 (Suppl. 1), 65–67. Wittkowsky, A.K., 2005. Why warfarin and heparin need to overlap when treating acute venous thromboembolism. Dis. Mon. 51, 112–115. Yamazaki, M., Asakura, H., Aoshima, K., Saito, M., Jokaji, H., Uotani, C., Kumabashiri, I., Morishita, E., Ikeda, T., Matsuda, T., 1994. Effects of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against experimental disseminated intravascular coagulation in rats. Thromb. Haemost. 72, 392–396. Zafar, M.U., Vorchheimer, D.A., Gaztanaga, J., Velez, M., Yadegar, D., Moreno, P.R., Kunitada, S., Pagan, J., Fuster, V., Badimon, J.J., 2007. Antithrombotic effects of factor Xa inhibition with DU-176b: Phase-I study of an oral, direct factor Xa inhibitor using an ex-vivo flow chamber. Thromb. Haemost. 98, 883–888.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular Pharmacology
Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats Taketoshi Furugohri ⁎, Toshio Fukuda, Naoki Tsuji, Akemi Kita, Yoshiyuki Morishima, Toshiro Shibano Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan
a r t i c l e
i n f o
Article history: Received 19 December 2011 Received in revised form 26 March 2012 Accepted 5 April 2012 Available online 21 April 2012 Keywords: Edoxaban Factor Xa inhibitor Tissue factor Hypercoagulation Thrombin inhibitor
a b s t r a c t 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 10 min after tissue factor injection. Platelet numbers, thrombin–antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1 mg/kg, i.v.) suppressed platelet consumption and thrombin–antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1 mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. In addition, although melagatran (3 mg/kg, i.v.) improved coagulation status when tissue factor was given 5 min after the drug administration, and 2, 4 and 8 h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15–234 ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190 ng/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. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Factor Xa plays an important role in the blood coagulation cascade, serving as the juncture between the intrinsic and extrinsic system leading to the generation of thrombin, thus factor Xa is an attractive target for the prevention and treatment of thromboembolic diseases (Ansell, 2007). Thrombin also has a key role in the blood coagulation cascade (Weitz, 2007). Therefore, several oral direct factor Xa inhibitors and an oral direct thrombin inhibitor have been launched. But, there are some concerns about thrombogenesis by direct thrombin inhibitors. Compared to warfarin/placebo, the elevated risk of arterial cardiovascular events in patients treated with ximelagatran (FDA, 2004), a prodrug of direct thrombin inhibitor melagatran, and dabigatran (Connolly et al., 2009) were reported. We (Furugohri et al., 2005) and other group (Perzborn et al., 2008a) have previously demonstrated that a direct thrombin inhibitor, melagatran, induces a paradoxical activation of coagulation pathway in a rat model of tissue factor-induced hypercoagulation. Low-dose
⁎ Corresponding author. Tel.: + 81 3 3492 3131; fax: + 81 3 5436 8587. E-mail address: [email protected] (T. Furugohri). 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.04.031
oral administration of melagatran enhances platelet consumption and thrombin–antithrombin complex generation, suggesting that this paradoxical phenomenon may be implicated in coagulation activation observed with direct thrombin inhibitors in clinical trials. We presented the precise mechanism of this paradoxical activation of coagulation by the direct thrombin inhibitor in our in vitro studies (Furugohri et al., 2011; Morishima et al., 2006). In short, direct thrombin inhibitors increase the activation of coagulation by suppression of the thrombininduced negative-feedback system through the inhibition of protein C activation. The same result was reported by other group (Perzborn and Harwardt, 2008b). However, the precise mechanism underlying the paradoxical coagulation activation in vivo by melagatran remains to be fully elucidated. Thus, in this study, we determined the relationship between the plasma concentrations of melagatran and the coagulation status by changing treatment conditions such as doses of the compound and timing of coagulation induction after the administration of melagatran. In terms of the paradoxical coagulation activation by other anticoagulants, the effects of edoxaban (the free form of edoxaban tosilate hydrate: Japanese Accepted Name), a recently approved direct factor Xa inhibitor in Japan, on tissue factor-induced hypercoagulation are not being investigated, even though we have shown that a factor Xa
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inhibitor, DX-9065a, does not cause the aggravation of coagulation status induced by tissue factor. Moreover, it is not examined whether a different type of anticoagulant heparin, which exerts an antithrombin-dependent inhibition of thrombin and factor Xa, induces hypercoagulation in rats treated with tissue factor. Therefore, we directly compared the potential of edoxaban and heparin with that of melagatran to aggravate tissue factor-induced coagulation in rats. 2. Materials and methods 2.1. Reagents and drugs Edoxaban and melagatran were synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Heparin sodium was purchased from Novo Nordisk A/S (Copenhagen, Denmark). Tissue factor (Thromboplastin C Plus) and Enzygnost TAT micro were purchased from Siemens AG (Munich, Germany). Halothane was from Takeda Pharmaceutical (Osaka, Japan) and thiopental sodium was from Mitsubishi Tanabe Pharma (Osaka, Japan). 2.2. Animals Animal facilities, animal care and study programs were in accordance with the in-house guidelines of the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd. Eight or nine-week-old male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan) and maintained on an 8:00 am/8:00 pm light/dark schedule, temperature (23 ± 2 °C) and humidity (55 ± 20%). Rats were housed 5–6 per cage and food and water were available ad libitum. They were acclimated for 1 or 2 weeks. 2.3. Tissue factor-induced hypercoagulation model Rats (232–284 g) were anesthetized with thiopental (100 mg/kg, i.p.). Coagulation was induced by the injection of 2.8 U/kg tissue factor into the femoral vein. Blood samples were collected into plastic syringe containing citrate solution (9 volumes of blood to 1 volume of 3.13% sodium citrate solution) 10 min after tissue factor injection. Immediately after blood collection, platelet number was measured using an automatic hematology analyzer MEK-6358 (Nihon Kohden, Tokyo, Japan). Then, blood samples were centrifuged at 1500 ×g for 10 min at 4 °C and plasma samples were prepared. Plasma was stored at −70 °C until the measurement of the following parameters: thrombin–antithrombin complex and plasma concentrations of drugs. Thrombin-antithrombin complex concentrations were assayed according to the previous study with Enzygnost TAT micro (Morishima et al., 1997). 2.4. Drug treatment 2.4.1. Dose-dependent effects of a direct thrombin inhibitor, heparin and a factor Xa inhibitor on hypercoagulation Under the thiopental (100 mg/kg, i.p.) anesthesia, melagatran at doses of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1 mg/kg or heparin at doses of 0.1, 0.3, 1, 10 and 100 U/kg was injected into the jugular vein of rats. Hypercoagulation was induced 5 min after drug administration. Edoxaban (0.1, 0.5 and 2.5 mg/kg) was orally administered by gavage to fasted rats, and then the rats were anesthetized with thiopental. Hypercoagulation was induced 30 min after drug administration. 2.4.2. Time-dependent effects of direct thrombin inhibitor and factor Xa inhibitor after drug treatment on hypercoagulation The experiment protocol is shown in Fig. 1. Under the halothane (2–3%) anesthesia, melagatran (3 mg/kg) or edoxaban (0.3 mg/kg) was injected into the jugular vein of rats. After their administration, the rats were awoken except 5 min after dosing. Hypercoagulation was induced 5 min, 2, 4, 8 and 16 h after dosing under the thiopental anesthesia.
Fig. 1. The experimental protocol of time-dependent experiments.
2.5. Measurement of plasma concentration The plasma concentrations of drugs were measured according to the previous study (Furugohri et al., 2008; Zafar et al., 2007) by liquid chromatography-tandem mass spectrometry analysis. Lower limit of quantitation is shown Table 1–4. 2.6. Statistical analysis Analyses were performed using EXSAS ver.7.10 (ARM SYTEX, Osaka, Japan) based on SAS release 8.2 (SAS Institute Japan, Tokyo, Japan). Data are expressed as means ± S.E.M. unless otherwise noted and statistical significance was measured at the level of P b 0.05. Comparison of the platelet number and thrombin–antithrombin complex concentration between groups were analyzed by t-test or Dunnett multiple comparison method. Mortality rate of rats up to 60 min after tissue factor injection was analyzed by Fisher test. 3. Results 3.1. Dose-dependent effects of melagatran, heparin and edoxaban on hypercoagulation 3.1.1. Melagatran In the sham group, platelet number and thrombin–antithrombin complex concentration were 79.2 ± 2.1 × 10 4 cells/μl and 3.6 ± 0.4 ng/ ml, respectively (Fig. 2). Intravenous injection of tissue factor significantly reduced platelet number to 47.5 ± 2.3 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin–antithrombin complex to 341.9 ± 37.7 ng/ml (P b 0.001) (Fig. 2). Compared to the tissue factor-treated control group, a high dose of melagatran (1 mg/kg, i.v.) given 5 min before the hypercoagulation induction suppressed platelet consumption (68.0 ± 3.1 × 10 4 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (152.5 ± 6.0 ng/ml, P b 0.01). The plasma concentration of melagatran was 1190±182 ng/ml (Table 1). Lower doses of melagatran (0.01, 0.03 and 0.1 mg/kg, Table 1 Dose-dependent effect of melagatran on mortality in tissue factor-induced hypercoagulation rats and plasma melagatran concentrations. Treatment Control Melagatran
Dose (mg/kg, i.v.)
Mortality
Concentration (ng/ml)
0.001 0.003 0.01 0.03 0.1 0.3 1
0/6 0/6 3/6 4/6 4/6 3/6 0/6 0/6
1.3 ± 0.2 4.5 ± 1.2 22 ± 0 81 ± 6 234 ± 31 454 ± 31 1190 ± 182
Concentrations represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml.
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Table 2 Plasma edoxaban concentrations after orally administration in a tissue factor-induced hypercoagulation model. Treatment Edoxaban
Dose (mg/kg, p.o.)
Concentration (ng/ml)
0.1 0.5 2.5
b 5.0 16.2 ± 5.0 163 ± 41
Data represent the means ± S.D. Lower limit of quantitation is 5.0 ng/ml.
Table 4 Plasma edoxaban concentrations after i.v. administration in a tissue factor-induced hypercoagulation model. Treatment
Time after i.v.
Concentration (ng/ml)
Edoxaban 0.3 mg/kg, i.v.
5 min 2h 4h 8h 16 h
43 ± 4 4.2 ± 0.9 1.4 ± 0.3 b 1.0 b 1.0
Data represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml.
i.v.) significantly enhanced platelet consumption (9.1 ± 2.0, 6.4 ± 0.5, 10.3 ± 1.1 ×104 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (753.3 ± 61.4, 868.6± 49.4, 918.1 ± 13.5 ng/ml, P b 0.001). The range of mean plasma melagatran concentrations at which melagatran aggravated hypercoagulation was 22–234 ng/ml (Table 1). In this aggravating range, melagatran showed the tendency to increase mortality at 0.01 and 0.03 mg/kg (P = 0.06) (Table 1). Very low doses (0.001 and 0.003 mg/kg, i.v.) of melagatran did not affect coagulation. 3.1.2. Heparin In the sham group, platelet number and thrombin–antithrombin complex concentration were 74.1±3.0×104 cells/μl and 7.3±1.5 ng/ml, respectively (Fig. 3). Compared to the sham group intravenous injection of tissue factor significantly reduced platelet number to 40.6 ± 2.2 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin– antithrombin complex to 826.6 ± 89.4 ng/ml (P b 0.001). Heparin at 100 U/kg, i.v. inhibited platelet consumption (68.9 ± 1.6 × 10 4 cells/μl, P b 0.01) and thrombin–antithrombin complex generation (198.0 ± 23.8 ng/ml, P b 0.001) compared to those in the control group (Fig. 3). Lower doses of heparin (0.1, 0.3, 1 and 10 U/kg, i.v.) have no effects on platelet consumption and thrombin–antithrombin complex generation.
3.2.1. Melagatran Intravenous injection of tissue factor significantly reduced platelet number from 82.0 ± 1.9 × 10 4 cells/μl to 48.2 ± 2.7 × 10 4 cells/μl (P b 0.001) and increased concentration of thrombin–antithrombin complex from 5.0 ± 0.5 ng/ml to 384.7 ± 44.7 ng/ml (P b 0.001) compared to those in the sham group (Fig. 5). Melagatran inhibited platelet consumption (80.6±1.7×104 cells/μl, Pb 0.001) and thrombin– antithrombin complex generation (48.9±1.0 ng/ml, Pb 0.001) when hypercoagulation was induced 5 min after the drug administration (Fig. 5). These data indicated that melagatran effectively inhibited hypercoagulation at this time point with a mean plasma concentration of 3987 ng/ml. Melagatran, however, enhanced platelet consumption (5.7±0.4, 10.6±0.6, 23.1±4.3×104 cells/μl, Pb 0.001) and thrombin– antithrombin complex generation (836.1±21.5, 756.1±8.9, 603.0± 60.9 ng/ml, Pb 0.001) (Fig. 5), when coagulation was induced 2, 4 and 8 h after dosing. Significant numbers of rats died when coagulation was
3.1.3. Edoxaban Edoxaban (0.1, 0.5 and 2.5 mg/kg, p.o.) inhibited platelet consumption (47.8±2.8, 50.2±1.2, 56.9±2.4×104 cells/μl, Pb 0.05, Pb 0.01, Pb 0.001) and thrombin–antithrombin complex generation (173.1±22.0, 108.3± 17.2, 38.6±3.2 ng/ml, Pb 0.05, Pb 0.001, Pb 0.001) compared to those in the control group (Fig. 4). Mean plasma concentrations of edoxaban were b5.0, 16.2 and 163 ng/ml, respectively (Table 2). 3.2. Time-dependent effects of melagatran and edoxaban after drug treatment on hypercoagulation The time-dependent effects after the injection of melagatran (3 mg/kg, i.v.) or edoxaban (0.3 mg/kg, i.v.) on hypercoagulation were evaluated in a tissue factor-induced rat hypercoagulation model.
Table 3 Time-dependent effect after melagatran treatment on mortality in tissue factorinduced hypercoagulation rats and plasma melagatran concentrations. Treatment
Time after i.v.
Mortality
Concentration (ng/ml)
Control Melagatran 3 mg/kg, i.v.
5 min
0/8 0/8
3987 ± 338
2h 4h 8h 16 h
a
7/8 7/8a 3/7 0/8
126 ± 12 55 ± 15 15 ± 11 1.2 ± 1.2
The experiment protocol is shown in Fig. 1. Melagatran (3 mg/kg) was injected into the jugular vein of rats. Hypercoagulation was induced 5 min, 2, 4, 8 and 16 h after melagatran administration. Concentrations represent the means ± S.D. Lower limit of quantitation is 1.0 ng/ml. a Indicates statistically significant differences vs control values (P b 0.01).
Fig. 2. Effects of melagatran on tissue factor-induced hypercoagulation in rats. Melagatran at 1 mg/kg, i.v. suppressed platelet consumption (A) and thrombin– antithrombin complex generation (B) compared to the control group. Lower doses of melagatran (0.01–0.1 mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. Data represent means ± S.E.M. (n = 3–6). ### P b 0.001 vs. sham. ** P b 0.01, *** P b 0.001 vs. control.
T. Furugohri et al. / European Journal of Pharmacology 686 (2012) 74–80
Fig. 3. Effects of heparin on tissue factor-induced hypercoagulation in rats. Heparin at 100 U/kg, i.v. suppressed platelet consumption (A) and thrombin–antithrombin complex generation (B) compared to the control group. Lower doses of heparin (0.1– 10 U/kg, i.v.) have no effects on platelet consumption and thrombin–antithrombin complex generation.Data represent means ± S.E.M. (n = 5–6). ### P b 0.001 vs. sham. ** P b 0.01, *** P b 0.001 vs. control.
induced 2 and 4 h after dosing of melagatran (Table 3), probably due to respiratory distress provoked by pulmonary embolism. These findings demonstrated that melagatran aggravated hypercoagulation at these time points. After 16 h, the effects of melagatran towards inhibition or enhancement of coagulation disappeared. The range of plasma melagatran concentrations at which melagatran aggravated intravascular coagulation was 15–126 ng/ml (Table 3). 3.2.2. Edoxaban Edoxaban inhibited platelet consumption (84.3 ± 6.1 × 10 4 cells/μl, P b 0.001) and thrombin–antithrombin complex generation (26.2 ± 1.5 ng/ml, P b 0.001) compared to those in the control group (platelet number: 59.1±3.5× 104 cells/μl, thrombin–antithrombin complex concentration: 315.6±45.7 ng/ml), when hypercoagulation was induced 5 min after dosing (Fig. 6). At this time, plasma concentration of edoxaban was 43 ± 4 ng/ml (Table 4). Edoxaban had no effects on platelet number and significantly inhibited thrombin–antithrombin complex generation 2–16 h after drug administration (plasma concentration of edoxaban: b1.0–4.2 ng/ml, Table 4). Unlike melagatran, edoxaban did not show any exacerbation of tissue factor-induced hypercoagulation at any time points. 3.3. Relationship between plasma melagatran concentration and platelet number or thrombin–antithrombin complex concentration To determine the relationship between plasma melagatran concentration and platelet number or thrombin–antithrombin complex
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Fig. 4. Effects of edoxaban on tissue factor-induced hypercoagulation in rats. Edoxaban dose-dependently inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) compared to the control group. Data repressent means± S.E.M. (n= 6). ### P b 0.001 vs. sham. * P b 0.05, ** P b 0.01, *** P b 0.001 vs. control.
concentration, platelet numbers and thrombin–antithrombin complex concentrations obtained from dose- and time-dependent studies were plotted against plasma melagatran concentrations (Fig. 7). Higher concentrations (>454 ng/ml) of melagatran inhibited platelet consumption and thrombin–antithrombin complex generation. Lower concentrations (15–234 ng/ml) of melagatran aggravated hypercoagulation regardless of the different treatment conditions. These results indicated that aggravation of hypercoagulability by melagatran depends on its plasma concentrations at the induction of hypercoagulation. On the other hand, edoxaban inhibited but not aggravated hypercoagulation, regardless of its plasma concentrations. 4. Discussion Hypercoagulability after cessation of anticoagulant treatment has been concerned. We have shown that low-dose administration of a direct thrombin inhibitor melagatran, but not a prototype direct factor Xa inhibitor (DX-9065a), significantly worsens the coagulation status induced by tissue factor injection. In this study, we clarified the relationship between the plasma concentrations of melagatran and aggravation or improvement of coagulation status. Furthermore, we directly compared the potential of a novel oral factor Xa inhibitor, edoxaban with a Ki value of 0.561 nM (Furugohri et al., 2008), and an antithrombin-dependent anticoagulant, heparin, with that of a direct thrombin inhibitor, melagatran, to aggravate tissue factor-induced coagulation in rats. Tissue factor is a critical factor responsible for the formation of arterial thrombi, which is the cause of myocardial infarction and ischemic stroke. Tissue factor is a membrane-bound protein that is
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Fig. 5. Time-dependent effects after melagatran treatment on hypercoagulation. Melagatran inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) when hypercoagulation was induced 5 min after the drug administration. However, melagatran enhanced platelet consumption and thrombin–antithrombin complex generation, when hypercoagulation was induced 2, 4 and 8 h after dosing.Data represent means ± S.E.M. (n= 5–8). ### P b 0.001 vs. sham. *** P b 0.001 vs. control.
Fig. 6. Time-dependent effects after edoxaban treatment on hypercoagulation. Edoxaban inhibited platelet consumption (A) and thrombin–antithrombin complex generation (B) just after the drug administration (5 min). Edoxaban did not exert any deleterious effects and had no effects on platelet number and significantly inhibited thrombin–antithrombin complex generation 2–16 h after drug administration.Data represent means± S.E.M. (n= 6). ## P b 0.01, ### P b 0.001 vs. sham. * P b 0.05, *** P b 0.001 vs. control.
not normally expressed on vascular cells which are in contact with blood. Under pathological circumstances, however, tissue factor expression is induced in cells such as monocytes and endothelial cells and tissue factor is localized in the lipid core within the atherosclerotic lesions of the coronary (Hathcock, 2004) and carotid (Jander et al., 2001) arteries. Tissue factor initiates the extrinsic pathway of coagulation and contribute to thrombus formation after the plaque rupture leading to the onset of coronary artery diseases and ischemic stroke. Therefore, we used tissue factor as a trigger of hypercoagulation in our model to mimic the initiation of thrombus formation at the site of atherosclerotic plaques (Asakura et al., 2002; Dickneite et al., 1995; Hara et al., 1995; Yamazaki et al., 1994). Our study clearly demonstrates that the effects of melagatran on coagulation depended on the plasma drug concentration. The coagulation status was improved at high melagatran concentrations (1190 ng/ml or more) and aggravated at lower concentrations (15–234 ng/ml) in both dose-dependent and time-dependent models. Therefore, we speculate that when the plasma concentrations of the direct thrombin inhibitor decline below therapeutic ranges, the paradoxical effect (enhancement of coagulation activity) might occur. On the other hand, a direct factor Xa inhibitor, edoxaban, and an antithrombin-dependent anticoagulant, heparin, did not exert any deleterious effects, regardless of its plasma concentration or at any time points under this examination conditions. This phenomenon in vivo is well matched with that observed in our in vitro studies (Furugohri et al., 2011), in which low concentrations (4–315 ng/ml) of melagatran enhances thrombin generation in human plasma whereas at high concentration (630 ng/ml) the compound
inhibits it. In consistent with the results of the present study, direct factor Xa inhibitors and antithrombin-dependent anticoagulants do not enhance thrombin generation in our in vitro studies. The elevated risks of arterial cardiovascular events in patients treated with direct thrombin inhibitors were reported. Compared to warfarin/ placebo in patients undergoing orthopaedic surgery, ximelagatran, a prodrug of direct thrombin inhibitor melagatran, significantly elevates the risk of arterial cardiovascular events (FDA, 2004). Moreover, although it is marginally, another direct thrombin inhibitor, dabigatran, also increases the rate of myocardial infarction compared to warfarin (0.74%/year vs. 0.53%/year) in the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) study (Connolly et al., 2009). To the contrary, the symptoms of myocardial infarction were not observed in clinical studies with direct factor Xa inhibitors: Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET-AF) study (Patel et al., 2011) and Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) study (Granger et al., 2011). Thus, hypercoagulation observed in our in vitro and in vivo studies might contribute to clinical cardiovascular events like myocardial infarction associated with anticoagulant therapies with direct thrombin inhibitors. We clearly pointed out the precise mechanism of enhancement of thrombin generation by the direct thrombin inhibitor in our in vitro study (Furugohri et al., 2011). In short, direct thrombin inhibitors increase thrombin generation by suppression of the thrombin-induced negative-feedback system through the inhibition of protein C
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enhance thrombin generation in our in vitro study. Edoxaban is a highly selective factor Xa inhibitor and has no effect on serine protease activity of activated protein C. Heparin is not a selective thrombin inhibitor, which has inhibitory effects on multiple activated coagulation factors including thrombin, factor Xa, factor IXa and protein C activation via antithrombin (Mattsson et al., 2001). These differences in mechanism of action between factor Xa inhibitors/heparin and a direct thrombin inhibitor may result in the different regulation of blood coagulation. Further studies may be required to clarify the distinctions between these anticoagulants in vivo. In conclusion, we have shown that at certain concentrations a direct thrombin inhibitor, melagatran, aggravates coagulation status initiated by the injection of tissue factor. The inhibition or activation of coagulation clearly depends on the plasma concentration of melagatran. This phenomenon may implicate the coagulation activation observed with direct thrombin inhibitors in the clinical trials. On the other hand, a factor Xa inhibitor, edoxaban, and heparin does not exert any deleterious effects on tissue factor-induced hypercoagulation. Therefore, direct factor Xa inhibitors are more useful than direct thrombin inhibitors in terms of lower possibility of the activation of coagulation pathway. References
Fig. 7. Relationship between plasma concentration of melagatran and platelet number (A) or thrombin–antithrombin complex (B) in a tissue factor-induced hypercoagulation model in rats. The data of platelet numbers and thrombin–antithrombin complex concentrations in dose- and time-dependent studies were plotted against plasma melagatran concentrations. Higher concentrations (>454 ng/ml) of melagatran inhibited platelet consumption and thrombin–antithrombin complex generation. Lower concentrations (15–234 ng/ml) of melagatran enhanced platelet consumption and thrombin– antithrombin complex generation and increased mortality. Circles are data from the dosedependent study. Squares are from the time-dependent study. Open and closed symbols indicate survival and dead rats, respectively.
activation. Protein C is activated by the thrombin-thrombomodulin complex and activated protein C acts as a negative regulator of the coagulation pathway (Dahlback and Villoutreix, 2003; Esmon, 2003). Activated protein C exerts a potent anticoagulant effect via proteolysis of factor Va and VIIIa (negative feedback). Melagatran inhibits the activation of protein C through the inhibition of thrombomodulinbound thrombin (Mattsson et al., 2001). Therefore, it is likely that the inhibition of the negative feedback loop by melagatran may enhance tissue factor-induced hypercoagulation in vivo. This idea is also supported by a finding that relative deficiency in protein C is one drive for the hypercoagulable state during initiation of treatment with vitamin K antagonists (Wittkowsky, 2005). The higher concentration of thrombin inhibitors could be enough to directly inhibit thrombin activity and suppressed thrombin generation through the inhibition of the positive-feedback activation of factor V, VII, VIII and XI, which are converted to active forms by thrombin. Since the positive feedback reactions by thrombin play an important role in thrombin generation (He et al., 2001), the inhibition of the reactions by the higher concentration of thrombin inhibitors may predominate over the thrombin generation enhanced by the inhibition of protein C activation. In contrast, we demonstrated that edoxaban and heparin does not
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