AJCP / Original Article
Laboratory Measurements of the Oral Direct Factor Xa Inhibitor Edoxaban Comparison of Prothrombin Time, Activated Partial Thromboplastin Time, and Thrombin Generation Assay Yoshiyuki Morishima, PhD, and Chikako Kamisato From Biological Research Laboratories, R&D Division, Daiichi Sankyo, Tokyo, Japan. Key Words: Activated partial thromboplastin time; Coagulation assay; Direct factor Xa inhibitor; Monitoring; Prothrombin time; Thrombin generation assay Am J Clin Pathol February 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
ABSTRACT Objectives: Edoxaban, an oral direct factor Xa inhibitor, does not require routine monitoring. However, assessment of the anticoagulant effects may be required in certain situations. Methods: We investigated the effects of edoxaban on prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin generation using human platelet-poor plasma (PPP) or platelet-rich plasma (PRP). Results: Edoxaban concentration-dependently prolonged PT and aPTT. There was a considerable variation in the magnitude of PT prolongation among the reagents used. The variability in aPTT prolongation among the reagents was smaller than that of PT. Edoxaban concentrationdependently inhibited thrombin generation, with a more potent effect seen in PPP than in PRP. Thrombin generation assay was three times more sensitive to edoxaban than PT and aPTT. Conclusions: PT had disadvantages of a large variability among different PT reagents. aPTT could be used as a conventional and convenient test with a smaller variation among reagents. Thrombin generation was the most sensitive assay.
© American Society for Clinical Pathology
Direct oral anticoagulants (DOACs) such as factor Xa (FXa) and thrombin inhibitors are now available for the prophylaxis and treatment of thromboembolic diseases in substitution for vitamin K antagonists (VKAs) or heparins.1-3 A major advantage of DOACs over VKAs or unfractionated heparin is the unnecessity of routine and frequent monitoring aimed to gauge their anticoagulant activities, due to their predictable and consistent pharmacokinetic and pharmacodynamic profiles. However, sensitive laboratory assays may be required to measure the plasma concentrations or anticoagulant activities of DOACs in certain situations such as an overdose, severe bleeding, urgent surgery, or issues with noncompliance.4,5 Edoxaban (Lixiana; Daiichi Sankyo, Tokyo, Japan) is an oral direct FXa inhibitor with a Ki value of 0.561 nmol/L6 and a rapid onset of action.7 Edoxaban has been shown to be effective for the prophylaxis of venous thromboembolism (VTE) in patients after orthopedic surgery8-10 and as effective as warfarin, with a lower incidence of bleeding, for the prevention of stroke in patients with atrial fibrillation and for the treatment and secondary prevention of symptomatic VTE.11,12 In these phase III clinical studies, the doses of edoxaban were 30 and 60 mg once daily. The peak and trough plasma concentrations of 60 mg edoxaban were approximately 250 ng/mL (1-3 hours after administration) and 25 ng/mL (24 hours after administration), respectively.7,13 Edoxaban has been launched only in Japan at present and has not been approved for use in the United States and Europe yet. To measure the anticoagulant activity of edoxaban, clinical studies have used standard plasma clotting time assays such as prothrombin time (PT) and activated partial thromboplastin time (aPTT). Prolongation of PT and aPTT correlates closely with plasma concentrations of edoxaban in
Am J Clin Pathol 2015;143:241-247 241 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
❚Table 1❚ PT Reagents Evaluated PT Reagent
Manufacturer
Source
ISI
Control PT, s
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus
Sysmex (Kobe, Japan) Instrumentation Laboratory (Lexington, MA) Sysmex Instrumentation Laboratory Sysmex Instrumentation Laboratory Kyowa Medex (Tokyo, Japan)
Rabbit brain Rabbit brain
1.76 1.1
17.3 20.8
Recombinant human tissue factor Recombinant human tissue factor Human placental tissue factor Rabbit brain Rabbit brain
0.93 0.88 1.18 1.45 1.9
11.5 14.5 18.7 20.4 14.3
Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
ISI, international sensitivity index; PT, prothrombin time.
healthy individuals.7 However, it is well known that clotting times vary depending on the reagents and instruments used. Therefore, for monitoring of VKAs, PT is commonly normalized by converting to the international normalized ratio (INR).14 Thrombin generation assay (TGA), by means of the automated calibrated thrombogram, can measure not only the time of initiation of the coagulation response but also the amount of thrombin generated after the stimulation as a global coagulation assay.15 Thrombin generation can be used to evaluate the activities of anticoagulants both in plateletpoor plasma (PPP) and platelet-rich plasma (PRP).16,17 An in vitro study of the effects of edoxaban on clotting times and thrombin generation in PPP was reported by Samama et al.18 In the present study, we determined the effects of edoxaban on PT and aPTT in vitro to investigate if there is variability in responses of edoxaban among several different reagents that were not used in the study by Samama et al.18 We also examined the effect of edoxaban on thrombin generation not only in PPP but also in PRP to clarify which sample is suitable for the assay. In addition, we compared the sensitivity of these assays to detect the anticoagulant effects of edoxaban.
Materials and Methods Human Plasma Human blood was collected by venipuncture from healthy volunteers into syringes containing a one-tenth volume of 3.8% sodium citrate solution (Sysmex, Kobe, Japan). All procedures were performed with the permission of the ethics committees of the research institutes of Daiichi Sankyo. PRP was separated by centrifugation at 150g for 10 minutes at room temperature. PPP was obtained from the residues removed from PRP by centrifugation at 2,000g for 10 minutes at room temperature. In some experiments, pooled human plasma obtained from George King BioMedical (Overland Park, KS) was used.
242 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Materials Edoxaban tosilate hydrate (Japanese accepted name) was synthesized by Daiichi Sankyo. Saline and distilled water were purchased from Otsuka Pharmaceutical Factory (Naruto, Japan). Dimethyl sulfoxide (DMSO) was purchased from Nacalai Tesque (Kyoto, Japan). PT and aPTT reagents used in this study are listed in ❚Table 1❚ and ❚Table 2❚. PPPReagents, PRP-Reagents, FluCa-kit (Fluo-substrate and Fluo-buffer), and Thrombin Calibrator were purchased from Thrombinoscope BV (Maastricht, the Netherlands). Measurements of PT and aPTT Edoxaban solution dissolved in a 40% DMSO-saline solution (12 μL) was mixed with 1,188 μL plasma. PT was measured with a microcoagulometer Amelung KC-10A micro (Trinity Biotech Plc., Bray, Ireland) as follows. Plasma containing edoxaban at concentrations of 50 to 400 ng/mL or 5.5 to 548 ng/mL (50 μL) were preincubated at 37°C for 1 minute. Coagulation was initiated by the addition of 100 μL PT reagents, and clotting time was measured. Concentrations of edoxaban required to double PT (PT-CT2) were calculated. aPTT was measured with the microcoagulometer as follows. Plasma containing edoxaban at concentrations of 50 to 600 ng/mL (50 μL), 25 μL saline, and 50 μL aPTT reagents was mixed and preincubated at 37°C for 5 minutes. Coagulation was initiated by the addition of 25 μL of a 50-mmol/L CaCl2 solution, and clotting time was measured. Thrombin Generation Assay Thrombin generation was assayed by means of the Calibrated Automated Thrombogram with a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific, Waltham, MA) and the thrombinoscope software (Thrombinoscope BV). The assay was performed as follows: 75 μL PPP or PRP and 5 μL edoxaban solution (concentrations in plasma: 10-3,000 nmol/L or 5.5-1,640 ng/mL) were pipetted into the well of a 96-well microtiter plate, together with 20 μL PPP-Reagent (5 pmol/L tissue factor and 4 mmol/L phospholipids) or PRP-Reagent (1 pmol/L tissue factor).
© American Society for Clinical Pathology
AJCP / Original Article
❚Table 2❚ aPTT Reagents Evaluated aPTT Reagent
Manufacturer
Phospholipid
Platelin LS II Thrombocheck APTT HemosIL APTT-SP
Kyowa Medex (Tokyo, Japan) Sysmex (Kobe, Japan) Mitsubishi Chemical Medience Corporation (Tokyo, Japan) Roche Diagnostics K.K. (Tokyo, Japan) Sysmex Sysmex Roche Diagnostics Roche Diagnostics
Phospholipids from porcine and chicken Silica Cephalin from rabbit brain Ellagic acid Synthetic phospholipids Silica
38.1 26.8 37.3
Cephalin
Celite
41.3
Cephalin from soybeans Synthetic phospholipids Cephalin Cephalin
Ellagic acid Ellagic acid Silica Polyphenol
32.5 32.1 43.4 32.8
STA APTT Thrombocheck APTT(S) Thrombocheck APTT-SLA PTT LA RD STA Cephascreen (APTT)
Activator
Control aPTT, s
aPTT, activated partial thromboplastin time.
After 5 minutes of preincubation at 37°C, the reaction was started by the addition of 20 μL FluCa-kit. The fluorescence was measured for 120 minutes at 37°C (excitation, 390 nm; emission, 460 nm). To calculate the concentration of thrombin, we used 20 μL Thrombin Calibrator together with 75 μL plasma and 5 μL distilled water. The following five parameters of TGA were analyzed: lag time, maximum concentration of thrombin (peak), time to peak (ttPeak), endogenous thrombin potential (ETP), and mean thrombin generation rate (mRate). Concentrations of edoxaban required to reach 50% of inhibition (IC50) of peak, ETP, and mRate and concentrations required to double (CT2) lag time and ttPeak were calculated. Statistical Analysis Statistical analyses were performed by using SAS release 8.2 (SAS Institute, Cary, NC). The data represent mean ± standard error of the mean. In the TGA, 95% confidence intervals of IC50 and CT2 were calculated. The statistical significance of the data was analyzed by a parametric Dunnett multiple-comparison test. The statistical significance level was P less than .05. IC50 and CT2 were calculated by linear regression analysis.
Results Effect on PT PT values in control plasma (in the absence of edoxaban) using seven PT reagents are shown in Table 1. Edoxaban prolonged PT in a concentration-dependent manner. To compare the responses of seven different PT reagents with edoxaban, we calculated PT ratios to control PT value and PT-CT2 values for each reagent. PT ratio was linearly correlated with plasma concentrations of edoxaban across all PT reagents ❚Figure 1A❚. However, there was considerable variation in the magnitude of PT prolongation among the reagents used. The most and the least sensitive reagents were
© American Society for Clinical Pathology
HemosIL PT-Fibrinogen HS (Instrumentation Laboratory, Lexington, MA) and Dade Innovin (Sysmex), respectively (Figure 1A) ❚Table 3❚. Since the magnitude of PT prolongation by VKAs also varies depending on PT reagents and is normalized to INR, PT ratio was converted to INR using the international sensitivity index (ISI). Correction of PT ratio by edoxaban to INR did not reduce the variability in the PT response due to reagents ❚Figure 1B❚. Effect on aPTT aPTT values in control plasma using eight PT reagents are shown in Table 2. Edoxaban also prolonged aPTT in a concentration-dependent manner, and the curvilinear correlation was observed between plasma edoxaban concentrations and aPTT ratios to control aPTT across eight different aPTT reagents ❚Figure 2❚. The greatest sensitivity was observed with Thrombocheck APTT(S) (Sysmex). However, the other seven aPTT reagents produced similar aPTT prolongation. Comparison of PT and aPTT aPTT ratios at a therapeutic plasma edoxaban concentration of 200 ng/mL were 1.4 to 1.5 with the seven aPTT reagents (except for Thrombocheck APTT(S) [Sysmex], with an aPTT ratio of 1.74), whereas PT ratios at the same plasma concentration were 1.5 to 2.3, indicating that the magnitude of responses to edoxaban was greater with PT than with aPTT. The minimum concentrations of edoxaban needed to induce statistically significant prolongation of PT and aPTT using HemosIL PT-Fibrinogen HS Plus (Instrumentation Laboratory) and STA APTT (Roche Diagnostics K.K., Tokyo, Japan) were 55 and 50 ng/mL, respectively ❚Figure 3❚. Effect on Thrombin Generation Since TGA can be performed using both PPP and PRP, we determined the effect of edoxaban on thrombin generation in PPP and PRP and calculated IC50 or CT2 values for each parameter of thrombin generation. In both plasma samples, edoxaban inhibited thrombin generation in
Am J Clin Pathol 2015;143:241-247 243 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
A
3.5
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
6.0
3.0
5.0
2.5
4.0 INR
PT (Ratio to Control)
B
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
2.0 1.5
3.0 2.0
1.0 0
100
200
300
400
1.0
500
0
Plasma Edoxaban Concentration (ng/mL)
100
200
300
400
500
Plasma Edoxaban Concentration (ng/mL)
❚Figure 1❚ Effect of edoxaban on prothrombin time (PT) measured with seven PT reagents. A, PT ratio of human plasma samples spiked with edoxaban. B, Conversion to the international normalized ratio (INR). Data represent mean of duplicate measurement of pooled plasma. For manufacturers of the reagents, see Table 1. ❚Table 3❚ Concentrations of Edoxaban Required to Double PT PT-CT2, ng/mL
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
314 182 406 265 237 163 294
PT, prothrombin time; PT-CT2, concentrations of anticoagulants required to double PT. a For manufacturers, see Tables 1 and 2.
a concentration-dependent manner. Edoxaban prolonged the initiation phase of thrombin generation, as demonstrated by prolongation of lag time ❚Figure 4❚. Edoxaban also delayed and suppressed the propagation phase of thrombin generation, as shown by a prolongation of ttPeak and a decrease in mRate and peak. In total, edoxaban decreased ETP. The effects of edoxaban on peak, mRate, and ttPeak were more potent in PPP compared with PRP ❚Table 4❚. In PPP, the most sensitive parameter to detect the effect of edoxaban was mRate, with an IC50 of 0.047 μmol/L (Table 4). Threefold higher concentrations of edoxaban (0.139-0.188 μmol/L) were required to either double lag time or ttPeak or inhibit peak 50%. In this regard, the IC50 values for ETP were 30 times greater than that for mRate. In comparison with PT and aPTT, TGA was more sensitive to edoxaban. The minimum concentration of edoxaban needed to induce statistically significant inhibition or prolongation of thrombin generation parameters was 16.4 ng/mL (Figure 4), which was three times lower than that for PT or aPTT.
244 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
3.0 aPTT (Ratio to Control)
PT Reagenta
Platelin LS II Thrombocheck APTT HemosIL APTT-SP STA APTT Thrombocheck APTT(S) Thrombocheck APTT-SLA PTT-LA RD STA Cephascreen (APTT)
2.5
2.0
1.5
1.0 0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
❚Figure 2❚ Activated partial thromboplastin time (aPTT) ratio of human plasma samples spiked with edoxaban measured with eight aPTT reagents. Data represent mean ± standard error of the mean (n = 3). For manufacturers of the reagents, see Table 2.
Discussion Edoxaban is a direct FXa inhibitor that suppresses the coagulation pathway by inhibiting the generation of thrombin.6 To determine the anticoagulant effects of edoxaban, we performed the classic clotting time assays, PT and aPTT, and the newer global coagulation assay, TGA. PT and aPTT measure the time to initiate the clot formation after the stimulation of the extrinsic and intrinsic coagulation
© American Society for Clinical Pathology
AJCP / Original Article
A
B
60
100 ***
50
80
***
30
aPTT (s)
PT (s)
40 *** **
20
*
***
***
***
40 20
10 0
60
***
0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
0
0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
❚Figure 3❚ Effects of edoxaban on prothrombin time (PT) (A) measured with the HemosIL PT-Fibrinogen HS Plus (Instrumentation Laboratory, Lexington, MA) and activated partial thromboplastin time (aPTT) (B) measured with the STA APTT (Roche Diagnostics K.K., Tokyo, Japan). Data represent mean ± standard error of the mean (n = 12 or 3). *P < .05, **P < .01, ***P < .001 (Dunnett test). Solid line, mean; dotted line, 95% confidence interval.
pathways, respectively. In contrast, TGA measures not only the time to initiate the thrombin generation but also the amount of thrombin formed after stimulation with tissue factor. Edoxaban prolonged PT, aPTT, and time-based parameters of thrombin generation (lag time and ttPeak) and suppressed the rate and amount of thrombin generation (mRate, peak, and ETP) in a concentration-dependent manner. PT prolongation by edoxaban largely varied depending on PT reagents. In the case of VKAs, the variation in PT responses due to the thromboplastin reagents can be normalized to the INR.14 However, for edoxaban, considerable variation was still observed after the conversion of PT ratios to the INR using the ISI. Similar observations have been reported for edoxaban18 and the other FXa inhibitors, rivaroxaban and apixaban.19,20 In the present study, we determined PT prolongation by edoxaban using different PT reagents and a different coagulometer than Samama et al.18 Differential interactions between edoxaban and phospholipids or thromboplastin in these PT reagents might be the cause of the observed variability. aPTT was considered less sensitive to edoxaban than PT when CT2 values were compared.6 The present study indicates that the extent of aPTT prolongation was actually smaller (1.4- to 1.5-fold at a therapeutic plasma concentration of 200 ng/mL) than that of PT prolongation (1.5- to 2.3-fold at 200 ng/mL). However, the present study reveals that the minimum concentration needed to induce statistically significant prolongation was similar for both aPTT and PT. The advantage of aPTT over PT is the less variability between the different reagents. This advantage has been previously reported using four aPTT reagents.18 We expanded the data using eight reagents. One reagent (Thrombocheck APTT(S); Sysmex) was an outlier, but the other seven reagents produced similar aPTT ratios, whereas the normal aPTT values differed from each reagent.
© American Society for Clinical Pathology
The limitation of this study is that we used only a semiautomatic 10-channel microcoagulometer to measure PT and aPTT. Since automatic coagulometers are commonly used in clinical laboratories and clotting times might differ depending on analyzers, the PT and aPTT responses to edoxaban should be determined in detail using automatic coagulometers and corresponding reagents. We determined the effect of edoxaban on thrombin generation in PPP and PRP. Phospholipids are added as a scaffold of coagulation reaction in PPP, whereas the coagulation response occurs on the platelet membranes in PRP. Therefore, measurement in PRP might be more relevant to the physiologic condition. However, our data indicate that there is no benefit of using PRP as samples for TGA to evaluate the anticoagulant activity of edoxaban due to a lower sensitivity compared with PPP. Among five parameters of thrombin generation, mRate is the most sensitive to edoxaban. The least sensitive parameter was ETP. The minimum concentration needed to induce statistically significant effects on mRate, peak, lag time, and ttPeak was one-third of that for PT and aPTT. Specifically, mRate and peak are expected to detect the effect of edoxaban at the trough level (approximately 25 ng/mL)13 at a therapeutic dose of 60 mg once daily. Our previous clinical pharmacology study demonstrates that mRate and peak actually decreased 24 hours after the administration of 60 mg edoxaban compared with baseline values.21 It has been reported that another promising laboratory measurement of direct FXa inhibitors is an anti–FXa-based chromogenic assay using calibrators.22-25 The assay is a more sensitive and specific method than PT or aPTT for the measurement of plasma concentrations of FXa inhibitors. Edoxaban does not require routine and frequent monitoring of anticoagulant activity.8-12 However, laboratory
Am J Clin Pathol 2015;143:241-247 245 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
A
B
350
2,000
250
ETP (nmol/L min)
Peak (nmol/L)
300 ***
200 ***
150 100
***
**
1,000
***
***
500
***
50 0 Control
1,500
*** 10
100
0 Control
1,000
D
100
***
15 80 ** 60 40
***
20 0 Control
*** 10
***
*** 1,000
100
35
***
30 ***
20 ***
15
*** *** ***
0 Control
***
10
100
1,000
Edoxaban (ng/mL)
40
25
10
5
Edoxaban (ng/mL)
Time to Peak (min)
1,000
20
Lag Time (min)
Mean Thrombin Generation Rate (nmol/L per min)
120
E
100
Edoxaban (ng/mL)
Edoxaban (ng/mL)
C
10
❚Figure 4❚ Effect of edoxaban on the parameters of thrombin generation assay in human platelet-poor plasma. A, Maximum concentration of thrombin (peak). B, Endogenous thrombin potential (ETP). C, Mean thrombin generation rate. D, Lag time. E, Time to peak. Data represent mean ± standard error of the mean (n = 12). **P < .01, ***P < .001 (Dunnett test). Solid line, mean; dotted line, 95% confidence interval.
***
10
***
5 0 Control
10
100
1,000
Edoxaban (ng/mL)
❚Table 4❚ IC50 and CT2 Values of Edoxaban for Parameters of Thrombin Generation Assay in Human PPP and PRPa Sample
Peak IC50, μmol/L
mRate IC50, μmol/L
ETP IC50, μmol/L
Lag Time CT2, μmol/L
ttPeak CT2, μmol/L
PPP PRP
0.145 (0.128-0.161) 0.305 (0.229-0.382)
0.047 (0.041-0.054) 0.230 (0.166-0.294)
1.39 (0.971-1.81) 0.636 (0.447-0.825)
0.188 (0.176-0.201) 0.191 (0.160-0.223)
0.139 (0.117-0.161) 0.588 (0.386-0.790)
CT2, concentrations of anticoagulants required to double time; ETP, endogenous thrombin potential; IC50, concentrations required for 50% inhibition; mRate, mean thrombin generation rate; peak, maximum concentration of thrombin; PPP, platelet-poor plasma; PRP, platelet-rich plasma; ttPeak, time to peak. a Values in parentheses are 95% confidence intervals.
246 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
© American Society for Clinical Pathology
AJCP / Original Article
assays that measure the anticoagulant activity of edoxaban may be valuable in certain situations such as an overdose, severe bleeding, urgent surgery, or issues with noncompliance. The present study concludes that PT has disadvantages of a large variability among the different reagents that is not corrected by using the INR and a lack of sensitivity to detect the activity of edoxaban at the trough level. Although aPTT is less sensitive than PT, it could be used as a conventional and convenient diagnostic test at peak plasma levels or in overdose situations because of a small variability in response using different reagents. The TGA, especially peak and mRate, is the most sensitive to edoxaban, suggesting the detection of the edoxaban activity at the trough level. However, further validation of these methods is required in clinical settings, such as using plasma samples of patients who receive edoxaban. Address reprint requests to Dr Morishima, Biological Research Laboratories, R&D Division, Daiichi Sankyo, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan; morishima.yoshiyuki.t4@ daiichisankyo.co.jp. Disclosure: All authors are employees of Daiichi Sankyo. Acknowledgments: The authors thank Yoko Shiozaki for technical assistance and Yuko Honda for editorial assistance. Additional editorial support was provided by AlphaBioCom, LLC, King of Prussia, PA, and funded by Daiichi Sankyo.
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9. Fuji T, Fujita S, Tachibana S, et al. Efficacy and safety of edoxaban versus enoxaparin for the prevention of venous thromboembolism following total hip arthroplasty: STARS J-V trial. ASH Annual Meeting Abstracts. 2010;116:3320. 10. Fuji T, Fujita S, Tachibana S, et al. Edoxaban versus enoxaparin for the prevention of venous thromboembolism: pooled analysis of venous thromboembolism and bleeding from STARS E-3 and STARS J-V. ASH Annual Meeting Abstracts. 2011;118:208. 11. Giugliano RP, Ruff CT, Braunwald E, et al. Edoxaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2013;369:2093-2104. 12. Hokusai-VTE Investigators, Büller HR, Décousus H, et al. Edoxaban versus warfarin for the treatment of symptomatic venous thromboembolism. N Engl J Med. 2013;369:1406-1415. 13. Weitz JI, Connolly SJ, Patel I, et al. Randomised, parallelgroup, multicentre, multinational phase 2 study comparing edoxaban, an oral factor Xa inhibitor, with warfarin for stroke prevention in patients with atrial fibrillation. Thromb Haemost. 2010;104:633-641. 14. Hirsh J. Optimal intensity and monitoring warfarin. Am J Cardiol. 1995;75:39B-42B. 15. Hemker HC, Giesen P, AlDieri R, et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002;32:249-253. 16. Gerotziafas GT, Petropoulou AD, Verdy E, et al. Effect of the anti–factor Xa and anti–factor IIa activities of low-molecularweight heparins upon the phases of thrombin generation. J Thromb Haemost. 2007;5:955-962. 17. Samama MM, Le Flem L, Guinet C, et al. Three different patterns of calibrated automated thrombogram obtained with six different anticoagulants. J Thromb Haemost. 2007;5:2554-2556. 18. Samama MM, Mendell J, Guinet C, et al. In vitro study of the anticoagulant effects of edoxaban and its effect on thrombin generation in comparison to fondaparinux. Thromb Res. 2012;129:e77-e82. 19. Samama MM, Martinoli JL, LeFlem L, et al. Assessment of laboratory assays to measure rivaroxaban—an oral, direct factor Xa inhibitor. Thromb Haemost. 2010;103:815-825. 20. Barrett YC, Wang Z, Frost C, et al. Clinical laboratory measurement of direct factor Xa inhibitors: anti-Xa assay is preferable to prothrombin time assay. Thromb Haemost. 2010;104:1263-1271. 21. Samama MM, Kunitada S, Oursin A, et al. Comparison of a direct factor Xa inhibitor, edoxaban, with dalteparin and ximelagatran: a randomised controlled trial in healthy elderly adults. Thromb Res. 2010;126:e286-e293. 22. Barrett YC, Wang Z, Frost C, et al. Clinical laboratory measurement of direct factor Xa inhibitors: anti-Xa assay is preferable to prothrombin time assay. Thromb Haemost. 2010;104:1263-1271. 23. Samama MM, Contant G, Spiro TE, et al. Evaluation of the anti–factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations using calibrators and controls. Thromb Haemost. 2012;107:379-387. 24. Gouin-Thibault I, Flaujac C, Delavenne X, et al. Assessment of apixaban plasma levels by laboratory tests: suitability of three anti-Xa assays. A multicentre French GEHT study. Thromb Haemost. 2014;111:240-248. 25. Francart SJ, Hawes EM, Deal AM, et al. Performance of coagulation tests in patients on therapeutic doses of rivaroxaban: a cross-sectional pharmacodynamic study based on peak and trough plasma levels. Thromb Haemost. 2014;111:1133-1140.
Am J Clin Pathol 2015;143:241-247 247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Laboratory Measurements of the Oral Direct Factor Xa Inhibitor Edoxaban Comparison of Prothrombin Time, Activated Partial Thromboplastin Time, and Thrombin Generation Assay Yoshiyuki Morishima, PhD, and Chikako Kamisato From Biological Research Laboratories, R&D Division, Daiichi Sankyo, Tokyo, Japan. Key Words: Activated partial thromboplastin time; Coagulation assay; Direct factor Xa inhibitor; Monitoring; Prothrombin time; Thrombin generation assay Am J Clin Pathol February 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
ABSTRACT Objectives: Edoxaban, an oral direct factor Xa inhibitor, does not require routine monitoring. However, assessment of the anticoagulant effects may be required in certain situations. Methods: We investigated the effects of edoxaban on prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin generation using human platelet-poor plasma (PPP) or platelet-rich plasma (PRP). Results: Edoxaban concentration-dependently prolonged PT and aPTT. There was a considerable variation in the magnitude of PT prolongation among the reagents used. The variability in aPTT prolongation among the reagents was smaller than that of PT. Edoxaban concentrationdependently inhibited thrombin generation, with a more potent effect seen in PPP than in PRP. Thrombin generation assay was three times more sensitive to edoxaban than PT and aPTT. Conclusions: PT had disadvantages of a large variability among different PT reagents. aPTT could be used as a conventional and convenient test with a smaller variation among reagents. Thrombin generation was the most sensitive assay.
© American Society for Clinical Pathology
Direct oral anticoagulants (DOACs) such as factor Xa (FXa) and thrombin inhibitors are now available for the prophylaxis and treatment of thromboembolic diseases in substitution for vitamin K antagonists (VKAs) or heparins.1-3 A major advantage of DOACs over VKAs or unfractionated heparin is the unnecessity of routine and frequent monitoring aimed to gauge their anticoagulant activities, due to their predictable and consistent pharmacokinetic and pharmacodynamic profiles. However, sensitive laboratory assays may be required to measure the plasma concentrations or anticoagulant activities of DOACs in certain situations such as an overdose, severe bleeding, urgent surgery, or issues with noncompliance.4,5 Edoxaban (Lixiana; Daiichi Sankyo, Tokyo, Japan) is an oral direct FXa inhibitor with a Ki value of 0.561 nmol/L6 and a rapid onset of action.7 Edoxaban has been shown to be effective for the prophylaxis of venous thromboembolism (VTE) in patients after orthopedic surgery8-10 and as effective as warfarin, with a lower incidence of bleeding, for the prevention of stroke in patients with atrial fibrillation and for the treatment and secondary prevention of symptomatic VTE.11,12 In these phase III clinical studies, the doses of edoxaban were 30 and 60 mg once daily. The peak and trough plasma concentrations of 60 mg edoxaban were approximately 250 ng/mL (1-3 hours after administration) and 25 ng/mL (24 hours after administration), respectively.7,13 Edoxaban has been launched only in Japan at present and has not been approved for use in the United States and Europe yet. To measure the anticoagulant activity of edoxaban, clinical studies have used standard plasma clotting time assays such as prothrombin time (PT) and activated partial thromboplastin time (aPTT). Prolongation of PT and aPTT correlates closely with plasma concentrations of edoxaban in
Am J Clin Pathol 2015;143:241-247 241 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
❚Table 1❚ PT Reagents Evaluated PT Reagent
Manufacturer
Source
ISI
Control PT, s
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus
Sysmex (Kobe, Japan) Instrumentation Laboratory (Lexington, MA) Sysmex Instrumentation Laboratory Sysmex Instrumentation Laboratory Kyowa Medex (Tokyo, Japan)
Rabbit brain Rabbit brain
1.76 1.1
17.3 20.8
Recombinant human tissue factor Recombinant human tissue factor Human placental tissue factor Rabbit brain Rabbit brain
0.93 0.88 1.18 1.45 1.9
11.5 14.5 18.7 20.4 14.3
Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
ISI, international sensitivity index; PT, prothrombin time.
healthy individuals.7 However, it is well known that clotting times vary depending on the reagents and instruments used. Therefore, for monitoring of VKAs, PT is commonly normalized by converting to the international normalized ratio (INR).14 Thrombin generation assay (TGA), by means of the automated calibrated thrombogram, can measure not only the time of initiation of the coagulation response but also the amount of thrombin generated after the stimulation as a global coagulation assay.15 Thrombin generation can be used to evaluate the activities of anticoagulants both in plateletpoor plasma (PPP) and platelet-rich plasma (PRP).16,17 An in vitro study of the effects of edoxaban on clotting times and thrombin generation in PPP was reported by Samama et al.18 In the present study, we determined the effects of edoxaban on PT and aPTT in vitro to investigate if there is variability in responses of edoxaban among several different reagents that were not used in the study by Samama et al.18 We also examined the effect of edoxaban on thrombin generation not only in PPP but also in PRP to clarify which sample is suitable for the assay. In addition, we compared the sensitivity of these assays to detect the anticoagulant effects of edoxaban.
Materials and Methods Human Plasma Human blood was collected by venipuncture from healthy volunteers into syringes containing a one-tenth volume of 3.8% sodium citrate solution (Sysmex, Kobe, Japan). All procedures were performed with the permission of the ethics committees of the research institutes of Daiichi Sankyo. PRP was separated by centrifugation at 150g for 10 minutes at room temperature. PPP was obtained from the residues removed from PRP by centrifugation at 2,000g for 10 minutes at room temperature. In some experiments, pooled human plasma obtained from George King BioMedical (Overland Park, KS) was used.
242 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Materials Edoxaban tosilate hydrate (Japanese accepted name) was synthesized by Daiichi Sankyo. Saline and distilled water were purchased from Otsuka Pharmaceutical Factory (Naruto, Japan). Dimethyl sulfoxide (DMSO) was purchased from Nacalai Tesque (Kyoto, Japan). PT and aPTT reagents used in this study are listed in ❚Table 1❚ and ❚Table 2❚. PPPReagents, PRP-Reagents, FluCa-kit (Fluo-substrate and Fluo-buffer), and Thrombin Calibrator were purchased from Thrombinoscope BV (Maastricht, the Netherlands). Measurements of PT and aPTT Edoxaban solution dissolved in a 40% DMSO-saline solution (12 μL) was mixed with 1,188 μL plasma. PT was measured with a microcoagulometer Amelung KC-10A micro (Trinity Biotech Plc., Bray, Ireland) as follows. Plasma containing edoxaban at concentrations of 50 to 400 ng/mL or 5.5 to 548 ng/mL (50 μL) were preincubated at 37°C for 1 minute. Coagulation was initiated by the addition of 100 μL PT reagents, and clotting time was measured. Concentrations of edoxaban required to double PT (PT-CT2) were calculated. aPTT was measured with the microcoagulometer as follows. Plasma containing edoxaban at concentrations of 50 to 600 ng/mL (50 μL), 25 μL saline, and 50 μL aPTT reagents was mixed and preincubated at 37°C for 5 minutes. Coagulation was initiated by the addition of 25 μL of a 50-mmol/L CaCl2 solution, and clotting time was measured. Thrombin Generation Assay Thrombin generation was assayed by means of the Calibrated Automated Thrombogram with a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific, Waltham, MA) and the thrombinoscope software (Thrombinoscope BV). The assay was performed as follows: 75 μL PPP or PRP and 5 μL edoxaban solution (concentrations in plasma: 10-3,000 nmol/L or 5.5-1,640 ng/mL) were pipetted into the well of a 96-well microtiter plate, together with 20 μL PPP-Reagent (5 pmol/L tissue factor and 4 mmol/L phospholipids) or PRP-Reagent (1 pmol/L tissue factor).
© American Society for Clinical Pathology
AJCP / Original Article
❚Table 2❚ aPTT Reagents Evaluated aPTT Reagent
Manufacturer
Phospholipid
Platelin LS II Thrombocheck APTT HemosIL APTT-SP
Kyowa Medex (Tokyo, Japan) Sysmex (Kobe, Japan) Mitsubishi Chemical Medience Corporation (Tokyo, Japan) Roche Diagnostics K.K. (Tokyo, Japan) Sysmex Sysmex Roche Diagnostics Roche Diagnostics
Phospholipids from porcine and chicken Silica Cephalin from rabbit brain Ellagic acid Synthetic phospholipids Silica
38.1 26.8 37.3
Cephalin
Celite
41.3
Cephalin from soybeans Synthetic phospholipids Cephalin Cephalin
Ellagic acid Ellagic acid Silica Polyphenol
32.5 32.1 43.4 32.8
STA APTT Thrombocheck APTT(S) Thrombocheck APTT-SLA PTT LA RD STA Cephascreen (APTT)
Activator
Control aPTT, s
aPTT, activated partial thromboplastin time.
After 5 minutes of preincubation at 37°C, the reaction was started by the addition of 20 μL FluCa-kit. The fluorescence was measured for 120 minutes at 37°C (excitation, 390 nm; emission, 460 nm). To calculate the concentration of thrombin, we used 20 μL Thrombin Calibrator together with 75 μL plasma and 5 μL distilled water. The following five parameters of TGA were analyzed: lag time, maximum concentration of thrombin (peak), time to peak (ttPeak), endogenous thrombin potential (ETP), and mean thrombin generation rate (mRate). Concentrations of edoxaban required to reach 50% of inhibition (IC50) of peak, ETP, and mRate and concentrations required to double (CT2) lag time and ttPeak were calculated. Statistical Analysis Statistical analyses were performed by using SAS release 8.2 (SAS Institute, Cary, NC). The data represent mean ± standard error of the mean. In the TGA, 95% confidence intervals of IC50 and CT2 were calculated. The statistical significance of the data was analyzed by a parametric Dunnett multiple-comparison test. The statistical significance level was P less than .05. IC50 and CT2 were calculated by linear regression analysis.
Results Effect on PT PT values in control plasma (in the absence of edoxaban) using seven PT reagents are shown in Table 1. Edoxaban prolonged PT in a concentration-dependent manner. To compare the responses of seven different PT reagents with edoxaban, we calculated PT ratios to control PT value and PT-CT2 values for each reagent. PT ratio was linearly correlated with plasma concentrations of edoxaban across all PT reagents ❚Figure 1A❚. However, there was considerable variation in the magnitude of PT prolongation among the reagents used. The most and the least sensitive reagents were
© American Society for Clinical Pathology
HemosIL PT-Fibrinogen HS (Instrumentation Laboratory, Lexington, MA) and Dade Innovin (Sysmex), respectively (Figure 1A) ❚Table 3❚. Since the magnitude of PT prolongation by VKAs also varies depending on PT reagents and is normalized to INR, PT ratio was converted to INR using the international sensitivity index (ISI). Correction of PT ratio by edoxaban to INR did not reduce the variability in the PT response due to reagents ❚Figure 1B❚. Effect on aPTT aPTT values in control plasma using eight PT reagents are shown in Table 2. Edoxaban also prolonged aPTT in a concentration-dependent manner, and the curvilinear correlation was observed between plasma edoxaban concentrations and aPTT ratios to control aPTT across eight different aPTT reagents ❚Figure 2❚. The greatest sensitivity was observed with Thrombocheck APTT(S) (Sysmex). However, the other seven aPTT reagents produced similar aPTT prolongation. Comparison of PT and aPTT aPTT ratios at a therapeutic plasma edoxaban concentration of 200 ng/mL were 1.4 to 1.5 with the seven aPTT reagents (except for Thrombocheck APTT(S) [Sysmex], with an aPTT ratio of 1.74), whereas PT ratios at the same plasma concentration were 1.5 to 2.3, indicating that the magnitude of responses to edoxaban was greater with PT than with aPTT. The minimum concentrations of edoxaban needed to induce statistically significant prolongation of PT and aPTT using HemosIL PT-Fibrinogen HS Plus (Instrumentation Laboratory) and STA APTT (Roche Diagnostics K.K., Tokyo, Japan) were 55 and 50 ng/mL, respectively ❚Figure 3❚. Effect on Thrombin Generation Since TGA can be performed using both PPP and PRP, we determined the effect of edoxaban on thrombin generation in PPP and PRP and calculated IC50 or CT2 values for each parameter of thrombin generation. In both plasma samples, edoxaban inhibited thrombin generation in
Am J Clin Pathol 2015;143:241-247 243 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
A
3.5
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
6.0
3.0
5.0
2.5
4.0 INR
PT (Ratio to Control)
B
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
2.0 1.5
3.0 2.0
1.0 0
100
200
300
400
1.0
500
0
Plasma Edoxaban Concentration (ng/mL)
100
200
300
400
500
Plasma Edoxaban Concentration (ng/mL)
❚Figure 1❚ Effect of edoxaban on prothrombin time (PT) measured with seven PT reagents. A, PT ratio of human plasma samples spiked with edoxaban. B, Conversion to the international normalized ratio (INR). Data represent mean of duplicate measurement of pooled plasma. For manufacturers of the reagents, see Table 1. ❚Table 3❚ Concentrations of Edoxaban Required to Double PT PT-CT2, ng/mL
Thromboplastin C Plus HemosIL PT-Fibrinogen HS Plus Dade Innovin Hemoliance RecombiPlasTin Thromborel S HemosIL PT-Fibrinogen HS Simplastin Excel
314 182 406 265 237 163 294
PT, prothrombin time; PT-CT2, concentrations of anticoagulants required to double PT. a For manufacturers, see Tables 1 and 2.
a concentration-dependent manner. Edoxaban prolonged the initiation phase of thrombin generation, as demonstrated by prolongation of lag time ❚Figure 4❚. Edoxaban also delayed and suppressed the propagation phase of thrombin generation, as shown by a prolongation of ttPeak and a decrease in mRate and peak. In total, edoxaban decreased ETP. The effects of edoxaban on peak, mRate, and ttPeak were more potent in PPP compared with PRP ❚Table 4❚. In PPP, the most sensitive parameter to detect the effect of edoxaban was mRate, with an IC50 of 0.047 μmol/L (Table 4). Threefold higher concentrations of edoxaban (0.139-0.188 μmol/L) were required to either double lag time or ttPeak or inhibit peak 50%. In this regard, the IC50 values for ETP were 30 times greater than that for mRate. In comparison with PT and aPTT, TGA was more sensitive to edoxaban. The minimum concentration of edoxaban needed to induce statistically significant inhibition or prolongation of thrombin generation parameters was 16.4 ng/mL (Figure 4), which was three times lower than that for PT or aPTT.
244 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
3.0 aPTT (Ratio to Control)
PT Reagenta
Platelin LS II Thrombocheck APTT HemosIL APTT-SP STA APTT Thrombocheck APTT(S) Thrombocheck APTT-SLA PTT-LA RD STA Cephascreen (APTT)
2.5
2.0
1.5
1.0 0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
❚Figure 2❚ Activated partial thromboplastin time (aPTT) ratio of human plasma samples spiked with edoxaban measured with eight aPTT reagents. Data represent mean ± standard error of the mean (n = 3). For manufacturers of the reagents, see Table 2.
Discussion Edoxaban is a direct FXa inhibitor that suppresses the coagulation pathway by inhibiting the generation of thrombin.6 To determine the anticoagulant effects of edoxaban, we performed the classic clotting time assays, PT and aPTT, and the newer global coagulation assay, TGA. PT and aPTT measure the time to initiate the clot formation after the stimulation of the extrinsic and intrinsic coagulation
© American Society for Clinical Pathology
AJCP / Original Article
A
B
60
100 ***
50
80
***
30
aPTT (s)
PT (s)
40 *** **
20
*
***
***
***
40 20
10 0
60
***
0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
0
0
100
200
300
400
500
600
Plasma Edoxaban Concentration (ng/mL)
❚Figure 3❚ Effects of edoxaban on prothrombin time (PT) (A) measured with the HemosIL PT-Fibrinogen HS Plus (Instrumentation Laboratory, Lexington, MA) and activated partial thromboplastin time (aPTT) (B) measured with the STA APTT (Roche Diagnostics K.K., Tokyo, Japan). Data represent mean ± standard error of the mean (n = 12 or 3). *P < .05, **P < .01, ***P < .001 (Dunnett test). Solid line, mean; dotted line, 95% confidence interval.
pathways, respectively. In contrast, TGA measures not only the time to initiate the thrombin generation but also the amount of thrombin formed after stimulation with tissue factor. Edoxaban prolonged PT, aPTT, and time-based parameters of thrombin generation (lag time and ttPeak) and suppressed the rate and amount of thrombin generation (mRate, peak, and ETP) in a concentration-dependent manner. PT prolongation by edoxaban largely varied depending on PT reagents. In the case of VKAs, the variation in PT responses due to the thromboplastin reagents can be normalized to the INR.14 However, for edoxaban, considerable variation was still observed after the conversion of PT ratios to the INR using the ISI. Similar observations have been reported for edoxaban18 and the other FXa inhibitors, rivaroxaban and apixaban.19,20 In the present study, we determined PT prolongation by edoxaban using different PT reagents and a different coagulometer than Samama et al.18 Differential interactions between edoxaban and phospholipids or thromboplastin in these PT reagents might be the cause of the observed variability. aPTT was considered less sensitive to edoxaban than PT when CT2 values were compared.6 The present study indicates that the extent of aPTT prolongation was actually smaller (1.4- to 1.5-fold at a therapeutic plasma concentration of 200 ng/mL) than that of PT prolongation (1.5- to 2.3-fold at 200 ng/mL). However, the present study reveals that the minimum concentration needed to induce statistically significant prolongation was similar for both aPTT and PT. The advantage of aPTT over PT is the less variability between the different reagents. This advantage has been previously reported using four aPTT reagents.18 We expanded the data using eight reagents. One reagent (Thrombocheck APTT(S); Sysmex) was an outlier, but the other seven reagents produced similar aPTT ratios, whereas the normal aPTT values differed from each reagent.
© American Society for Clinical Pathology
The limitation of this study is that we used only a semiautomatic 10-channel microcoagulometer to measure PT and aPTT. Since automatic coagulometers are commonly used in clinical laboratories and clotting times might differ depending on analyzers, the PT and aPTT responses to edoxaban should be determined in detail using automatic coagulometers and corresponding reagents. We determined the effect of edoxaban on thrombin generation in PPP and PRP. Phospholipids are added as a scaffold of coagulation reaction in PPP, whereas the coagulation response occurs on the platelet membranes in PRP. Therefore, measurement in PRP might be more relevant to the physiologic condition. However, our data indicate that there is no benefit of using PRP as samples for TGA to evaluate the anticoagulant activity of edoxaban due to a lower sensitivity compared with PPP. Among five parameters of thrombin generation, mRate is the most sensitive to edoxaban. The least sensitive parameter was ETP. The minimum concentration needed to induce statistically significant effects on mRate, peak, lag time, and ttPeak was one-third of that for PT and aPTT. Specifically, mRate and peak are expected to detect the effect of edoxaban at the trough level (approximately 25 ng/mL)13 at a therapeutic dose of 60 mg once daily. Our previous clinical pharmacology study demonstrates that mRate and peak actually decreased 24 hours after the administration of 60 mg edoxaban compared with baseline values.21 It has been reported that another promising laboratory measurement of direct FXa inhibitors is an anti–FXa-based chromogenic assay using calibrators.22-25 The assay is a more sensitive and specific method than PT or aPTT for the measurement of plasma concentrations of FXa inhibitors. Edoxaban does not require routine and frequent monitoring of anticoagulant activity.8-12 However, laboratory
Am J Clin Pathol 2015;143:241-247 245 DOI: 10.1309/AJCPQ2NJD3PXFTUG
Morishima and Kamisato / Laboratory Measurements of Edoxaban
A
B
350
2,000
250
ETP (nmol/L min)
Peak (nmol/L)
300 ***
200 ***
150 100
***
**
1,000
***
***
500
***
50 0 Control
1,500
*** 10
100
0 Control
1,000
D
100
***
15 80 ** 60 40
***
20 0 Control
*** 10
***
*** 1,000
100
35
***
30 ***
20 ***
15
*** *** ***
0 Control
***
10
100
1,000
Edoxaban (ng/mL)
40
25
10
5
Edoxaban (ng/mL)
Time to Peak (min)
1,000
20
Lag Time (min)
Mean Thrombin Generation Rate (nmol/L per min)
120
E
100
Edoxaban (ng/mL)
Edoxaban (ng/mL)
C
10
❚Figure 4❚ Effect of edoxaban on the parameters of thrombin generation assay in human platelet-poor plasma. A, Maximum concentration of thrombin (peak). B, Endogenous thrombin potential (ETP). C, Mean thrombin generation rate. D, Lag time. E, Time to peak. Data represent mean ± standard error of the mean (n = 12). **P < .01, ***P < .001 (Dunnett test). Solid line, mean; dotted line, 95% confidence interval.
***
10
***
5 0 Control
10
100
1,000
Edoxaban (ng/mL)
❚Table 4❚ IC50 and CT2 Values of Edoxaban for Parameters of Thrombin Generation Assay in Human PPP and PRPa Sample
Peak IC50, μmol/L
mRate IC50, μmol/L
ETP IC50, μmol/L
Lag Time CT2, μmol/L
ttPeak CT2, μmol/L
PPP PRP
0.145 (0.128-0.161) 0.305 (0.229-0.382)
0.047 (0.041-0.054) 0.230 (0.166-0.294)
1.39 (0.971-1.81) 0.636 (0.447-0.825)
0.188 (0.176-0.201) 0.191 (0.160-0.223)
0.139 (0.117-0.161) 0.588 (0.386-0.790)
CT2, concentrations of anticoagulants required to double time; ETP, endogenous thrombin potential; IC50, concentrations required for 50% inhibition; mRate, mean thrombin generation rate; peak, maximum concentration of thrombin; PPP, platelet-poor plasma; PRP, platelet-rich plasma; ttPeak, time to peak. a Values in parentheses are 95% confidence intervals.
246 Am J Clin Pathol 2015;143:241-247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
© American Society for Clinical Pathology
AJCP / Original Article
assays that measure the anticoagulant activity of edoxaban may be valuable in certain situations such as an overdose, severe bleeding, urgent surgery, or issues with noncompliance. The present study concludes that PT has disadvantages of a large variability among the different reagents that is not corrected by using the INR and a lack of sensitivity to detect the activity of edoxaban at the trough level. Although aPTT is less sensitive than PT, it could be used as a conventional and convenient diagnostic test at peak plasma levels or in overdose situations because of a small variability in response using different reagents. The TGA, especially peak and mRate, is the most sensitive to edoxaban, suggesting the detection of the edoxaban activity at the trough level. However, further validation of these methods is required in clinical settings, such as using plasma samples of patients who receive edoxaban. Address reprint requests to Dr Morishima, Biological Research Laboratories, R&D Division, Daiichi Sankyo, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan; morishima.yoshiyuki.t4@ daiichisankyo.co.jp. Disclosure: All authors are employees of Daiichi Sankyo. Acknowledgments: The authors thank Yoko Shiozaki for technical assistance and Yuko Honda for editorial assistance. Additional editorial support was provided by AlphaBioCom, LLC, King of Prussia, PA, and funded by Daiichi Sankyo.
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Am J Clin Pathol 2015;143:241-247 247 DOI: 10.1309/AJCPQ2NJD3PXFTUG
