Thrombosis Research 127 (2011) 457 465. Contents lists available at ScienceDirect. Thrombosis Research

Thrombosis Research 127 (2011) 457 465 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres Regular Article Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: Two observational studies in patients undergoing total hip or total knee replacement Geneviève Freyburger a,, Gérard Macouillard b, Sylvie Labrouche a, François Sztark b a Laboratoire d'hématologie, Hôpital Pellegrin, CHU de Bordeaux, 33076 Bordeaux, France b Service d'anesthésie Réanimation 1, Hôpital Pellegrin, CHU de Bordeaux, 33076 Bordeaux, France article info abstract Article history: Received 4 October 2010 Received in revised form 24 December 2010 Accepted 3 January 2011 Available online 31 January 2011 Keywords: Dabigatran Rivaroxaban Coagulation Thrombin Generation Orthopedic surgery Introduction: Dabigatran and rivaroxaban have recently been added to the armamentarium for thromboprophylaxis in orthopedic surgery. Although this is their first licensed indication, others will soon follow. Owing to their claimed predictable anticoagulant response that dispenses with the need for monitoring coagulation, their effects are poorly described in routine cases. However, interpreting blood coagulation results and evaluating whether a treatment is properly targeted in the case of untoward incidents will become a common concern for clinicians. Methods: Eighty patients undergoing total hip or knee replacement were included in two studies. Forty of them received dabigatran (study 1) and 40 rivaroxaban (study 2). Blood samples (n=176 and 166) were taken preoperatively and twice a week from the first postoperative day. Results: Dabigatran increased apttr about two-fold and PT about 1.2-fold, and it was mostly an initiationphase modulator of thrombin generation. Mean circulating concentrations as measured by a diluted thrombin time were 105±85 ng/ml at T max in samples from patients receiving the full dosing. They depended significantly on renal function, body weight and gender. Rivaroxaban increased apttr and PTr around 1.5 fold and modified the initiation and amplification phases of thrombin generation, with a lowered and prolonged thrombin burst. Mean circulating concentrations as measured by an antixa test were 117 ±78 ng/ml at T max. With both drugs, routine coagulation tests, thrombin generation curves and functionally determined concentrations exhibited high interindividual variability. Conclusion: Routine coagulation tests are altered in patients receiving dabigatran or rivaroxaban, but their alterations poorly reflect the circulating concentrations as determined by functional approaches. 2011 Elsevier Ltd. All rights reserved. Introduction Two oral anticoagulants recently received full market approval for the prevention of venous thromboembolism after total hip replacement (THR) or total knee replacement (TKR) surgery, dabigatran (Pradaxa from Boehringer Ingelheim) and rivaroxaban (Xarelto from Bayer). They are specific and direct inhibitors of either thrombin or factor Xa (FXa) respectively, and are referred to hereafter as DOACs (Direct Oral AntiCoagulants). Beside their proven efficacy and safety [1 6], the new DOACs are likely to take more than half of the anticoagulant market share between now and 2014 [7]. Their oral route is highly appreciable for patients who receive up to 6 weeks of daily anticoagulant treatment, and the cost-effectiveness of these Abbreviations: od, omne in die (once daily); PT, Prothrombin Time; aptt, activated Prothrombin Time; TGT, Thrombin Generation Test; DOACs, Direct Oral AntiCoagulants; C max, Maximum plasma concentration; T max, Time after administration when C max is reached. Corresponding author. Tel.: +33 5 57 820 206; fax: +33 5 56 796 020. E-mail address: Genevieve.freyburger@chu-bordeaux.fr (G. Freyburger). treatments has been demonstrated in elective hip and knee surgery [8 11]. Owing to their claimed predictive effects and the absence of the need for monitoring, there is no systematic report of laboratory data from the pivotal studies. The transition from research studies to patient care, whose clinical conditions are more heterogeneous, raises the question of how do laboratory tests react to the new treatments. Although laboratory tests are not required to monitor new DOACs, some patients receiving such treatments will inevitably have intercurrent coagulation assessment. The coagulation pattern in patients receiving these drugs needs to be well-recognized by the laboratory and the clinicians to interpret what alterations are potentially drug-induced or not. Moreover, in the presence of a hemorrhagic event in a DOAC-treated patient, the circulating concentration of the drug must be known in order to interpret any alterations in the coagulation tests and to decide how the patient is to be managed. The present study was conducted on behalf of our institution's COMEDIMS (i.e. Committee for Medicine and Sterile Medical Devices) before referencing the new DOACs in the hospital formulary. Its aim was to gather experience on laboratory coagulation tests in patients receiving DOACs in order to anticipate the management 0049-3848/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.01.001

458 G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 of the hopefully scarce patients with possible untoward effects, among the increasing number of patients that will benefit from these new treatments in the near future. Methods Patients Eighty patients were included in two subsequent studies. Half of them received dabigatran etexilate (study 1, dabigatran or Pradaxa from Boehringer Ingelheim, from 2009/05/29 to 2009/09/15) and half of them received rivaroxaban (study 2, rivaroxaban or Xarelto from Bayer, from 2009/09/15 to 2010/01/21). Dabigatran was given at 220 mg (2 110 mg) od (once daily) in 30 patients (15 males and 15 females, mean age respectively 56±12 and 67±8 years), while 10 patients (3 males and 7 females, mean age respectively 82±3 and 80±5 years) older than 75 years, with a creatinine clearance between 30 and 50 ml/min or treated by amiodarone, received a lower dosage of 150 mg od (2 75 mg). The first dabigatran dose was halved and given 1-4 hours after surgery in all patients. Rivaroxaban was given at 10 mg dosing in all patients whatever their age, provided that creatinine clearance was higher than 30 ml/ min and that they were not treated by azole antifungals or by the protease inhibitor ritonavir. This study included 14 males and 26 females, mean age 62±14 years. The first dose was given 6-10 hours after surgery and then every day at 8 o'clock. The two patient groups were very comparable in weight, renal function and gender. Only age was slightly different between the two groups (66±13 years in dabigatran-treated patients vs 61±14 years in rivaroxaban-treated patients, pb0.008). Patients gave their informed consent to participate in the study. Laboratory variables Blood samples were taken before surgery (T0), on the day of surgery (T1) and twice a week in the following period (T2 to T4), 2 hours±10 minutes after drug ingestion under nurse supervision. Maximum plasma concentration (C max ) was targeted in order to obtain noticeable pharmacodynamic effects. The number of samples obtained was 176 in study 1 and 166 in study 2. Hemogram was performed on a CELL-DYN Sapphire from Abbott Laboratories (Abbott Park, Illinois, U.S.A), and coagulation parameters on an ACL Top from Instrumentation Laboratory (IL, Bedford, MA, USA). Activated partial thromboplastin clotting time (aptt) was measured using HemosIL APTT-SP from IL, prothrombin time (PT) using HemosIL RecombiPlasTin 2G from IL. D-dimer was measured using the VIDAS DDimer Exclusion test (biomérieux SA, Marcy l'etoile, France). Thrombin Generation Test (TGT) was carried out on a Thrombinoscope system (Synapse BV, Maastricht, the Netherlands). The endogenous thrombin potential (ETP, in nm x sec, i.e. the surface under the curve) reflects the whole amount of thrombin generated as a function of time, whatever the shape of the thrombin curve, and the height of the peak (PK in nm). Kinetic parameters included lag time (LT), time to peak (TPK) and start tail (ST), all in seconds. Velocity index (in nm/sec, calculated by PK/ TPK LT) reflects the highest rate of thrombin formation per minute. Data from patients receiving dabigatran were reprocessed using a locally developed software [12]. Specific dabigatran and rivaroxaban measurement tests were also carried out. In the absence of commercially available standards and controls for the two drugs, home-made calibrators and controls were prepared from a mixture of the appropriately reconstituted active substance in a normal plasma pool. The CRYOcheck Pooled Normal Plasma from Cryopep (Parc 2000, Montpellier, France) was spiked with increasing concentrations of dabigatran (BIBR 953 ZW) and rivaroxaban (Bay 56-7939). Drugs (10 mg) were obtained free of charge from Bayer HealthCare and from Boehringer Ingelheim, respectively. Both were reconstituted in DMSO according to the providers instructions. A concentration range from 0 to 400 ng/ml was used for calibrations of both DOACs, prepared from 990 μl of plasma aliquots spiked with 10 μl of DMSO containing increasing concentrations of the drugs. As reference tests for measuring DOAC concentrations, we used the ecarin time, which has already been described as a universal method to quantify direct thrombin inhibitors [13], and the Biophen DiXal from Hyphen Biomed (Neuville-sur-Oise, France) which is used to direct FXa inhibitors and was used according to the provider's instructions. Clotting ecarin time was measured after mixing 75 μl 4 u/ ml ecarin (echis carinatus snake venom from Sigma-Aldrich, St. Louis, MO, USA) and 75 μl of plasma sample. A linear regression was observed with an R 2 =0.99 between clotting time (range 60-400 sec.) and dabigatran concentration (range 0-900 ng/ml). We also developed locally two DOAC measuring tests available in real time since they use our routine reagents. In the dabigatran dedicated diluted Thrombin Time (dtt), 75 μl of plasma diluted 1/3 in a normal pool of human plasmas (HemosIL Normal Control from IL) was incubated for 90 sec. at 37 C and 75 μl purified bovine thrombin (Thrombin Time from IL) was added. Coagulation times (20-320 sec.) are linearly correlated to the dabigatran concentration (range 0-400 ng/ ml, R 2 N0.99). For the rivaroxaban-dedicated antixa test (XaraXa), 75 μl of plasma diluted 1/3 in Diluent Factor from IL was mixed with 67 μl of S-2732 from Chromogenix (Coamatic Heparin, IL). After 60 sec incubation, FXa was added (Coamatic Heparin, IL) and absorbance change at 405 nm was recorded after 20 sec. and for 40 sec. Over a 0-400 ng/ml range of concentrations, the slope of the absorbance as a function of time was directly proportional to the rivaroxaban concentration (R 2 N0.99). To estimate the within-run and between-run coefficients of variation (CV) of dtt and XaraXa test, a pool of normal plasmas was spiked with dabigatran or rivaroxaban to achieve a therapeutic plasma concentration of 170 ng/ml. Within-run CVs were calculated from the results measured 10 times. Run-to-run CVs were calculated from the results measured on the 9 following days using different aliquots of the same pool of spiked plasma conserved before measurement at -80 C. Serum creatinine was measured using the Beckman Coulter reagent on an Olympus AU 5400 (Beckman Coulter, Brea, CA USA) and creatinine clearance was calculated using the Cockcroft & Gault formula. Samples estimates were described by their arithmetic means and standard deviations. Median values of age, renal function and body weight were used to split variables in subgroups of comparable size (top half versus bottom half). Unpaired Student's t-test and paired Student's t-test were compared differences between groups of data, when normality was confirmed by a skewness statistic of less than 2 standard errors of skewness (ses), calculated by using the formula from Tabachnick and Fidell [14]. The Wilcoxon signed-rank test was otherwise used for comparison of data. Strength of the relationship between two parameters was studied by a linear regression model fitted using the least square approach. Results Patients: demographic data For both studies, mean hospital stay was 11±1 days for THR and 14±1 days for TKR, This is similar to the duration of hospital stay observed in our ward for enoxaparin-treated patients. Two patients experienced DVT during the studies, one in each treatment group. No bleeding was reported. In study 1, significantly older patients were found as expected in the patients receiving the dabigatran 150 mg od regimen (80±4 vs 61±11 years in the group receiving 220 mg od, pb0.0001). Mean

G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 459 body weight was 74±14 kg and renal function was normal (clearance higher than 50 ml/min) in all patients, except in four patients in the dabigatran 150 mg od group (respectively 34, 38, 48 and 50 ml/min). Mean clearance values were 96 ± 32 ml/min in the dabigatran 220 mg od group (lowest 10% percentile, 67) and 51±10 ml/min in the dabigatran 150 mg od group (lowest 10% percentile, 38). In study 2, mean age was 62±14 years, body weight was 73±13 and renal function was normal but in one patient (49 ml/min) and mean clearance values were 96±27 ml/min (lowest 10% percentile, 67). Study 1: routine parameters, dtt-determined concentrations and effects on thrombin generation in dabigatran-treated patients Table 1 shows the mean results and standard deviations for PT, aptt, and D-dimer according to the sampling times. Using the paired Student's t-test, all parameters were significantly altered after receiving dabigatran when compared with the pre-therapeutic time T0, apart from the D-dimer, which was significantly increased at the first post-operative time only (pb0.001). Mean aptt ratio was more altered than PT ratio, and exhibited the highest standard deviations. Fig. 1 shows the calibration curve obtained with the test developed locally to determine circulating concentrations. The dtt was highly correlated with the reference ecarin test (R 2 =0.978). Within-run CV values were 2.8% (n=10) while between-run CV values were 5.0% (n=9). Fig. 2 shows the mean drug concentrations determined by dtt in patients as a function of sampling time. Dabigatran was given at half the dose at T1, and the mean plateauing concentration was only reached from the T3 sampling time i.e. from the sixth day post-surgery, with a significant difference between mean values at T2 and T3 (pb0.003 at the Wilkoxon signed rank test). The mean concentration observed in samples taken from the second postoperative day was 105±85 ng/ml (n=94), with a maximum value of 356 ng/ml observed in a 53 year-old woman with 52 ml/min clearance and a 65 kg body weight at day 6. Very high standard deviations were found due to a wide inter-individual variability in the results according to the patients. This is further illustrated by Fig. 3, which shows the dtt-determined concentrations of dabigatran on sampling days in individual patients as well as the aptt obtained at the corresponding sampling days. Very low concentrations (b30 ng/ml) were occasionally measured up to day 15, and these low concentrations were in line with normal PT and aptt values (ratios less than 1.1 and 1.2 respectively). To investigate the involvement of clinical parameters in the relatively unpredictable concentrations observed, dtt-determined concentrations of dabigatran from 94 samples taken at full dosing time (from T2) were dichotomized according to the median value of the following clinical parameters: age, renal function, body weight and gender. This made it possible to calculate the mean circulating concentrations in the lowest and highest half of patients according to their age, renal function and body weight and in males and females. Table 2 shows that dabigatran concentrations were significantly higher in patients with lowest clearance (in spite of dose adjustment in patients with lower clearance values), lowest body weight, and in females. Dabigatran concentrations higher than 210 ng/ml were always encountered in samples from patients with a body weight less than 70 kg (mean value=62 kg, 14 samples), while, they never exceeded 90 ng/ml in those with a body weight higher than 90 kg (mean=46 ng, 14 samples). As body weight and gender are not independent factors, we restricted the comparison of concentrations in both genders separately. The difference in concentrations as a function of body weight remained significant in men and women (p=0.027 and p=0.006 respectively). Dabigatran concentrations determined by dtt were better correlated with apttr (R 2 =0.622) than with PTr (R 2 =0.347). Interestingly, the relationship between dabigatran circulating concentrations and aptt was improved when observed in isolated patients, with a mean individual coefficient R 2 =0.894±0.130. Individual modulating factors thus regulated in each patient his/her own sensitivity to the drug with regards to aptt.factors able to influence individual sensitivity of aptt to the dabigatran concentrations are numerous [15]. In a preliminary approach, factors VIII, V and antithrombin were measured in 38 randomly selected samples presenting detectable dabigatran concentrations. Mean factor V was significantly lower when aptt ratios were higher (over 2.25 in the last quartile of patients: 76±20% versus 100 ±22%, p b0.01). The difference in factor VIII did not reach significance although a difference in the mean was noted (135±57% versus 167±69%). Antithrombin was not found to be related to the sensitivity of the aptt ratio to dabigatran concentration. In the presence of dabigatran, the thrombin generation experiment was confounded by direct interaction of the drug with the calibrator. In the Thrombinoscope method, the calibrator slope makes it possible to translate fluorescence units into nanomoles of thrombin. In the presence of dabigatran, the calibrator (thrombin trapped in a cage of alpha-2 macroglobulin) is inhibited by the seeping of the small drug into the cage; the transformation of the fluorescent substrate by the calibrator is collapsed by this interference. A false increase in thrombin generation can thus be artificially inferred. Interestingly, the calibrator slope was highly correlated to the circulating dtt-determined concentration of dabigatran (R 2 =0.902). TGT tests in dabigatran-treated patients thus require a correction of the calibration curve. We reprocessed the TGT data from each Table 1 Routine coagulation parameters. Mean routine coagulation parameters and standard deviations in patients receiving dabigatran (study 1) or rivaroxaban (study 2) at the different sampling times (before surgery, T0, on the day of surgery, T1, and twice a week in the following period, T2 to T4).

460 G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 Fig. 1. Methods for dabigatran and rivaroxaban measurement. Calibration curves of the locally developed methods for dabigatran (dtt, a) and rivaroxaban (XaraXa; b) measurement in patients samples. c represents the relationship between results obtained in dabigatran-treated patients with the diluted thrombin time (dtt) and with the reference ecarin time. d represents the relationship between results obtained in rivaroxaban-treated patients with the XaraXa method and the reference Hyphen DiXal method. Fig. 2. Mean dabigatran and rivaroxaban concentrations (± standard deviations) as a function of sampling time. The Wilcoxon signed-rank test was used to test the repeated measurements in each treatment group.

G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 461 Fig. 3. Concentrations and routine coagulation tests, individual data. Dabigatran and rivaroxaban concentrations. Results in routine coagulation parameters (apttr for study 1 and PTr for study 2) in individual patients. The bold lines represent the floating mean. Day of sampling is represented on the X axis. dabigatran-treated patient by using the calibration curve obtained from his/her pretherapeutic sample taken at T0. The intra-individual CV of the calibrator slope at different times of sampling was 8% in orthopedic patients not treated with dabigatran (n=40), demonstrating a relative stability of the slope in individuals. The use of the pretherapeutic sample for TGT calibration is thus an efficient way to approximate thrombin generation in dabigatran-treated samples from a given patient. This method was used thereafter. Unlike quantitative parameters (peak, ETP), kinetic parameters (lag time, time t peak and start tail) are not modified by the calibrator curve value. TGT was carried out in all patients in whom plasma aliquots were available (161/176 samples). Table 3 summarizes these modifications in dabigatran-treated patients at full dose (from T2) when compared with the results of TGT data at the pre-therapeutic time (T0). The ETP decreased less than 15% while older anticoagulants (heparins and vitamin K antagonists) are known to decrease the ETP much more [18]. Dabigatran delayed thrombin outbreak, as reflected by prolonged lag time and altered routine coagulation tests. However, lag time was poorly related with PT and aptt ratios: R 2 =0.382 and 0.355 respectively. The effect on lag-time was highly variable between subjects, as shown by the high CV value in Table 3, and seemed to increase over the sampling times during our study period, while the dtt-determined concentrations and effects on PT and aptt plateaued from T3 (see Figs. 2-3). Circulating dtt-determined concentrations of dabigatran were correlated with lag time, (R 2 =0.593) but not with ETP and peak (R 2 = 0.084 and 0.016 respectively, nn100). All patients exhibited the same pattern but the degree of alteration varied much. Fig. 4 represents the TGT curves from two representative individual patients with different response intensity to the treatment, and the mean curves reconstructed by pooling the individual results at subsequent time intervals after surgery. The therapeutic samples exhibited a progressive delay of the thrombin outbreak without modifying notably the subsequent profile of thrombin generation. Study 2: routine parameters, antixa-determined concentrations and effects on thrombin generation in rivaroxaban-treated patients PT and aptt ratios were significantly and similarly altered by rivaroxaban at all sampling time, while D-dimer was significantly increased at T1 only (Table 1). PTr exhibited a better correlation with antixa-determined concentrations of rivaroxaban (R 2 =0.745) than apttr (R 2 =0.468). The PTr correlation was further improved in individual patients (mean individual coefficient of correlation R 2 =0.860±0.167), demonstrating that mixing the samples from all the rivaroxaban-treated patients introduces a variability factor due to an unpredictable individual susceptibility of PTr to circulating concentrations of rivaroxaban. The one stage antixa test developed locally by using our routine reagent (XaraXa) was highly correlated with the two stage commercial test devoted to laboratory measurement of direct factor Xa inhibitors and

462 G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 Table 2 Clinical factors influencing drug concentrations. Dabigatran and rivaroxaban mean concentrations (± standard deviation, ng/ml) in samples from patients from T2 to T4 (from the second day postoperatively and full dosing for study 1) dichotomized according to the median value of age (years), creatinine clearance (ml/minute), body weight (kg) and according to gender. Median values are given in black boxes: similar numbers of patients are found below and above the median value and mean drug concentrations were compared in 52 samples from females (F) versus 42 from males(m) for dabigatran, and 64 samples from females versus 33 from males for rivaroxaban. Unpaired t-test compares the alterations observed with the two drugs. especially rivaroxaban(dixal from Hyphen). Fig. 1 exhibits the good correlation between both tests in samples from rivaroxaban-treated patients (R 2 =0.965). Fig. 2 shows the progressive increase in XaraXadetermined concentrations of rivaroxaban from T1 to T3. Due to the large inter-individual variability (see standard deviations) differences between mean concentrations at the different sampling time were not significant. The mean concentration observed in samples taken from the second postoperative day was 117±78 ng/ml (n=97), while the highest concentration found during our study was 425 ng/ml in a 55 years old woman with 57 ml/mn clearance and a 58 kg body weight at day 10. Rivaroxaban concentrations follow up in individual patients is represented in Fig. 3: very low values were occasionally measured during all the follow up period, and were associated with normal PT values. Table 2 demonstrates that XaraXa-determined concentration of rivaroxaban results were not different with regard to age, renal function, body weight and gender, as studied by dichotomizing the patients according to the median value of the quantitative parameters or according to the gender. TGT results in rivaroxaban-treated patients at the different sampling times are summarized in Table 3 (160/166 samples were available). The overall generation of thrombin reflected by the ETP was modestly decreased (~15%), but the kinetic of thrombin appearance and decrease was profoundly altered. Thrombin outbreak was delayed, as reflected by prolonged lag time which was highly correlated with the routine coagulation tests, PT and aptt ratios (R 2 =0.810 and R 2 =0.751 respectively). Rivaroxaban slowed down the thrombin burst efficiently with a lower maximum thrombin concentration (decreased peak) that lasted longer (time to peak was markedly lengthened as well as the start tail, which reflects the delayed end of the process). With the highest XaraXa-determined concentrations of rivaroxaban, the thrombin peak was characteristically prolonged by a plateau that differed from the regular bell-shaped curves observed with other anticoagulant drugs. Interestingly, rivaroxaban concentrations were also correlated with the other kinetic parameters, i.e. time of peak and start tail (R 2 =0.629 and 0.755 respectively, nn100) while it was less significantly correlated with the overall quantity of thrombin generated (ETP, R 2 =0.304, nn100). Fig. 4 represents the curves from two representative patients and the mean TGT curves calculated from the whole patients group results at subsequent time intervals after surgery, by merging from 29 to 35 individual curves according to the time. The first therapeutic sample was frequently less altered than the following ones, while the subsequent samples exhibited a progressively increasing effect of rivaroxaban. Discussion Our studies investigated how coagulation tests perform in samples from patients receiving the new DOACs in a routine setting. Although much is already known in the literature, mostly from preclinical, dose finding phase IIa or in vitro studies [19 22], each laboratory has to define its own reagents reactivity to DOAC in order to interpret coagulation testing properly, whatever the reason for exploring the patient. The imminent prescription of DOACs for new indications such as atrial fibrillation will require the inclusion of DOACs-induced alterations in the decision trees for exploring coagulation, exactly as vitamin K antagonists and heparins are included. PT and aptt are both prolonged in patients receiving DOACs, aptt being more sensitive to dabigatran and PT to rivaroxaban. Our routine reagents exhibited reactivity towards both drugs, in line with previous findings [19 22], the Recombiplastin Table 3 Parameters of thrombin generation test. All data from the pre-therapeutic time are pooled in a unique T0. Asterisks express the significance of the unpaired t-test carried out between samples taken at T0 and samples taken from T2 to T4 (from the second day postoperatively and full dosing for study 1) in studies 1 and 2 (* pb0.01, *** pb0.0001).

G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 463 from IL used in our study having been described as one of the most sensitive reagents to rivaroxaban [22]. According to Stangier, aptt may be unsuitable for accurately quantifying the anticoagulant effect of dabigatran but provides a qualitative assessment of the anticoagulant activity [23], and according to Laux, the PT may be suitable for assessing drug exposure, if it was necessary, in special cases [24]. However, variation in PT and aptt responses for a given DOAC regimen is very high, as shown by the CV in Table 1. Therefore, these tests are hardly suitable for evaluating the circulating concentration of the drugs. Each patient's plasma composition highly influences the individual reactivity of routine coagulation tests to DOAC concentrations. This point deserves to be assessed by further experimental studies. In our study, factor V was one of these factors that could be identified. Another reason for avoiding PT and aptt as evaluation tools for DOAC exposure is that they may be confounded by other previously unknown or acquired alterations of coagulation. Nevertheless, functionally-determined Fig. 4. TGT in DOACs-treated patients. TGT curves observed in two representative individual patients receiving dabigatran (study 1) or rivaroxaban (study 2) as a function of sampling times (subsequent samples are represented from bold to regular and to dotted lines). In dabigatran-treated patients, one patient comes from the 220 mg dosing group and one patient from the 150 mg od dosing group. At the bottom, mean T1 to T4 curves are represented. Mean values were calculated by pooling the thrombin concentrations data as a function of time after merging the data according to the different periods of time (before surgery T0, the day of surgery following the first dose T1, second to fourth day T2, end of first week T3 and from the second week T4). A progressive delay and decrease in the mean curves is observed with both DOACs.

464 G. Freyburger et al. / Thrombosis Research 127 (2011) 457 465 concentrations of dabigatran and rivaroxaban can be measured by focusing the tests specifically on the activity of the target enzyme. Although some convenient and reliable tests are already commercially available to measure DOACs [22,25], we chose to develop and validate new tests for dabigatran and rivaroxaban measurement in order to use the reagents routinely available on our automated coagulation systems. Commercial titrated plasmas in dabigatran and rivaroxaban for calibrations and controls should rapidly become available and make it possible to adapt these methods more easily. Study 1, dtt-determined concentration of dabigatran For dabigatran, published concentrations in normal volunteers and orthopedic patients (Bistro Ib) are 107±72 ng/ml and 76±49 ng/ml respectively, following a single-dose administration of a 150-mg capsule [26,27]. These results are in line with the mean 62±68 ng/ml concentration observed after the first intake of 110 mg (or 75 mg in patients at risk) observed in our study. A high interindividual variability in orthopedic patients was reported, with CV greater than 65% for the C max [27]. In our study the variability in dabigatran concentration at full dosing was 81%; restricting the data to the 30 patients receiving 220 mg od did not improve variability (CV=0.91% in this group). In the pharmacokinetic study by Stangier, C max was demonstrated to occur between 1 and 24 hours following drug administration. The 81% CV observed in our study at T1 is thus in line with the 65% CV described by Stangier. The latter reported that stable concentrations in healthy volunteers were reached within 3 days of administration [19]. In the orthopedic patients of our study, steady state was achieved later from the sixth day (see Fig. 2). Age, gender, renal function, hepatic function (involved in prodrug transformation) and weight have already been described to influence circulating concentrations of dabigatran. Age and renal function, which were taken into account by the adapted 150 mg dose of dabigatran, were respectively not or poorly related to circulating concentrations of the drug. In contrast, body weight and gender exhibited a strong influence on dtt-determined concentration of dabigatran in our patients. According to the European Medicines Agency report, body weight had a minor effect on the plasma clearance of dabigatran resulting in higher exposure in patients with low body weight [28]. However, no detailed data are available from the dabigatran pivotal studies, and our results seem to indicate a significant rather than a minor effect of body weight. Stangier reported a 48% increase in the mean AUC0-24 in females when compared to males in the 59 patients in the Bistro Ib study [27], while our study shows a 61% increase in the C max (concentration 2 hours after dosing) in females. Special attention should thus be paid to detecting early a potential overdosing of dabigatran in women with a low body weight and borderline renal function. The effects dabigatran on the TGT have already been described in plasma spiked with the drug and from phase I and phase II studies, but no results in patients receiving the drugs in vivo in a routine setting have yet been published to our knowledge. In vitro, the thrombin burst is delayed but mainly unaltered [16]. We demonstrated a previously unreported dampening effect of calibrator in the presence of dabigatran, which dramatically and falsely increases the thrombin concentrations measured (peak, ETP, velocity) when using the Thrombinoscope software and the interest in reprocessing patients data by using the pretherapeutic slope value in each individual patient. The individual variability in patients curves cannot be suspected from in vitro spiking. Although expressed at different intensities depending on individual characteristics that are still to be recognized, dabigatran is clearly an initiation-phase modulator. Study 2, XaraXa-determined concentration of rivaroxaban Pharmacokinetic data concerning circulating rivaroxaban concentrations have been published in healthy volunteers [20] and in patients undergoing total hip replacement [29] receiving the 10 mg dosing. Mean C max was determined in 8 volunteers receiving a 10 mg rivaroxaban single dose and was found to be 141±22 ng/ml. Phase II studies reported a moderate effect of renal function, age (largely due to reduced renal function) and body weight in patients undergoing total hip replacement, while gender was reported to have virtually no effect on the pharmacokinetics and pharmacodynamics of rivaroxaban. Median predicted rivaroxaban concentration after 10 mg od at steady state was 124 ng/ml, which is very similar to the 117±84 ng/ ml mean concentration found in our study. Neither age, creatinine clearance, body weight nor gender influenced XaraXa-determined concentrations of rivaroxaban in our study. Rivaroxaban has been described in vitro as increasing the lag time and decreasing the velocity and ETP: the thrombin burst is delayed, lowered and prolonged [17]. We confirmed these data ex vivo, This suggests a continuous effect of the drug in the prothrombinase complex to slow down the thrombin burst but without much modification of the overall quantity produced at the end of the process (the ETP). Limitations of our studies Because the aim of our two studies was to observe the druginduced alterations of coagulation parameters and not to compare the drugs, they were not randomized. Drug concentrations were determined by using calibrated and targeted coagulation tests rather than liquid chromatography tandem mass spectrometry methods. This was done for practical reasons of availability of the tests in an emergency context, and because direct correlations had been previously described between LC-MS/MS-determined concentrations and functional coagulation tests [21,31]. T max was determined since trough values are not properly studied by coagulation tests owing to their poor sensitivity to residual anticoagulant concentration. Variability in the T max may have played a role in the variability in coagulation parameters as samples were taken 2 h after drug ingestion, while the real T max has been shown in orthopedic patients to be 2.3-2.9 h for dabigatran and within 1-2 h for rivaroxaban at steady state and to exhibit a greater variability on the first postoperative day [23,30]. Although both DOACs have a low potential for drug-drug interactions in comparison to vitamin K antagonists, coprescribed drugs were not the focus of our studies, although they may have contributed to the wide interindividual variations observed. A recent review addresses the latter question [32]. Conclusion Although the efficiency and safety of the DOACs in preventing thrombosis in orthopedic patients was demonstrated in a large number of patients, the widely held view about their predictable dose-response, thereby eliminating the need for routine laboratory monitoring, should be mitigated on the basis of our results. The high inter-individual variability in response to fixed doses (or two adapted doses in the case of dabigatran) for every patient illustrates the potential risk of observing extremely low or extremely high functionally determined concentrations. Whatever the reason why this variability is observed, further work will be required to infer the potential role of this inter-individual variability on the risk of thrombosis or hemorrhage. Conflict of interest statement None. Acknowledgments We thank Nadine Capdessus for excellent technical assistance.

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