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Introduction: Lupus anticoagulant (LA) investigation in patients on anticoagulant therapy is problematic. Rivaroxaban in particular causes significant interference, prolonging both LA screening and confirmation tests, and falsely raising LA screen/confirm ratios, leading to potential false identification of LA. The Russell Viper Venom Time (RVVT) assay, key to the investigation of LA, is especially sensitive to rivaroxaban., Materials and Methods: We assessed cross laboratory (n = 82) testing of four samples to investigate whether rivaroxaban induced interference in LA testing could be neutralised. Testing was performed blind to sample type. The samples comprised: (A) A pool of normal plasma (LA-negative control); (B) sample A spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (LA 'false' positive sample); (C) sample B subsequently treated with a commercial 'DOAC-neutraliser' (DOAC Stop); (D) sample B treated with andexanet alfa (200 μg/mL)., Results: As expected, the rivaroxaban-spiked sample (B) caused prolongation of most LA-tests, and also generated a falsely prolonged RVVT screen/confirm ratio (median 1.37, compared to 0.97 for sample A). The sample (C) treated with DOAC Stop evidenced a correction in LA-test clotting times, as well as neutralising the false positive LA (median RVVT screen/confirm ratio of 0.99). Although the andexanet alfa treated sample (D) also yielded a low median RVVT screen/confirm ratio of 0.88, it did not fully correct LA-test clotting times. Consistent with test findings, all laboratories interpreted samples A and C as being LA-negative. For sample B (rivaroxaban), 45.3% identified this as LA positive, and 38.7% identified LA interference. Most (61.3%) also identified sample D as LA negative, with the remainder (38.7%) identifying LA interference., Conclusions: DOAC Stop was able to neutralise the false LA activity induced by rivaroxaban, both in terms of clot-times and LA ratios. In contrast, whilst andexanet alfa negated the rivaroxaban-prolonged LA-ratio, it did not fully correct clot-times, leaving some residual LA interference, and requiring additional testing to investigate prolonged clotting times., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Essentials Currently, DOACs are given at fixed doses and do not require laboratory monitoring. Direct oral anticoagulant-specific measurements were performed at trough and peak. Patients who developed bleeding events showed higher DOAC plasma levels at peak. This study suggests the need of a more accurate DOAC dose assessment., Background: Direct oral anticoagulants (DOACs) are administered at fixed dose. The aim of the study was to evaluate the relationship between DOAC C-trough or C-peak plasma levels and bleeding complications in patients with non-valvular atrial fibrillation (NVAF)., Methods: Five hundred sixty five consecutive naive NVAF patients were enrolled. The DOAC measurements at C-trough and at C-peak (available in 411 patients) were performed at steady state, within the first month of treatment. Major bleeding (MB), clinically relevant non-major bleeding (CRNMB), and minor bleeding (MinB), occurring during 1 year of follow-up after blood sampling, were recorded. For each DOAC, interval of C-trough and C-peak levels was subdivided into four equal classes and results were attributed to these classes; the median values of results were also calculated., Results: Two hundred eight patients were on apixaban, 185 on dabigatran, and 172 on rivaroxaban. For 1-[qqqdeletezzz] year follow up for all patients, we observed: 19 MB (3.36%), 6 CRNMB (1.06%), and 47 MinB (8.31%). The prevalence of bleeding patients with anticoagulant levels in the upper classes of C-peak activity (II + III + IV) was higher than that in the lowest class. Normalized results of C-peak levels were higher in patients with bleeding than in those without bleeding., Conclusions: Bleeding complications during DOAC treatment were more frequent among atrial fibrillation (AF) patients with higher C-peak anticoagulant levels. In addition to a previous study that showed an increased risk of thrombotic complications in the patients with low C-trough levels, this study seems to indicate that patients with NVAF on DOACs would need a more accurate definition of their optimal therapeutic window., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

A Micellar Chromatographic method to determine rivaroxaban in plasma and urine has been developed. The samples were dissolved in the mobile phase (SDS 0.05 M - 1-propanol 12.5%, phosphate buffered at pH 7) and 20 μL directly injected, avoiding the extraction and purification steps. Using a C18 column and running under isocratic mode at 1 mL/min, analyte was eluted without interference from the matrix in <6.0 min. The detection absorbance wavelength was set to 250 nm. The procedure was validated by Food and Drug Administration guidelines in terms of: system suitability, calibration range (0.05-5 mg/L), linearity, sensitivity, robustness, carry-over effect, specificity, accuracy (-11.1 to 4.2%), precision (<19.9%), stability and analysis of incurred samples. The method was found reliable, practical, easy-to-conduct, rapid, relatively eco-friendly, safe, inexpensive, widely available and with a high sample throughput. The method was applied to the analysis of incurred samples, including incurred sample reanalysis, to verify that the instrumentation works correctly. In addition, the constants of the different partition equilibria occurring in the column were elucidated in order to have a better comprehension of the theoretical aspects of the retention mechanism. A moderately strong association between rivaroxaban and the stationary phase and the micelles was found, weakened by short chain alcohol., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Essentials Direct oral anticoagulants (DOAC) are used for stroke and venous thromboembolism prevention. We report a new assay that measures anti-factor Xa DOAC levels in plasma and whole blood. Rivaroxaban and apixaban can be accurately quantified below trough levels. The ease and accuracy of the assay demonstrate its potential for point-of-care applications., Background: Rivaroxaban and apixaban are the most commonly used anti-factor (F) Xa direct oral anticoagulants (DOAC), with indications for prevention of stroke in nonvalvular atrial fibrillation as well as treatment and prevention of venous thromboembolism. However, lacking is accessibility to a detection method that is able to quantify low levels of anti-FXa DOACs., Objective: We report a new assay that measures anti-FXa DOAC levels in plasma and whole blood., Methods: This is achieved by the use of a prothrombin derivative that is labeled with a fluorescent probe (Flu-II), which then acts as the macromolecular substrate to measure residual FXa activity. The Flu-II cleavage is then initiated by the addition of a solution containing FXa, FVa, and phospholipid vesicles composed of 75% PC and 25% PS (PCPS) vesicles with calcium, in the presence of hirudin to prevent feedback activity by the native thrombin generated. The Flu-II cleavage is monitored by fluorescence in real time where the initial rate of fluorescence change is inversely proportional to DOAC levels., Results: In plasma systems, the assay demonstrates dose-response between 0 and 5 nmol/L rivaroxaban and between 0 and 10 nmol/L apixaban. Corn trypsin inhibitor did not affect this assay. With individual plasma samples, the assay showed excellent consistency and reproducibility. From 2 μL of whole blood, the assay showed dose-response between 0 and 2 nmol/L of DOACs in the final mixture of 100 μL, thus representing up to 100 nmol/L in circulating blood., Conclusion: The assay is ideal for rapidly and accurately measuring DOAC levels in plasma and blood, demonstrating its potential for point-of-care applications., (© 2019 International Society on Thrombosis and Haemostasis.)

Background : The prothrombin time may be used to monitor the plasma concentration of rivaroxaban. However, there is variability in the responsiveness of rivaroxaban to different thromboplastins. We aimed to develop a rivaroxaban-monitoring method using the prothrombin time to reduce the differences in the sensitivity among reagents. Methods : Rivaroxaban-spiked pooled normal plasma at a 0-1000 ng/ml concentration was used to generate a rivaroxaban-adjusted sensitivity index (SI) values, and was tested with three thromboplastins. The warfarin-adjusted international sensitivity index (ISI-warfarin), rivaroxaban-adjusted sensitivity index (SI-rivaroxaban), international normalized ratio (INR) calculated with ISI-warfarin, normalized ratio (NR) calculated with SI-rivaroxaban, and their coefficient of variances (CVs) were compared. The NR-rivaroxaban value was compared with the results of an anti-Xa assay. Results : The ISI-warfarin and SI-rivaroxaban using different thromboplastins were 1.02 and 1.88, respectively, with Thromborel S, 0.90 and 1.00 using Recombiplastin 2G, and 1.30 and 1.15 using Neoplastin CI-plus. Between-thromboplastin variability expressed as CV were 6.3%-25.1% when expressed as INR-warfarin and 1.7%-4.7% when expressed as NR-rivaroxaban. CVs for the NR-rivaroxaban with another laboratory were significantly lower than those for INR-warfarin. Anti-Xa assay v NR-rivaroxaban correlation coefficients were 0.97-0.99. Conclusion : Using a rivaroxaban-specific NR effectively minimises inter-thromboplastin variability. By utilizing a NR-rivaroxaban, standardized prothrombin time results could be rapidly obtained, especially useful in standardizing the therapeutic effect of rivaroxaban.

Background: Apixaban and rivaroxaban are approved for the prevention and treatment of deep vein thrombosis (DVT), pulmonary embolism (PE), and embolic stroke in atrial fibrillation (AF) patients. The aim of this study was to find appropriate methods of monitoring the anticoagulant effects of are direct oral anticoagulants (DOACs) and establish on-therapy ranges using conventional tests., Methods: A total of 184 samples were collected from 91 patients receiving DOACs. Concentrations of apixaban and rivaroxaban in plasma were accessed by an anti-factor Xa chromogenic assay. PT, APTT, antithrombin, D-dimer, dRVVT screen/confirm, FDP, and fibrinogen levels were measured. On-therapy ranges were calculated by substituting previously reported trough plasma concentrations of DOACs., Results: Anti-factor Xa chromogenic assay-based DOACs levels were 26.0-279.5 (115.9 ± 56.5) ng/mL for apixaban at 2.5 mg BID, 19.9-565.1 (205.3 ± 162.4) ng/mL for apixaban at 5 mg BID, 2.3-395.3 (205.3 ± 162.4) ng/mL for rivaroxaban at 15 mg OD, 3.6-494.8 (119.6 ± 95.1) ng/mL for rivaroxaban at 20 mg OD, and 9.6-431.4 (140.8 ± 113.6) ng/mL for rivaroxaban at 15 mg BID. PT (%), antithrombin, and dRVVT confirm tests showed good correlation with plasma apixaban levels. Plasma rivaroxaban concentrations were correlated well with PT (sec), PT (%),and dRVVT confirm results. On-therapy ranges established for dRVVT confirm test by linear regression were as follows: 1.32-1.52 for apixaban 2.5 mg BID, 1.12-1.75 for apixaban 5 mg BID, 1.11-1.78 for rivaroxaban 15 mg OD, 1.09-1.64 for rivaroxaban 20 mg OD, and 1.22-1.81 for rivaroxaban 20 mg BID., Conclusions: Apixaban concentrations were well correlated with PT (%), antithrombin, and dRVVT confirm test. Rivaroxaban concentrations showed good correlation with PT (sec), PT (%), and dRVVT confirm test., (© 2019 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.)

Background: Rapid detection of the anticoagulant effect of oral factor Xa (FXa) inhibitors may be essential in several emergency clinical situations. Specific assays quantifying the drugs are performed in plasma and require a turnaround time that is too long to be useful in emergency situations. Rotational thromboelastometry (ROTEM) is a whole blood coagulation assay of blood viscoelasticity and could be of interest for FXa inhibitor detection in emergency. However, conventional ROTEM reagents only detect high amounts of inhibitors., Objective: The aim of this study was first to assess the effect of whole blood components on the viscoelastic measurement of the effects of FXa inhibitors, and second to evaluate whether a modified ROTEM, triggered with a low amount of tissue factor and a saturating amount of phospholipid vesicles, can reliably detect low levels of FXa inhibitor activity in whole blood., Design: Diagnostic test study., Settings: A university research laboratory. From November 2014 to April 2016., Patients: Sixty-six patients: 30 treated with rivaroxaban, 17 with apixaban and 19 without treatment., Intervention: ROTEM was triggered with 2.5 pmol l of tissue factor and 10 μmol l of phospholipid vesicles., Main Outcome Measures: Modified ROTEM parameters were measured in different experimental conditions: platelet-poor plasma (PPP), platelet-rich plasma, PPP supplemented with fibrinogen and reconstituted whole blood with various haematocrit levels adjusted between 30 and 60%. Modified ROTEM was further validated using whole blood from patients who were either treated or not treated with FXa inhibitors., Results: Modified ROTEM allowed detection of as little as 25 ng ml FXa inhibitors in PPP, with at least a 1.4-fold increase of the clotting time (P ≤ 0.02). Neither changes of fibrinogen concentration nor variations of platelet count or haematocrit precluded FXa inhibitor detection. A lengthened modified ROTEM clotting time of more than 197 s allowed detection of FXa inhibitor concentrations above 30 ng ml in whole blood with 90% sensitivity and 85% specificity., Conclusion: Modified ROTEM may be applicable in emergency situations for the detection of FXa inhibitors in whole blood.

Purpose: Rivaroxaban is a substrate for ABCB1 transporter and is commonly used in patients undergoing hip or knee replacement surgery for thromboprophylaxis. The objective of this study was to develop a population pharmacokinetic-pharmacodynamic (PK-PD) model to investigate the influence of ABCB1 gene expression and polymorphism on rivaroxaban exposure and anticoagulation effects., Methods: Five blood samples per patient were collected during 5 days after the surgery for the determination of rivaroxaban concentration in plasma and for determination of prothrombin time and partial thromboplastin time. Non-linear mixed effects model was used for a population PK-PD analysis and for testing covariate effects., Results: A one-compartment PK model with first-order absorption adequately described the pharmacokinetic data. The typical oral clearance (CL/F) was 6.12 L/h (relative standard error, 15.8%) and was associated with ABCB1 expression. Compared to base line before the surgery, a significant ABCB1 downregulation was observed 5 days after the surgery (p < 0.001). Prothrombin time and partial thromboplastin time were both linearly associated to the logarithm of the rivaroxaban plasma concentration., Conclusions: We confirmed that variable rivaroxaban CL/F is associated with ABCB1 expression, which is in accordance with previous studies on P-glycoprotein involvement in rivaroxaban PK. Furthermore, we observed the downregulation of ABCB1 expression after the surgery. The cause remains unclear and further research is needed to explain the underlying mechanisms.

The aim of the study was to evaluate the diagnostic accuracy of thromboelastometry for assessing rivaroxaban concentrations. The accuracy of thromboelastometry was compared with the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method, which is the gold standard for drug plasma monitoring (the reference standard). Forty-six clinically stable patients were treated with 10, 15, or 20 mg of rivaroxaban once daily (OD group) or 15 mg twice a day (BID group) (no particular indication for treatment). Patient samples were collected 2 h after the use of the medication (peak) and 2 h before the next dose (trough). The rivaroxaban plasma concentrations were determined via HPLC-MS/MS, and thromboelastometry was performed using a ROTEM® delta analyzer. There were significant prolongations in clotting time (CT) for the 10, 15, and 20 mg of rivaroxaban treatments in the OD groups. In the 15 mg BID group, the responses at the peak and trough times were similar. At the peak times, there was a positive correlation between the plasma concentration of rivaroxaban and CT (Spearman correlation rho=0.788, P<0.001) and clot formation time (rho=0.784, P<0.001), and a negative correlation for alpha angle (rho=-0.771, P<0.001), amplitude after 5 min (rho=-0.763, P<0.001), and amplitude after 10 min (rho=-0.680, P<0.001). The CT presented higher specificity and sensitivity using the cut-off determined by the receiver characteristics curve. ROTEM has potential as screening tool to measure possible bleeding risk associated with rivaroxaban plasma levels.

There are no clear and consistent guidelines on how to utilize DOAC assays, and reports on the use of DOAC levels in clinical practice is limited. The objective of this study was to analyze why DOAC levels are ordered, how the results affect clinical decision-making, and to determine if DOAC assays are utilized appropriately. This was a retrospective chart review study analyzing 150 dabigatran, rivaroxaban, and apixaban levels performed at a single institution. The majority of DOAC assays were ordered in situations or special patient populations where confirming absence or detecting presence of drug may be useful. The most common indication for ordering assays was prior to an invasive procedure. Most DOAC levels were timed appropriately but peak levels were most likely to be incorrectly ordered. Clinical decisions following level results depended on indication for ordering and were most commonly used to determine whether or not to proceed with an invasive procedure. The results of our study suggest while DOAC assays are generally ordered for useful indications, there is still a lack of understanding of when levels should be drawn and how to interpret DOAC assay results.

Background: Oral anti-Xa inhibitors have demonstrated noninferiority to vitamin K antagonists (VKAs) for the prevention of stroke in patients with atrial fibrillation and recurrent venous thromboembolism. They are associated with a decrease in major bleeding. In contrast with VKA, no coagulation monitoring is required. However, in clinical practice, determination of drug concentration is sometimes necessary., Objective: The objective of this study was to evaluate a low-molecular-weight heparin (LMWH) calibrated anti-Xa assay for the quantification of rivaroxaban and apixaban plasma concentration in emergency., Methods: The anti-Xa plasma concentration of rivaroxaban and apixaban were measured in emergency in 210 patients using STA anti-Xa liquid assay. For each plasma concentration <150 ng/mL of rivaroxaban or apixaban, an anti-Xa assay calibrated with LMWH was performed., Results: We demonstrated a significant correlation between LMWH anti-Xa activity and rivaroxaban ( R 2 = 0.947) or apixaban ( R 2 = 0.959) concentration and a significant correlation between rivaroxaban and apixaban plasma concentration ( R 2 = 0.972). A LMWH anti-Xa activity <0.50 IU/mL could exclude a plasma concentration of rivaroxaban and apixaban >30 ng/mL and indicate the feasibility of invasive procedure. Conclusion and Relevance: In the absence of a specific test, LMWH-calibrated anti-Xa assay could be used to determine the presence and evaluate the plasma concentration of oral anti-Xa inhibitors. However, these initial findings require confirmation using other chromogenic calibrated oral anti-Xa assays.

Background and Objectives: There have been no animal experiments and clinical studies on the pharmacokinetic interaction between rivaroxaban and enalapril. To investigate whether a potential pharmacokinetic interaction is present between rivaroxaban and enalapril, a rapid and sensitive Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine the concentration of rivaroxaban and enalapril in rat plasma and was then applied to a pharmacokinetic interaction study., Methods: The analytes were separated on an Acquity UPLC BEH C18 chromatography column (2.1 × 50 mm, 1.7 μm) with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. The mass spectrometer was operated in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of 436.1 → 145.1 m/z for rivaroxaban, 377.3 → 234.2 m/z for enalapril and 285.2 → 193.1 m/z for diazepam (IS)., Results: The method was validated over the concentration range of 1.0-200 ng/mL for rivaroxaban and 0.5-100 ng/mL for enalapril. The intra- and inter-day precision and accuracy of the quality control (QC) samples exhibited relative standard deviations (RSD) < 9.4% and the accuracy values ranged from - 8.3 to 9.6%. After co-administration of rivaroxaban and enalapril, the maximum plasma concentration (C max ) and area under the systemic drug concentration-time curve from time 0 to infinity (AUC 0-∞ ) of rivaroxaban were significantly increased by 19.6% (p < 0.05) and 21.3% (p < 0.05), respectively. On the contrary, the plasma clearance rate (CL/F) of rivaroxaban and enalapril was significantly decreased by 17.8% (p < 0.05) and 23.8% (p < 0.05), respectively., Conclusions: The UPLC-MS/MS method was successfully applied to simultaneous determination of rivaroxaban and enalapril in rat plasma and applied to study the pharmacokinetic interaction between rivaroxaban and enalapril. The co-administration of rivaroxaban and enalapril resulted in a significant drug interaction in rats.

Background: The crushed-tablet rivaroxaban concentration has been previously reported to be lower than the non-crushed concentration. However, the rivaroxaban concentration of fine granules has not yet been investigated. The anticoagulation intensity of rivaroxaban with fine granules, tablets, and crushed tablets was compared in acute stroke patients to assess the efficacy of each form., Methods and Findings: Hospitalized patients over 75 years old with acute stroke who started taking rivaroxaban from April 2012 to September 2017 were included. Blood samples were drawn just before and 4 hours after taking rivaroxaban on a median of 5 days after treatment initiation for concentration measurements (C0h, C4h) based on an anti-factor Xa chromogenic assay. Of 114 patients (49 female, 83±5 years old), 97 had ischemic strokes, 9 had transient ischemic attacks, and 8 had intracerebral hemorrhages. Rivaroxaban was administered a median of 7 days after onset. Of these, 38 patients were given the 15 mg dose, and 76 were given the 10 mg dose. In the 15 mg dose group, C0h was significantly higher in the fine granule group than in the crushed tablet group, with no significant difference compared to the tablet group [C0h: 27.6±6.8 vs 4.0±4.1 (P = 0.01) vs. 33.3±25.2 ng/ml, (P = 0.51), respectively], as was C4h [223.0±66.6 vs 103.0±79.5 (P = 0.02) vs. 229.5±121.6 ng/ml (P = 0.88)]. In the 10 mg dose group, C0h was significantly higher in the fine granule group than in the crushed tablet group and comparable to that in the tablet group [23.2±7.9 vs 7.5±6.2 (P<0.01) vs 19.0±15.8 ng/ml, (P = 0.35)], as was C4h [150.7±85.4 vs 85.1±46.8 (P<0.01) vs 189.8±92.7 ng/ml (P = 0.18)]., Conclusions: The rivaroxaban concentration with fine granules was consistent with that in the tablet group and higher than that in the crushed tablet group., Competing Interests: Toyoda, Ihara, and Koga report honoraria for speaking from Bayer Yakuhin. This study was not funded by Bayer HealthCare Pharmaceuticals or Bayer Yakuhin. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

There is limited clinical experience with the use of coagulation concentrates to reverse the effect of direct oral anticoagulants. We assess the achievement of effective clinical hemostasis with the use of 4-factor prothrombin complex concentrate (PCC) in patients on apixaban or rivaroxaban presenting with major bleeding. A retrospective chart review was conducted at a tertiary referral medical center in the USA. We assess the achievement of clinical hemostasis using 4-factor PCC in patients on chronic apixaban or rivaroxaban therapy presenting with major bleeding. Clinical hemostasis was assessed by the International Society of Thrombosis and Hemostasis Scientific and Standardization Subcommittee criteria. A total of 29 patients are included in the study. The most common site of bleeding was intracranial hemorrhage (ICH) (72.4%), followed by gastrointestinal bleed (13.8%). Clinical hemostasis was achieved in 21 (72.4%) patients. Patients who did not achieve clinical hemostasis (27.6%) suffered from ICH, and all of them died during hospitalization except for two patients who were discharged with neurologic deterioration. One patient developed multiple brain infarctions after receiving 4-factor PCC. Sixteen patients (55.2%) were receiving concomitant medications that interact with apixaban and rivaroxaban and increase the risk of bleeding. Four-factor PCC appears to be effective in achieving clinical hemostasis in patients on apixaban or rivaroxaban presenting with major bleeding. It may be an alternative to patients who need anticoagulation reversal if the specific antidote, andexanet alfa, is not available.

There is a high degree of uncertainty regarding optimum care of patients with potential or known intake of oral anticoagulants and traumatic brain injury (TBI). Anticoagulation therapy aggravates the risk of intracerebral hemorrhage but, on the other hand, patients take anticoagulants because of an underlying prothrombotic risk, and this could be increased following trauma. Treatment decisions must be taken with due consideration of both these risks. An interdisciplinary group of Austrian experts was convened to develop recommendations for best clinical practice. The aim was to provide pragmatic, clear, and easy-to-follow clinical guidance for coagulation management in adult patients with TBI and potential or known intake of platelet inhibitors, vitamin K antagonists, or non-vitamin K antagonist oral anticoagulants. Diagnosis, coagulation testing, and reversal of anticoagulation were considered as key steps upon presentation. Post-trauma management (prophylaxis for thromboembolism and resumption of long-term anticoagulation therapy) was also explored. The lack of robust evidence on which to base treatment recommendations highlights the need for randomized controlled trials in this setting.

Direct Oral Anticoagulants (DOACs) available for the treatment and prevention of thromboembolic diseases include dabigatran, a direct thrombin (IIa) inhibitor, and apixaban, edoxaban and rivaroxaban, which are direct inhibitors of Stuart factor (Xa). DOACs have a different pharmacokinetic and pharmacodynamics profiles, with less probable drug-drug interactions than vitamin K antagonists. They do not require systematic therapeutic monitoring except in specific clinical situations such as emergency procedures or drug non-compliance. Furthermore, anticoagulant effects of DOACs could be impacted by renal impairment, drug-drug interactions, food interactions, or pharmacogenetic variability. In this context, we developed a method for simultaneous determination of dabigatran, rivaroxaban and apixaban in human plasma using high performance liquid chromatography coupled with a mass spectrometry assay and applied it to 26 patient samples. Our method presents a total run time of 5 min and extends from 25 to 1000 μg/L for apixaban and dabigatran; and from 5 to 1000 μg/L for rivaroxaban. Intra- and inter-assay accuracy were between -22.3 and 25.4%; and - 23.7 and 3.8%, respectively. Precision at low and high concentrations were below 17.5%. Frozen samples were stable up to 3 months. No significant cross-contamination was observed. In conclusion, our assay can be used during clinical studies and in daily routine practice for the management of specific clinical situations at reasonable cost., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The objectives of this study are to compare steady-state trough (Cmin,ss) and peak (Cmax,ss) concentrations of rivaroxaban between Asians and Caucasians and to evaluate the relationship between rivaroxaban concentrations and prothrombin time/international normalized ratio (PT/INR). Recruited patients were advised on the time to take rivaroxaban. Cmin,ss and PT/INR were taken when patients arrived. Cmax,ss and PT/INR were drawn between 2 and 4 h later after the patient took rivaroxaban with food. Thirty patients were included in the analyses: 57% (n = 17) males and 43% (n = 13) females, 77% (n = 23) on 20 mg and 23% (n = 7) on 15 mg. Median PT trough and PT peak are moderately correlated with Cmin,ss (r 2  = 0.43) and Cmax,ss (r 2  = 0.49), respectively. Patients on 15 mg have lower Cmin,ss and Cmax,ss versus Caucasians [12 ng/ml vs. 57 ng/ml (Cmin,ss); 87 ng/ml vs. 229 ng/ml (Cmax,ss), p < 0.01 for both]. Patients on 20 mg also have lower Cmin,ss and Cmax,ss versus Caucasians [14 ng/ml vs. 44 ng/ml (Cmin,ss); 101 ng/ml vs. 249 ng/ml (Cmax,ss), p < 0.01 for both]. Subgroup analysis shows patients with BMI ≥ 30 have lower Cmax,ss than patients with BMI < 30 [80.47 ng/ml vs. 124 (p = 0.014)]. Cmin,ss and Cmax,ss were lower in Singaporeans than Caucasians. This may have an impact on the effectiveness of rivaroxaban in Singaporeans. Patients with higher BMI may not benefit similarly as patients with lower BMI. Lastly, the Dade Innovin reagent's measure of PT/INR is not sensitive towards changes in rivaroxaban concentrations.

Background: Detection of new oral anticoagulant (NOAC) levels by screening, special and global tests, and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is important in clinical situations when the cause of bleeding needs to be determined., Methods: We compared a routine coagulation test, special function test for NOACs, global coagulation test, and an LC-MS/MS method that enables simultaneous determination of apixaban, dabigatran and rivaroxaban in human plasma within one analysis to determine the optimal indication of the comparison methods, including their limitations and interferences., Results: This study was conducted on a set of blood samples from 116 patients treated with NOACs. The results of both specific dilute thrombin time (dTT) tests for dabigatran provided the same results as the activated partial thromboplastin time (aPTT) screening test in comparison with LC-MS/MS as a reference. The dTT assay HemosIL® showed better results for low concentrations when compared to LC-MS/MS than dTT HYPHEN® as HemosIL® uses a non-linear calibration curve. Results of the specific anti-Xa assay yielded better results than the prothrombin time test in comparison with LC-MS/MS as a reference, especially for apixaban, but also for rivaroxaban. Our LC MS/MS method is simply feasible, but only in a specialized laboratory. The method is easy-to-use for the simultaneous determination of all dabigatran, apixaban and rivaroxaban by LC-MS/MS within three minutes with a concentration range of 1 to 500 µg/L without dilution., Conclusions: In the normal practice of the coagulation laboratory, it is advisable to use specific tests for NOAC determination as screening and global assays are not sufficiently specific. The dTT test is the optimal choice for dabigatran determination and for xabans to determine anti-Xa activity. The LC-MS/MS method is suitable as an arbitration method for serious conditions.

Background: In atrial fibrillation (AF) patients with coronary artery disease (CAD), anticoagulants are commonly used in combination with antiplatelet drugs. However, dual therapy can increase the risk of bleeding, and the potential therapeutic benefits must be weighed against this. Therefore, it is recommended that dual therapy is only used for a limited time, and that monotherapy with anticoagulants should start from 1 year after percutaneous coronary intervention (PCI). However, there is a lack of evidence on the use of monotherapy, in particular with direct oral anticoagulants, in this group of patients., Methods: The AFIRE Study is a multicenter, prospective, randomized, open-label, parallel group study conducted in patients aged ≥20 years with non-valvular AF (NVAF) and CAD. Patients who have undergone PCI or coronary artery bypass graft at least 1 year prior to enrollment, or those without significant coronary lesions requiring PCI (≥50% stenosis), will be included. Approximately 2200 participants will be randomized to receive either rivaroxaban monotherapy or rivaroxaban plus an antiplatelet drug (aspirin, clopidogrel, or prasugrel). The primary efficacy endpoints are the composite of cardiovascular events (stroke, non-central nervous system embolism, myocardial infarction, and unstable angina pectoris requiring revascularizations) and all-cause mortality. The primary safety endpoint is major bleeding as defined by the International Society on Thrombosis and Haemostasis criteria., Conclusions: This study will be the first to assess the efficacy and safety of rivaroxaban monotherapy in NVAF patients with stable CAD., (Copyright © 2018. Published by Elsevier B.V.)

Prothrombin time (PT) has been widely used for measuring anticoagulation intensity under rivaroxaban therapy, but precise information has not been well established yet. Consecutive 96 non-valvular atrial fibrillation (NVAF) under rivaroxaban between Jan/June, 2015 were recruited. Serum concentration (SC) and PT with 5 representative reagents available in Japan (Neoplastin Plus®, Thromborel S®, Thrombocheck PT®, Thrombocheck PT Plus®, and Recombiplastin®) at 2-4 hours after (peak) and before intake of rivaroxaban (trough) were measured at outpatient clinic in the cardiovascular institute (CVI ARO study 1). Nonlinear mixed-effects modelling was used to model the population pharmacokinetics and pharmacodynamics of rivaroxaban. An oral one-compartment model was employed to describe the population pharmacokinetics of rivaroxaban. The pharmacokinetics of rivaroxaban were affected by creatinine clearance, alanine aminotransferase, and use of CYP3A4 or P-gp inhibitors. PTs with 5 reagents were predicted by pharmacodinamic models with SC, hematocrit, serum albumin, and age, with medium predicting ability (highest/lowest R2 = 0.746/0.658 in Recombiplastin/Thromborel S, respectively). This population analysis in NVAF patients under rivaroxaban therapy demonstrated that pharmacokinetics of rivaroxaban was described by an oral one-compartment model with expected covariates, and can be assessed by PT with available reagents in Japan with medium predicting ability., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Introduction: Rivaroxaban concentrations were measured in 127 inpatient samples using an HPLC-MS/MS assay., Methods: We compared this measurement with a calibrated anti-Xa assay and performed PT, aPTT and dilute PT tests to assess the value of clot-based assays in clinical decision-making., Results: The correlation between the anti-Xa assay and the HPLC-MS/MS at therapeutic concentrations was strong (R 2  = 0.98). The PT, RecombiPlasTin 2G, and aPTT, Actin FS, showed a linear dose-response but poor correlation (R 2  = 0.32 and 0.44, respectively) and at dilutions of 1 in 150 to 1 in 750 the dilute PT assay also showed poor correlation with rivaroxaban concentrations measured by specific assays. A normal PT or aPTT alone did not identify a likely safe rivaroxaban concentration to allow surgery or invasive procedures, but the combination of normal PT and aPTT identified a group of patients with rivaroxaban levels less than 90 ng/mL. Combined normal PT and aPTT had specificity and sensitivity of 0.97 (95% CI 0.92-0.99) and 0.37 (95% CI 0.1-0.74) for a rivaroxaban concentration < 32 ng/mL., Conclusions: The PT and aPTT show poor correlation with rivaroxaban levels measured by calibrated anti-Xa and HPLC-MS/MS assays. A normal combined PT and APTT identified low rivaroxaban levels with high specificity but lacked sensitivity. The dPT assay at several dilutions could not be used to quantify rivaroxaban in clinical samples. The utility of these PT, aPTT and dilute PT assays in a clinical setting is very limited, and results generated must be interpreted with caution., (© 2018 John Wiley & Sons Ltd.)

Background: Direct oral anticoagulants (DOACs) are prescribed for anticoagulation in patients with atrial fibrillation and venous thromboembolic disease. Fixed doses are recommended, but measuring their serum drug concentrations as a basis for dose adjustments may be useful in some clinical settings., Methods: An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the analysis of the DOACs apixaban, dabigatran, edoxaban, and rivaroxaban in human serum was developed and validated. A 100-µL serum sample was handled using a pipetting robot. Protein precipitation was performed with 375 µL of 1% formic acid in acetonitrile (vol/vol), and phospholipid removal was performed using a Waters Ostro 96-well plate. The injection volume was 1 µL, and run time was 3.0 minutes., Results: The calibration range was 5-800 nmol/L. The between-day precision relative SDs were in the range of 3.3%-10%. Recoveries ranged from 85% to 105%, and matrix effects from 88% to 102%, when corrected with internal standard. Edoxaban was, in contrast to the other DOACs, unstable when stored for more than 6 hours at 30°C. The suitability of the method was demonstrated by analyzing routine samples from 345 patients undergoing anticoagulation treatment., Conclusions: The developed method fulfilled the set validation criteria, and its suitability was demonstrated in a routine setting. The instability of edoxaban may complicate the transport of routine samples to the laboratory.

Background: Rivaroxaban oral anticoagulant does not need laboratory monitoring, but in some situations plasma level measurement is useful. The objective of this paper was to verify analytical performance and compare two rivaroxaban calibrated anti Xa assays/coagulometer systems with specific or other branch calibrators., Methods: In 59 samples drawn at trough or peak from patients taking rivaroxaban, plasma levels were measured by HemosIL Liquid anti Xa in ACLTOP 300/500, and STA liquid Anti Xa in TCoag Destiny Plus. HemosIL and STA rivaroxaban calibrators and controls were used. CLSI guideline procedures EP15A3 for precision and trueness, EP6 for linearity, and EP9 for methods comparison were used., Results: Coefficient of variation within run and total precision (CVR and CVWL respectively) of plasmatic rivaroxaban were < 4.2 and < 4.85% and BIAS < 7.4 and < 6.5%, for HemosIL-ACL TOP and STA-Destiny systems, respectively. Linearity verification 8 - 525 ng/mL a Deming regression for methods comparison presented R 0.963, 0.968 and 0.982, with a mean CV 13.3% when using different systems and calibrations., Conclusions: The analytical performance of plasma rivaroxaban was acceptable in both systems, and results from reagent/coagulometer systems are comparable even when calibrating with different branch material.

Purpose: Patients treated with direct oral anticoagulants (DOACs) frequently undergo interventional procedures requiring temporary discontinuation of anticoagulant therapy. Little is known about remaining peri-procedural exposure to rivaroxaban in real-world patients., Methods: Fifty-six patients with rivaroxaban treatment and scheduled cardiac catheterization were included in this prospective, observational, and single-center study. Rivaroxaban concentrations were determined by LC-MS/MS and a chromogenic anti-Xa assay. Population pharmacokinetic modeling was carried out on LC-MS/MS concentration data using NONMEM software, and results were applied to Monte Carlo simulations to predict appropriate rivaroxaban discontinuation intervals., Results: Rivaroxaban concentrations ranged from

Essentials Direct oral anticoagulants (DOACs) do not require laboratory monitoring currently. DOAC specific measurements were performed at trough in patients with atrial fibrillation. Patients who developed thromboembolic events showed lower DOAC plasma levels. This study supports the concept of measuring DOAC levels at steady state., Summary: Background Direct oral anticoagulants (DOACs) are administered at fixed doses without the need for dose adjustment according to laboratory testing. High interindividual variability in drug blood levels has been shown with all DOACs. To evaluate a possible relationship between DOAC C-trough anticoagulant levels and thromboembolic events, 565 consecutive naive patients with atrial fibrillation (AF) were enrolled in this study performed within the START Laboratory Registry. Methods DOAC-specific measurements (diluted thrombin time or anti-activated factor II calibrated for dabigatran; anti-activated FX calibrated for rivaroxaban or apixaban) at C-trough were performed locally at steady state within 15-25 days after the start of treatment. For each DOAC, the interval of C-trough levels, from the limit of quantification to the highest value, was subdivided into four equal classes, and results were attributed to these classes; the median values of results were also calculated. Thromboembolic complications occurring during 1 year of follow-up were recorded. Results Thromboembolic events (1.8%) occurred in 10 patients who had baseline C-trough levels in the lowest class of drug levels. The incidence of thromboembolic events among patients with DOAC C-trough levels in the lowest level class was 2.4%, and that in the remaining groups was 0%. The patients with thrombotic complications also had a higher mean CHA 2 DS 2 -VASc score than that of the total patient population: 5.3 (95% confidence interval [CI] 4.3-6.3 versus 3.0 (95% CI 2.9-3.1). Conclusion In this study cohort, thrombotic complications occurred only in DOAC-treated AF patients who had very low C-trough levels, with a relatively high CHA 2 DS 2 -VASc score. Larger studies are warranted to confirm these preliminary observations., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Introduction: Rivaroxaban (RXA) is a direct oral factor Xa (Xa) antagonist with a short half-life and a fast onset and offset of effect. Before elective surgery, discontinuation is recommended with an interval of at least > 24 hours. In clinical practice, this is, however, not always sufficient to achieve a residual RXA plasma concentration deemed appropriate for surgery, defined as ≤ 50 mcg/L. Our study aimed at identifying factors associated with a higher-than-expected residual RXA plasma concentration in a large group of real-life patients., Materials and Methods: This retrospective single-centre study included all patients taking RXA between 2012 and 2016 where RXA plasma concentration was determined by pharmacodynamic anti-Xa assay (518 measurements in 368 patients). Medical records were reviewed. Residual RXA plasma concentrations were then compared with expected values according to a pharmacokinetic model., Results: Residual RXA plasma concentration was significantly higher-than-expected in patients with atrial fibrillation, impaired kidney function (glomerular filtration rate [GFR] < 60 mL/min), CYP3A4-, CYP2J2- and PGP-inhibitory co-medication including amiodarone. Impaired kidney function (odds ratio [OR], 2.22, 95% confidence interval [CI], 1.30-3.78, p  = 0.003) and concomitant amiodarone intake (OR, 1.97, 95% CI, 1.04-3.72, p  = 0.036) were significantly associated with RXA plasma concentrations > 50 mcg/L at 24 to 48 hours after the last RXA intake., Conclusion: In our group of real-life patients, impaired kidney function (GFR < 60 mL/min) and co-medication with amiodarone were independently associated with higher-than-expected residual RXA plasma concentrations. In these patients, standard intervals of RXA discontinuation may not always be sufficient before elective surgery and routine pre-operative determination of the residual RXA concentration could be advisable., Competing Interests: A.K. and A.S., B.S.: None. P.S.: Received honoraria for lecturing by Vifor Pharma (Munich, Germany). A.J.: Receives research grant support from independent research foundations and from Pfizer, New York, United States. D.S.: Donat R. Spahn's academic department receives grant support from the Swiss National Science Foundation, Berne, Switzerland, the Ministry of Health (Gesundheitsdirektion) of the Canton of Zurich, Switzerland, for Highly Specialized Medicine, the Swiss Society of Anaesthesiology and Reanimation (SGAR), Berne, Switzerland, the Swiss Foundation for Anaesthesia Research, Zurich, Switzerland, Bundesprogramm Chancengleichheit, Berne, Switzerland, CSL Behring, Berne, Switzerland, Vifor SA, Villars-sur-Glâne, Switzerland. Dr. Spahn was the chair of the ABC Faculty and is the co-chair of the ABC-Trauma Faculty, which both are managed by Physicians World Europe GmbH, Mannheim, Germany, and sponsored by unrestricted educational grants from Novo Nordisk Health Care AG, Zurich, Switzerland, CSL Behring GmbH, Marburg, Germany and LFB Biomédicaments, Courtaboeuf Cedex, France. In the past 5 years, Dr. Spahn has received honoraria or travel support for consulting or lecturing from the following companies and organizations: Danube University of Krems, Austria; U.S. Department of Defense, Washington, United States; European Society of Anaesthesiology, Brussels, Belgium; Baxter AG, Volketswil, Switzerland; Baxter S.p.A., Roma, Italy; Bayer (Schweiz) AG, Zürich, Switzerland; Bayer Pharma AG, Berlin, Germany; B. Braun Melsungen AG, Melsungen, Germany; Boehringer Ingelheim (Schweiz) GmbH, Basel, Switzerland; Bristol-Myers-Squibb, Rueil-Malmaison Cedex, France, and Baar, Switzerland; CSL Behring GmbH, Hattersheim am Main, Germany, and Berne, Switzerland; Curacyte AG, Munich, Germany; Daiichi Sankyo (Schweiz) AG, Thalwil, Switzerland; Ethicon Biosurgery, Somerville, New Jersey, United States; Fresenius SE, Bad Homburg v.d.H., Germany; Galenica AG, Bern, Switzerland (including Vifor SA, Villars-sur-Glâne, Switzerland); GlaxoSmithKline GmbH & Co. KG, Hamburg, Germany; Haemonetics, Braintree, Massachusetts, United States; Janssen-Cilag AG, Baar, Switzerland; Janssen-Cilag EMEA, Beerse, Belgium; LFB Biomédicaments, Courtaboeuf Cedex, France; Merck Sharp & Dohme AG, Luzern, Switzerland; Novo Nordisk A/S, Bagsvärd, Denmark; Octapharma AG, Lachen, Switzerland; Oxygen Biotherapeutics, Costa Mesa, California, United States; PAION Deutschland GmbH, Aachen, Germany; Pharmacosmos A/S, Holbaek, Denmark; Photonics Healthcare B.V., Utrecht, Netherlands; ratiopharm Arzneimittel Vertriebs-GmbH, Vienna, Austria; Roche Diagnostics International Ltd, Reinach, Switzerland; Roche Pharma (Schweiz) AG, Reinach, Switzerland; Sarstedt AG & Co., Sevelen, Switzerland, and Nümbrecht, Germany; Schering-Plough International, Inc., Kenilworth, New Jersey, United States; Tem International GmbH, Munich, Germany; Verum Diagnostica GmbH, Munich, Germany; Vifor Pharma Deutschland GmbH, Munich, Germany; Vifor Pharma Österreich GmbH, Vienna, Austria; Vifor (International) AG, St. Gallen, Switzerland. J.S.: Lecture honoraria and advisory honoraria from Baxter (Switzerland), Bayer (Switzerland), BMS Pfizer (Switzerland), Boehringer-Ingelheim (Switzerland), CSL Behring (Switzerland), Janssen-Cilag (Switzerland), Mitsubishi Pharma, Novo Nordisk (Switzerland), Octapharma (Switzerland), Siemens Healthineers (Switzerland)., (Schattauer GmbH Stuttgart.)

Pharmacokinetics and antithrombotic effects of the Factor Xa inhibitor rivaroxaban were studied in subjects with mild renal insufficiency concurrently taking the P-glycoprotein and moderate CYP3A inhibitor verapamil, a drug commonly administered to patients with hypertension, ischemic heart disease, or atrial fibrillation. Age-matched controls with normal renal function were studied concurrently. Subjects' overall mean age was 59 years. Mean creatinine clearance values in the 2 groups were 105 and 71 mL/min. After single 20-mg oral doses, rivaroxaban area under the curve (AUC) was increased by a factor of 1.11 (ratio of geometric means [RGM]) in mild renal insufficiency compared to controls. Verapamil coadministration independently increased AUC to the same extent in both the mild renal insufficiency and control groups (RGM, 1.39 and 1.43). Concurrent mild renal insufficiency and verapamil produced additive inhibition compared to controls without verapamil (RGM, 1.58). Prothrombin time (PT) prolongation and Factor Xa inhibition tracked plasma rivaroxaban, and were enhanced by verapamil. Concentration-response relationships for PT (linear) and Factor Xa inhibition (hyperbolic) were unaffected by renal function or verapamil. The absolute and relative increases in rivaroxaban AUC caused by verapamil in mild renal insufficiency subjects are potentially associated with an increased bleeding risk. Modification of recommended dosage may be required in this combination of circumstances to reduce risk to patients., (© 2017, The American College of Clinical Pharmacology.)

Objective: Information about rivaroxaban plasma level (RivLev) may guide treatment decisions in patients with acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH) taking rivaroxaban., Methods: In a multicenter registry-based study (Novel Oral Anticoagulants in Stroke Patients collaboration; ClinicalTrials.gov: NCT02353585) of patients with stroke while taking rivaroxaban, we compared RivLev in patients with AIS and ICH. We determined how many AIS patients had RivLev ≤ 100ng/ml, indicating possible eligibility for thrombolysis, and how many ICH patients had RivLev ≥ 75ng/ml, making them possibly eligible for the use of specific reversal agents. We explored factors associated with RivLev (Spearman correlation, regression models) and studied the sensitivity and specificity of international normalized ratio (INR) thresholds to substitute RivLev using cross tables and receiver operating characteristic curves., Results: Among 241 patients (median age = 80 years, interquartile range [IQR] = 73-84; median time from onset to admission = 2 hours, IQR = 1-4.5 hours; median RivLev = 89ng/ml, IQR = 31-194), 190 had AIS and 51 had ICH. RivLev was similar in AIS patients (82ng/ml, IQR = 30-202) and ICH patients (102ng/ml, IQR = 51-165; p = 0.24). Trough RivLev (≤137ng/ml) occurred in 126/190 (66.3%) AIS and 34/51 (66.7%) ICH patients. Among AIS patients, 108/190 (56.8%) had RivLev ≤ 100ng/ml. In ICH patients, 33/51 (64.7%) had RivLev ≥ 75ng/ml. RivLev was associated with rivaroxaban dosage, and inversely with renal function and time since last intake (each p < 0.05). INR ≤ 1.0 had a specificity of 98.9% and a sensitivity of 25.7% to predict RivLev ≤ 100ng/ml. INR ≥ 1.4 had a sensitivity of 59.3% and specificity of 90.1% to predict RivLev ≥ 75ng/ml., Interpretation: RivLev did not differ between patients with AIS and ICH. Half of the patients with AIS under rivaroxaban had a RivLev low enough to consider thrombolysis. In ICH patients, two-thirds had a RivLev high enough to meet the eligibility for the use of a specific reversal agent. INR thresholds perform poorly to inform treatment decisions in individual patients. Ann Neurol 2018;83:451-459., (© 2018 American Neurological Association.)

Background: There is no established method for monitoring the anticoagulant effects of apixaban and rivaroxaban. Linear correlation between serum levels and anti-Xa activity has been shown, with r 2 ranging from 0.88 to 0.99. However, there are minimal data in patients receiving apixaban 5 mg twice daily or rivaroxaban 20 mg once daily., Objective: To evaluate the anti-Xa activity and serum levels at those doses and compare the trough anti-Xa activity., Methods: This was a single-center prospective study,approved by the institutional review board. Patients on an inappropriate dose or receiving an interacting drug were excluded. Blood samples were drawn 0.5 to 3 hours before a dose for both agents, 2 to 3 hours after an apixaban dose, and 12 to 16 hours after a rivaroxaban dose. Anti-Xa activity and serum levels were determined, and correlation was done via regression analysis. Trough anti-Xa activity was compared using a t-test., Results: The study enrolled 88 patients receiving each drug. The r 2 values were 0.79 and 0.87 for apixaban and rivaroxaban, respectively. The mean trough anti-Xa activity was 1.79 ± 0.96 IU/mL for apixaban and 1.25 ± 0.88 IU for rivaroxaban ( P < 0.01). The trough sample was drawn a mean of 1.3 and 1.8 hours prior to the next dose for apixaban and rivaroxaban, respectively ( P < 0.01)., Conclusions: Good correlation was shown between anti-Xa activity and serum levels. The clinical utility of monitoring anti-Xa activity and the significance of the difference in trough anti-Xa activity for these agents remains to be established.

Essentials Tests for direct oral anticoagulants (DOACs) are not widely applied. These tests are perceived to be difficult to run and subjected to large between-lab variation. We carried out proficiency testing surveys for DOAC testing in Italy. Interlab variability was small and similar to that of the international normalised ratio., Summary: Background Tests for direct oral anticoagulants (DOACs) are not widely available. The perception that they are difficult to perform and are subject to large between-laboratory variation makes their implementation difficult. Aims We carried out proficiency-testing surveys for DOACs within the activity of the external quality-assessment scheme of the Italian Federation of Thrombosis Centers. Design Participants were provided with coded freeze-dried plasmas without or with graded concentrations of the three main DOACs, and asked to measure prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time and DOAC concentrations with dedicated tests. The results were centralized for statistical analysis. Results and conclusions All participants (n = 235) reported results for PT and APTT, and approximately one-third reported results for DOAC concentration. PT and APTT showed variable responsiveness to DOACs: PT was more responsive to rivaroxaban than to dabigatran or apixaban. APTT was more responsive to dabigatran than to rivaroxaban or apixaban. The thrombin time ratio (test/normal) was close to unity for plasmas without dabigatran, and was high (i.e. 7.6-fold or 15.4-fold longer than the plasma free from the drug) for plasmas containing dabigatran at low (i.e. 42 ng mL -1 ) or high (i.e. 182 ng mL -1 ) concentration. Dedicated tests were responsive to the respective drugs, and their interlaboratory variability was relatively small (overall coefficients of variation of 8.7%, 8.4% or 10.3% for dabigatran, rivaroxaban and apixaban, respectively) and was comparable to that observed within the same survey for the International Normalized Ratio (i.e. 11.4%). In conclusion, tests for DOAC measurement performed reasonably well in a national quality-control scheme. Regulatory authorities should urgently issue recommendations on their use, and clinical laboratories should make them available., (© 2018 International Society on Thrombosis and Haemostasis.)

Background: Rivaroxaban is metabolized in the liver via CYP3A4, the cytochrome involved in the metabolism of nearly 50% of all medications. Thus, its effective concentration depends on multiple pharmacologic parameters., Methods: The primary goal of our research was to study the correlation between the CYP3A family activity and the safety and efficacy of anticoagulant therapy with rivaroxaban in patients with deep vein thrombosis (DVT). Thirty one patients with DVT aged 21-83 years, 18 men and 13 women, received rivaroxaban (Xarelto) 30 mg/day for 21 days after diagnosis and 20 mg/day for the follow-up period of 6 months. During the study period, Doppler ultrasound was performed weekly to assess the clot dynamics and recanalization time., Results: We found a direct statistically reliable correlation between CYP3A4 activity and both peak and trough rivaroxaban levels. A correlation was also found between the initial clot length and the time to full recanalization r = 0.764 (0.554-0.883), p < 0.0001. No significant link was found between either the glomerular filtration rate and peak rivaroxaban concentrations or between CYP3A4 activity and the treatment effectiveness parameters. No connection between renal function and rivaroxaban concentration was established in our study, which agrees with the clinical trials data that allow unlimited rivaroxaban use in patients with glomerular filtration rate >30 mL/min., Conclusions: The direct link between the initial clot length and time to full recanalization that has been found means that patients with more advanced stages of thrombosis need more time to reach recanalization than their counterparts with a less severe condition.

Aims: The aim of the present study was to demonstrate evidence of reduced thrombin generation at the trough plasma rivaroxaban concentration., Methods: A single-centre, prospective, nonrandomized, drug-intervention, self-controlled study was conducted in 51 anticoagulation therapy-naïve patients with nonvalvular atrial fibrillation. Plasma rivaroxaban concentration was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and the anti-factor Xa chromogenic assay. Partial thrombin time (PT), protein C activity, and protein S antigen, prothrombin fragment 1 + 2 (F1 + 2), D-dimer, thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasminogen activator inhibitor-1 (PAI-1) and tissue factor pathway inhibitor (TFPI) levels were also measured at the trough steady state after 4 weeks of rivaroxaban treatment and compared with baseline., Results: Plasma concentrations obtained by the LC-MS/MS and anti-Xa assays were correlated (r = 0.841, P < 0.001). The mean concentration of rivaroxaban at the trough steady state was 23.6 ng ml -1 , at which F1 + 2, TAT and D-dimer had decreased from the baseline values (P < 0.0001, P = 0.029 and P < 0.005, respectively). PT was prolonged (+0.59 s, P < 0.0001). TFPI increased from baseline to the trough steady state in the first to third quartile groups (+0.79 pg ml -1 , P = 0.048). By contrast, PAI-1, protein C activity, protein S antigen and TM remained within the normal range at the trough steady state., Conclusions: Residual plasma rivaroxaban at the trough steady state may explain the antithrombin effect of rivaroxaban in patients with nonvalvular atrial fibrillation., (© 2017 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Essentials In this crossover study the anticoagulant effects of rivaroxaban and apixaban were compared. Healthy volunteers received rivaroxaban 20 mg once daily or apixaban 5 mg twice daily. Rivaroxaban was associated with more prolonged inhibition of thrombin generation than apixaban. Rivaroxaban induced a clear prolongation of prothrombin time and activated partial thromboplastin time., Summary: Background The anticoagulant actions of the oral direct activated factor Xa inhibitors, rivaroxaban and apixaban, have not previously been directly compared. Objectives To compare directly the steady-state pharmacokinetics and anticoagulant effects of rivaroxaban and apixaban at doses approved for stroke prevention in patients with non-valvular atrial fibrillation. Methods Twenty-four healthy Caucasian male volunteers were included in this open-label, two-period crossover, phase 1 study (EudraCT number: 2015-002612-32). Volunteers were randomized to receive rivaroxaban 20 mg once daily or apixaban 5 mg twice daily for 7 days, followed by a washout period of at least 7 days before they received the other treatment. Plasma concentrations and anticoagulant effects were measured at steady state and after drug discontinuation. Results Overall exposure was similar for both drugs: the geometric mean area under the plasma concentration-time curve for the 0-24-h interval was 1830 μg h L -1 for rivaroxaban and 1860 μg h L -1 for apixaban. Rivaroxaban was associated with greater inhibition of endogenous thrombin potential (geometric mean area under the curve relative to baseline during the 0-24-h interval: 15.5 h versus 17.5 h) and a more pronounced maximal prolongation relative to baseline of prothrombin time (PT) (1.66-fold versus 1.14-fold) and activated partial thromboplastin time (APTT) (1.43-fold versus 1.16-fold) at steady state than apixaban. Conclusions Despite similar exposure to both drugs, rivaroxaban 20 mg once daily was associated with greater and more sustained inhibition of thrombin generation than apixaban 5 mg twice daily. Sensitive PT and APTT assays can be used to estimate the anticoagulant effects of rivaroxaban., (© 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Dabigatran etexilate, rivaroxaban and apixaban (DOACs) are widely used and measurement of their concentration is desirable in certain clinical situations. Target-specific assays are available but limited information exists on their performance especially in their ability to accurately measure low and high concentrations., Aims: To define, in a multicenter study, the precision and accuracy of DOAC measurements in daily practice., Methods: 15 plasma samples (kindly provided by Hyphen-Biomed) spiked with 5 blinded concentrations of dabigatran, rivaroxaban or apixaban (targeted 0-40-100-250-500ng/mL, actual concentrations measured by HPLC-MS/MS), were sent to 30 haemostasis laboratories. DOAC concentration, PT and aPTT were measured once in each sample using local reagents. Interlaboratory precision was determined by its coefficient of variation (CV) and accuracy by its bias., Results: 464 DOAC measurements were performed in the 30 laboratories using 4 dabigatran and 5 rivaroxaban/apixaban calibrated assays on 3 analysers. Inter-laboratory CVs were below 18% for concentrations ≥100ng/mL, and higher for concentrations ~40ng/mL; biases were below 8% for all drugs and concentrations. In DOAC-free samples, concentrations were all below the lower limit of quantification except for one value (dabigatran: 35ng/mL). Depending on the concentrations, significant differences were found between reagents in rivaroxaban and apixaban concentration values. PT and aPTT ratios displayed a low sensitivity to apixaban., Conclusion: Our results suggest that calibrated DOAC assays allow the reliable measurement of a wide range of drug concentrations, even though improvement of their performances is necessary, especially for measuring low concentrations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Anti-Xa activity (AXA) in patients with nonvalvular atrial fibrillation (NVAF) and relationship to bleeding events remains unclear., Methods: We evaluated AXA in 94 patients at both trough and peak rivaroxaban concentrations. Rivaroxaban dosage was determined according to creatinine clearance (CrCl): 10 and 15mg once daily for patients with CrCl 15-49 and CrCl ≥50mL/min, respectively. AXA value distribution and its association with bleeding events were examined in enrolled subjects., Results: The mean peak AXA level was significantly higher than the mean trough level (1.98±0.81 vs. 0.16±0.15IU/mL; p<0.001). The peak AXA level significantly differed among patients with CrCl 15-29, 30-49, 50-79, and ≥80mL/min (2.51±0.83, 1.72±0.76, 2.05±0.82, and 1.66±0.51IU/mL, respectively; p=0.004). Major and non-major clinically relevant bleeding events occurred in 22 patients (23.4% and 14.6% per year, respectively). The mean peak AXA level was significantly higher in patients who experienced bleeding events than in those who did not (2.40±0.70 vs. 1.84±0.80IU/mL; p=0.001). A Cox multivariate analysis showed that the peak AXA level was independently related to the incidence of major and non-major clinically relevant bleeding events (p=0.012). Cumulative bleeding rates were significantly higher in patients with high peak AXA levels (p<0.001)., Conclusion: Peak AXA level was an independent predictor for bleeding events in Japanese NVAF patients receiving rivaroxaban., (Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russell's viper venom time - dRVVT or activated partial thromboplastin time - aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs - dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2-1.4 LA could not be ruled out. Plasma concentrations varied between 8-172 µg/l for DAB, 8-437 µg/l for RIV and 36-178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.

We sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (K s ) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2-6 hours (h) after and 20-25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (K s -12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for K s and CLT before rivaroxaban intake. At 2-6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (K s +38 %, and +37 %, CLT -25 % and -25 %, CLT-TAFI -20 % and -24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (K s -12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20-25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

Background: Assessment of the anticoagulant activity of direct oral anticoagulants (DOACs) is justified in special clinical situations. Here, we evaluated two independent extraction methods and developed a multi-analyte ultra-high performance liquid chromatography tandem mass (UHPLC-MS/MS) method for the quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma., Methods: Routine extraction based on protein precipitation with acetonitrile and subsequent centrifugation was compared to sample clean-up using commercial paramagnetic micro-particles and subsequent magnetic depletion. Stable isotope-labeled analogs of all analytes were employed as internal standards. The method was validated according to international guidelines in terms of linearity, precision, trueness, sensitivity, recovery and matrix effects. The performances of both extraction methods were assessed in clinical samples obtained from patients treated with either apixaban or rivaroxaban. Additionally, we report on a patient with nonadherence to rivaroxaban treatment and fulminant pulmonary embolism., Results: The method was linear from 2 to 500 ng/mL for all analytes, and quantification of DOACs was established within a run time of 2.0 min. Based on MS/MS analyte responses, relative matrix effects were better controlled for dabigatran after extraction with paramagnetic micro-particles. Internal standards fully compensated for recovery and matrix effects in all assays, yielding equivalent results for both methods. Apixaban and rivaroxaban concentrations determined in clinical samples after extraction with both methods were in good agreement (R2=0.990)., Conclusions: A rapid and accurate multi-component UHPLC-MS/MS method for the quantification of four DOACs in human plasma was established. Paramagnetic micro-particles appear suitable for clean-up of plasma samples for LC-MS/MS-based therapeutic drug monitoring purposes.

Essentials Accurate determination of anticoagulant plasma concentration is important in clinical practice. We studied the accuracy and consistency of anti-Xa assays for rivaroxaban in a multicentre study. In a range between 50 and 200 μg L -1 , anti-Xa activity correlated well with plasma concentrations. The clinical value might be limited by overestimation and intra- and inter-individual variation., Summary: Background Determining the plasma level of direct oral anticoagulants reliably is important in the work-up of complex clinical situations. Objectives To study the accuracy and consistency of anti-Xa assays for rivaroxaban plasma concentration in a prospective, multicenter evaluation study employing different reagents and analytical platforms. Methods Rivaroxaban 20 mg was administered once daily to 20 healthy volunteers and blood samples were taken at peak and trough levels (clinicaltrials.gov NCT01710267). Anti-Xa activity was determined in 10 major laboratories using different reagents and analyzers; corresponding rivaroxaban plasma concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Findings Overall Pearson's correlation coefficient of anti-Xa levels and HPLC-MS results was 0.99 for Biophen ® Heparin (95% CI, 0.99, 0.99), Biophen ® DiXaI (95% CI, 0.99, 0.99) and STA ® anti-Xa liquid (95% CI, 0.99, 1.00). Correlation was lower in rivaroxaban concentrations below 50 μg L -1 and above 200 μg L -1 . The overall bias of the Bland-Altman difference plot was 14.7 μg L -1 for Biophen Heparin, 17.9 μg L -1 for Biophen DiXal and 19.0 μg L -1 for STA anti-Xa liquid. Agreement between laboratories was high at peak level but limited at trough level. Conclusions Anti-Xa activity correlated well with rivaroxaban plasma concentrations, especially in a range between 50 and 200 μg L -1 . However, anti-Xa assays systematically overestimated rivaroxaban concentration as compared with HPLC-MS, particularly at higher concentrations. This overestimation, coupled with an apparent interindividual variation, might affect the interpretation of results in some situations., (© 2017 International Society on Thrombosis and Haemostasis.)

Background: In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE)., Methods: Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP 139-151 . Stable anticoagulation was maintained throughout the entire experiment. Mice without anticoagulation treated with the vehicle only were used as controls. The neurological deficit was recorded during the course of EAE, and histopathological analyses of inflammatory lesions were performed., Results: In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed., Conclusion: We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.

Objective: To quantify the impact of activated charcoal (AC) on rivaroxaban exposure in healthy volunteers., Methods: This was an open-label study with an incomplete cross-over design of single-dose rivaroxaban (40 mg) administered alone or with AC in 12 healthy volunteers. The study comprised three treatment periods in randomised sequence, one with rivaroxaban administered alone and two with AC given at 2, 5 or 8 h post-dose. Rivaroxaban plasma concentration was measured in blood samples drawn at 16 time points. The pharmacokinetic model of rivaroxaban alone or with AC administration was built using a non-linear mixed-effect modelling approach., Results: The pharmacokinetic model was based on a one-compartment model with an absorption rate described by the sum of three inverse Gaussian densities to reproduce multiphasic and prolonged absorption. The inclusion in the model of each AC administration schedule significantly improved objective function value. AC reduced the area under the rivaroxaban concentration-time curve by 43% when administered 2 h post-dose, by 31% when administered 5 h post-dose and by 29% when administered 8 h post-dose. Based on the estimated pharmacokinetic model, simulations suggested that AC might have an impact even after 8 h post-dose., Conclusion: AC administration significantly reduces exposure to rivaroxaban even if AC is administered 8 h after rivaroxaban. These results suggest that AC could be used in rivaroxaban overdose and accidental ingestion to antagonise absorption. CLINICALTRIAL., Gov Registration No: NCT02657512.

Background: The use of prothrombin complex concentrates and the role of plasma concentration of anticoagulants in the management of bleeding in patients treated with direct oral anticoagulants are still debated. Our aim was to describe management strategies and outcomes of severe bleeding events in patients treated with direct oral anticoagulants., Methods: We performed a prospective cohort study of 732 patients treated with dabigatran, rivaroxaban, or apixaban hospitalized for severe bleeding, included prospectively in the registry from June 2013 to November 2015., Results: Bleeding was gastrointestinal or intracranial in 37% (212 of 732) and 24% (141 of 732) of the cases, respectively. Creatinine clearance was lower than 60 ml/min in 61% (449 of 732) of the cases. The plasma concentration of direct oral anticoagulants was determined in 62% (452 of 732) of the cases and was lower than 50 ng/ml or higher than 400 ng/ml in 9.2% (41 of 452) and in 6.6% (30 of 452) of the cases, respectively. Activated or nonactivated prothrombin complex concentrates were administered in 38% of the cases (281 of 732). Mortality by day 30 was 14% (95% CI, 11 to 16)., Conclusions: Management of severe bleeding in patients treated with direct oral anticoagulants appears to be complex. The use of prothrombin complex concentrates differs depending on bleeding sites and direct oral anticoagulant plasma concentrations. Mortality differs according to bleeding sites and was similar to previous estimates.