Activity Of Direct Oral Anticoagulants Research Articles

The use of reduced DOAC doses in atrial fibrillation patients does not always lead to good anticoagulation levels and avoid adverse events

BackgroundThe MAS study (Blood Advances 2024) showed that a high proportion of Italian AF patients treated with direct oral anticoagulants (DOACs) receive reduced doses. This sub-analysis of MAS data aimed to analyze the effects of reduced (appropriate or not)- or standard-dose use on DOAC activity assessed at baseline and the occurrence of thrombotic or bleeding complications during follow-up. MethodsThe MAS study design, the methods for DOAC measurement, the results, and the adverse events during follow-up, are described in detail elsewhere. ResultsSeven hundred AF patients (42 % of the total 1657) received a reduced dose (considered inappropriate in 140 [20 %]). They were older, more frequently women, with lower body mass index (BMI), hemoglobin levels, and creatinine clearance. They more often had cerebral or cardiovascular diseases, were taking more medications, with higher scores for thrombotic or bleeding risk. Despite the use of low doses, 133 (19.0 %) patients had high standardized C-trough DOAC levels and experienced a high proportion of bleeding events (8.3 % per year). Conversely, some patients (4.7 %) had very low levels, resulting in a high incidence of thrombotic events (6.7 % per year). No difference was detected if the reduced dose was appropriate or not. ConclusionThe unpredictable, highly variable inter-individual anticoagulant effect of DOACs may lead to either too low or too high anticoagulant levels, increasing the risk of thrombotic or bleeding events. This is particularly relevant for patients with high-risk conditions, such as those chosen for reduced-dose treatment. Further studies are needed to investigate this important clinical issue.

Utility of thromboelastography to assess the effect of anticoagulation reversal in intracranial hemorrhage.

Intracranial hemorrhage (ICH) is a serious complication associated with oral anticoagulant use and is associated with significant morbidity and mortality. Although anticoagulation reversal agents are utilized as standard of care, practitioners are limited in their ability to assess degree of anticoagulation reversal for direct oral anticoagulants (DOACs). There is a clinical need identify biomarkers to assess anticoagulation status in patients with DOAC-associated ICH to ensure hemostatic efficacy of anticoagulation reversal agents in the acute setting. The purpose of this study was to assess the utility of thromboelastography (TEG) to assess the impact of anticoagulation reversal in patients presenting to the emergency department (ED) with DOAC-associated ICH. We conducted a prospective, observational cohort study in adult patients presenting to the ED with acute DOAC-associated ICH. Patients were excluded if last DOAC dose was >48 hours prior to hospital arrival, if they experienced polytrauma, were pregnant, incarcerated, had a history of hepatic failure or coagulopathy, or received anticoagulation reversal with products other than prothrombin complex concentrates (PCCs). We collected baseline TEG samples from participants prior to anticoagulation reversal, as well as 30-minutes, 12-hours, and 24-hours post-reversal. TEG samples were also collected from participants who transferred to our facility after reversal at ED presentation, as well as 12- and 24-hours post-reversal. Pre-reversal TEG was collected on 10 participants prior to DOAC reversal. A significant decrease in TEG R-time was observed at 30 minutes post-reversal. R-time increased at 12- and 24-hours to baseline levels. Significant changes were not observed in K-time, clot strength, maximum amplitude, or coagulation index. TEG R-time may be able to detect a change in anticoagulation activity of DOACs in ICH after anticoagulation reversal. R-time decreases acutely after anticoagulation reversal and rebounds at 12- and 24-hours post-reversal.

Detection of direct oral anticoagulants with the diluted Russel's viper venom time.

The activity of direct oral anticoagulants (DOAC) is important in acute clinical situations. Recent studies have suggested a strong influence of DOAC on the diluted Russel's Viper Venom Time (dRVVT). Therefore, it may be a suitable screening parameter for antithrombotic plasma activity of different DOAC. This prospective study aims to evaluate the sensitivity and specificity of dRVVT to detect residual DOAC activity at recommended plasma level thresholds. A total of 80 patients were recruited, with 20 each treated with one of the four approved DOAC (apixaban, edoxaban, rivaroxaban or dabigatran), respectively. Blood plasma was collected before (baseline), at plasma peak time, and 6 and 12 h after DOAC. DRVVT was measured using the screen (LA1) and confirm (LA2) assay for lupus anticoagulant and compared with DOAC plasma levels. A reference range was calculated based on the dRVVT values of 61 healthy blood donors. All DOAC significantly prolonged the dRVVT especially at higher DOAC plasma levels. The LA1 time ≥41 s had a sensitivity ≥98% to detect edoxaban, dabigatran and rivaroxaban plasma levels ≥30 ng/mL but it was only 87% for apixaban. Sensitivity was ≥98% for all DOAC with the LA2 assay ≥36 s. The negative predictive value of a DOAC plasma level <30 ng/mL and dRVVT LA2 <36 s was 99%. The dRVVT confirm assay (LA2) reliably detects residual DOAC plasma levels ≥30 ng/mL and could be useful to rapidly rule out relevant DOAC activity in emergency situations and to guide treatment decisions.

A small-molecule hemostatic agent for the reversal of direct oral anticoagulant–induced bleeding

BackgroundThe bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer. ObjectivesTo identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent. MethodsWe sought to identify a small procoagulant molecule by screening a chemical library with a plasma clotting assay. The selected molecule was assessed for its procoagulant properties and its ability to reverse the effects of the DOACs in a thrombin generation assay. Its activity as a DOAC reversal agent was also evaluated in a tail-clip bleeding assay in mice. ResultsThe hemostatic molecule (HeMo) dose-dependently promoted thrombin generation in plasma, with dose values effective in producing half-maximum response ranging between 3 and 5 μM, depending on the thrombin generation assay parameter considered. HeMo also restored impaired thrombin generation in DOAC-spiked plasma and reversed DOAC activity in the mouse bleeding model. HeMo significantly reduced apixaban-induced bleeding from 709 to 65 μL (vs 43 μL in controls; P < .01) and dabigatran-induced bleeding from 989 to 155 μL (vs 126 μL in controls; P < .01). ConclusionHeMo is a small-molecule procoagulant that can counterbalance hemostatic disruption by a thrombin inhibitor (dabigatran) or factor Xa inhibitors (apixaban and rivaroxaban). The compound’s effective clot formation and versatility make it a possible option for managing the inherent hemorrhagic risk during DOAC therapy.

Optimal management of cardiac surgery patients using direct oral anticoagulants: recommendations for clinical practice.

Literature is scarce on the management of patients using direct oral anticoagulants (DOACs) undergoing elective, urgent and emergency surgery. Therefore, we summarize the current evidence and provide literature-based recommendations for the management of patients on DOACs in the perioperative phase. A general literature review was conducted on the pharmacology of DOACs and for recommendations on the management of cardiac surgical patients on DOACs. Additionally, we performed a systematic review for studies on the use of direct DOAC reversal agents in the emergency cardiac surgical setting. When surgery is elective, the DOAC cessation strategy is relatively straightforward and should be adapted to the renal function. The same approach applies to urgent cases, but additional DOAC activity drug level monitoring tests may be useful. In emergency cases, idarucizumab can be safely administered to patients on dabigatran in any of the perioperative phases. However, andexanet alfa, which is not registered for perioperative use, should not be administered in the preoperative phase to reverse the effect of factor Xa inhibitors, as it may induce temporary heparin resistance. Finally, the administration of (activated) prothrombin complex concentrate may be considered in all patients on DOACs, and such concentrates are generally readily available. DOACs offer several advantages over vitamin K antagonists, but care must be taken in patients undergoing cardiac surgery. Although elective and urgent cases can be managed relatively straightforwardly, the management of emergency cases requires particular attention.

Monitoring the activity of direct oral anticoagulants in a patient with severe nephrotic syndrome and pulmonary embolism. Lessons for the clinical nephrologist.

Monitoring the activity of direct oral anticoagulants in a patient with severe nephrotic syndrome and pulmonary embolism. Lessons for the clinical nephrologist.

Monitoring of Anticoagulant Activity of Dabigatran and Rivaroxaban in the Presence of Heparins.

The routine monitoring of direct oral anticoagulants (DOACs) may be considered in patients with renal impairment, patients who are heavily obese, or patients requiring elective surgery. Using the heparin-binding copolymer (HBC) and polybrene, we aimed to develop a solution for monitoring the anticoagulant activity of DOACs in human plasma in the interfering presence of unfractionated heparin (UFH) and enoxaparin. The thrombin time (TT) and anti-factor Xa activity were monitored in pooled plasma from healthy volunteers. In these tests, plasma with dabigatran or rivaroxaban was mixed with UFH or enoxaparin and then incubated with HBC or polybrene, respectively. HBC and polybrene neutralized heparins and enabled monitoring of anticoagulant activity of dabigatran in the TT test. Both agents allowed for accurate measurement of anti-factor Xa activity in the plasma containing rivaroxaban and heparins in the concentration range reached in patients’ blood. Here, we present diagnostic tools that may improve the control of anticoagulation by eliminating the contamination of blood samples with heparins and enabling the monitoring of DOACs’ activity.

Detection of direct oral anticoagulants with the dilute Russels viper venom time – observations in a real life setting

Abstract Background The detection of direct oral anticoagulants (DOACs) in emergency settings still remains challenging, especially when dedicated anti-Xa or anti-IIa tests are not readily available or the specific DOAC is unknown. The dilute Russel's viper venom time (dRVVT) is widely known in lupus anticoagulant (LA) testing and may also provide helpful information regarding the residual antithrombotic activity of DOACs. Yet real world data describing the effect of DOACs on the dRVVT is scarce. Objective The study aimed to evaluate the sensitivity and specificity of dRVVT for different DOAC plasma levels. Methods A total of 80 patients were recruited – 20 patients for each approved DOAC (apixaban, edoxaban, rivaroxaban and dabigatran). Blood plasma was sampled before (baseline), 6 and 12 hours after DOAC-intake and at plasma peak time. DRVVT was measured using the LA1 screening assay for lupus anticoagulant. Plasma levels were measured by calibrated anti-Xa or anti-IIa tests. Additionally, activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured. Results All DOACs significantly prolonged the dRVVT. The effects were more pronounced at higher DOAC plasma levels. The area under the receiver operating characteristic (ROC) curve regarding a plasma level cut-off of 30 ng/ml was 0.92 (95% confidence interval (CI) 0.823 to 1.00) for apixaban, 0.97 (95% CI 0.902 to 1.00) for edoxaban, 0.87 (95% CI 0.649 to 1.00) for rivaroxaban and 0.96 (95% CI 0.871 to 1.00) for dabigatran. Conclusion A dose dependent effect of all approved direct oral anticoagulants (DOACs) on the dilute Russel's viper venom time (dRVVT) was documented in our real world setting, while relevant DOAC plasma levels did not affect standard coagulation tests. The negative predictive value for the dRVVT was high even at low DOAC plasma levels. The dRVVT could therefore be helpful to rule out relevant antithrombotic plasma activity caused of DOACs in emergency situations. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Universität Leipzig Figure 1. ROC curves

Direct oral anticoagulants in patients with nonvalvular atrial fibrillation and extreme body weight.

Limited clinical data exist describing the use of direct oral anticoagulants (DOACs) in patient with extreme body weight. Thus, the International Society of Thrombosis and Haemostasis (ISTH) recommends avoiding DOACs in patients with weight >120Kg, and on the contrary, no restrictions exist for underweight patients. To evaluate the effects of extreme body weight on DOAC activity and to compare the clinical outcomes of patients with an extreme body weight versus patients with a normal weight (61-119Kg) treated with DOACs. Single tertiary care Italian centre multidisciplinary registry including nonvalvular atrial fibrillation (NVAF) patients treated with DOACs. Based on weight, three subcohorts were defined: (i) underweight patients (≤60Kg); (ii) patients with a normal weight (61-119Kg, as control group); and (iii) overweight patients (≥120Kg). Primary efficacy endpoint was 2-year rate of thromboembolic events. Primary safety endpoint was 2-year rate of major bleeding. Event-free survival curves among groups were compared using Cox-Mantel test. 812 NVAF patients were included, 108 patients weighed ≤60Kg (13%, underweight), 688 weighed between 61 and 119Kg (85%, normal weight), and 16 weighed ≥120Kg (2%, overweight). In particular, among underweight patients, dabigatran was prescribed in 26% patients, apixaban in 27%, rivaroxaban in 28% and edoxaban in 22% ones. Instead, among overweight patients, 44% were treated with dabigatran, 25% with apixaban, 25% with rivaroxaban and 4% with edoxaban. Underweight patients were older, more frequently women, with lower creatinine clearance and a history of previous strokes, resulting in higher CHA2DS2-VASc score than in both remaining groups. Up to 2years, no statistically significant difference was observed between the three groups of weight for thromboembolic events (P=.765) and for overall bleeding (P=.125), but a trend towards decreased overall bleeding rates was noticed as weight increased (24.1% vs 16.7% vs 12.5%, respectively). In this tertiary care centre registry, 15% of patients treated with DOACs presented an extreme weight. Compared to patients with a normal weight, no significant rates of thromboembolic events were observed for underweight or overweight patients. A trend towards decreased overall bleeding frequency as weight increased was highlighted up to 2years. The present results should be considered as preliminary and hypothesis generating.

Direct Oral Anticoagulants Plasma Levels Measurement: Clinical Usefulness from Trials and Real-World Data.

Measurement of direct oral anticoagulants (DOACs) activity is not routinely necessary. Indeed, evaluation of DOACs plasmatic concentration is discouraged for the majority of patients, due to the lack of outcome data supporting this approach. Nevertheless, DOAC measurements may be useful in emergency situations such as serious bleeding events, need for urgent invasive procedures, and acute ischemic stroke or in managing anticoagulation in "special populations" not adequately studied in clinical trials, for example the very elderly or those at the extremes of body weight. The aim of this review is to describe and summarize the methods for DOACs activity evaluation and the settings in which their plasma level measurement may be indicated, analyzing indications from scientific societies and evidence from clinical trials, as well as real world data on the usefulness of DOACs plasma levels "monitoring."

Direct oral anticoagulants in point-of-care monitoring: an ex-vivo study.

Anticoagulatory activity of direct oral anticoagulants (DOACs) is not routinely measurable by point-of-care monitoring. Thus, the aim of this study was to evaluate the influence of dabigatran/rivaroxaban on point-of-care testing. Samples from 34 participants under DOAC therapy were drawn at two time points. Before ingestion and two-to-three hours afterwards. Thrombelastometric (ROTEM) and aggregometric (Multiplate) measurements were performed. Dabigatran and rivaroxaban plasma levels were determined. Dabigatran and rivaroxaban plasma levels showed significant correlations with clotting time (CT) in EXTEM (r=0.765, P<0.0001; r=0.689, P<0.0001) and INTEM (r=0.792, P<0.0001; r=0.595, P<0.001). A positive correlation was identified between dabigatran ingestion and maximum-clot-firmness (MCF) (r=0.354, P<0.05) in the EXTEM test, pronounced in the absence of concomitant antiplatelet therapy (r=0.709, P<0.05). EXTEM-MCF positively correlated with the TRAP test in aggregometry (0.662, P<0.05), an effect not observed in patients treated with antiplatelet therapy. Prolongation of CT-EXTEM and CT-INTEM indicates delayed initiation of clot formation. The CT-EXTEM seems to facilitate qualitative monitoring of dabigatran. In contrast, qualitative monitoring of rivaroxaban by CT-EXTEM may be limited as rivaroxaban may affect the measurement at therapeutic plasma levels. It seems that clot formation is faster/firmer in the presence of increased dabigatran plasma levels. This can be attributed to a non-dose-dependent effect via increased fibrin polymerization and second to a dose-dependent effect via increased platelet sensitivity to thrombin.

Remaining activity of temporary interrupted direct oral anticoagulants and its impact on intra-ablation heparinization in patients with atrial fibrillation: Comparisons across four drugs and two dose regimens.

Atrial fibrillation (AF) ablation with minimally interrupted direct oral anticoagulants (DOACs) may raise a concern about their remaining activity. We tested the residual activity of four different DOACs and its impact on intraprocedural heparinization in patients undergoing AF ablation. We measured the anti-factor Χa activity for rivaroxaban, apixaban, and edoxaban, and serum DOAC concentration for rivaroxaban, apixaban, and dabigatran, 24 hours after the last intake in patients undergoing AF ablation treated with standard or reduced doses of DOACs. The heparin requirement during the procedure was also measured. We enrolled 34 patients with rivaroxaban, 35 with apixaban, 32 with edoxaban, and 31 with dabigatran, and among them, 30 were treated with reduced doses. The anti-factor Χa activity was the highest in the apixaban group among the patients with standard doses. The DOAC concentration was paradoxically lower in patients with standard doses than in those with reduced doses among the patients with rivaroxaban (34.3 ± 19.8 vs 56.6 ± 7.7 ng/mL; P = .01) and dabigatran (12.6 ± 10.6 vs 23.4 ± 14.7 ng/mL; P = .03). The total heparin requirement per body surface area had significant correlations with the anti-factor Χa activity (r = -.36) and DOAC concentration (r = -.32). Two different multiple linear regression models (adjusted R2 = 0.56 and 0.6, respectively) revealed that the anti-factor Χa activity (β = -.28; P = .002) and DOAC concentration (β = -.38; P < .001) were independent determinants of the total heparin requirement. Factors determining residual DOAC activity may include its type and dose regimen, and it may influence the heparin requirement during AF ablation.

Direct oral anticoagulant plasma levels and thrombin generation on ST Genesia system

BackgroundMonitoring of anticoagulant activity of direct oral anticoagulants (DOACs) can be necessary in special situations. DOAC plasma levels have a high inter‐ and intraindividual variation and do not necessarily reflect the coagulation status of the patient. Thrombin generation (TG) is a global hemostatic assay with the capacity to overcome this limitation. The aim of this study was to show correlations between DOAC plasma levels and TG parameters using the fully automated ST Genesia system. MethodsA total of 380 blood samples (120 with apixaban, 79 with dabigatran, 79 with edoxaban, and 102 with rivaroxaban) from patients at different time points after DOAC intake were included in the analysis. DOAC plasma levels were analyzed using calibrated anti‐Xa or anti‐IIa tests. Thrombin generation was measured using the ST Genesia system and STG‐DrugScreen reagent. ResultsThere was a significant correlation between the drug levels of all DOACs and the TG parameters’ lag time and time to peak. Peak thrombin and velocity index show a negative correlation following an exponential regression curve with all anti‐Xa DOACs but not with dabigatran. Apart from a weak correlation with rivaroxaban, there was no correlation between drug levels of all other DOACs and endogenous thrombin potential. ConclusionTG parameters measured with ST Genesia correlate with the drug levels of anti‐Xa DOACs. Peak thrombin and velocity index are of special interest for the determination of residual anticoagulant effect at low drug levels. For dabigatran‐treated patients, only lag time shows a correlation with the dabigatran plasma levels.

Monitoring the Effects of Direct Oral Anticoagulants with a Novel Point-of-Care Sensor: Results of a Pilot Clinical Study

Introduction: Direct oral anticoagulants (DOACs) are a new class of medications that have been designed to provide direct inhibition of either coagulation factor IIa (FIIa, dabigatran) or FXa (rivaroxaban, apixaban, and edoxaban) and are rapidly replacing warfarin as the anticoagulant-of-choice for the treatment of most thrombotic disorders and atrial fibrillation. These newer agents present several advantages including predictable pharmacokinetic profile, wide therapeutic window, and shorter half-life. Despite these advantages, metabolism of DOACs is complex and requires enteric absorption and liver or renal clearance. Thus, the pervading maxim that DOACs do not require laboratory monitoring is simplistic and there are several situations in which assessment of the anticoagulant effect of DOACs will be useful. Indications for assessment of DOAC effects on the coagulation system include need for emergent surgery, unexpected thrombotic or hemorrhagic events, patients with extreme body weight, abrupt impairment of liver and kidney function, suspicion of DOAC overdose, poor compliance, or after administration of reversal agents. To date, no assays developed to measure DOAC activity have been approved by the Food and Drug Administration (FDA). Thus, there is an unmet need for a rapid coagulation assay that can measure the anticoagulant effect of DOACs in the laboratory or at the POC. We have recently developed a novel dielectric microsensor, termed ClotChip, that allows for the comprehensive assessment of blood hemostasis using a disposable microfluidic sensor at the POC (Fig 1A). The aim of this study was to assess the clinical utility of ClotChip at measuring the anticoagulant effects of DOACs in whole blood.Methods: We conducted a pilot clinical study at the Louis Stokes Cleveland VA Medical Center and accrued 127 individuals after IRB approval. Of these, 47 were healthy volunteers (not taking any anticoagulation or antiplatelet therapy) and the remaining participants were patients on DOACs (rivaroxaban: n=25, apixaban: n=47, dabigatran: n=8). Whole blood samples were collected in 3.2% sodium citrate. Samples from patients on DOACs were collected within 6 hours of rivaroxaban ingestion, between 3 - 5 hours post-morning dose of apixaban, or within 12 hours of dabigatran intake. All samples underwent whole blood measurement with ClotChip within 4 hours of collection as well as conventional plasma-based coagulation tests including PT, aPTT, drug specific anti-Xa assays or dilute thrombin time. The ClotChip is based on the electrical technique of dielectric spectroscopy (DS) to monitor the hemostatic process ex vivo in a low-cost (material cost < $1), small-sized (26mm × 9mm × 3mm), and disposable microfluidic sensor with miniscule sample volume (< 10µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. The time to reach a peak in permittivity (Tpeak), is a sensitive parameter to assess coagulation defects, and we hypothesized that Tpeak can effectively measure the anticoagulant effect of DOACs (Fig. 1B).Results: Using Gaussian fit method, mean Tpeak for the control group was determined to be 429 sec (239 - 619 sec, ±2 SD, Fig 1C). There was statistically significant prolongation in Tpeak for rivaroxaban (637 ± 24 sec., mean ± SEM, p < 0.0001), apixaban (593 ± 23 sec, mean ± SEM, p < 0.0001) and dabigatran (894 ± 82 sec, mean ± SEM, p < 0.0001) samples (Fig 1C). An ROC curve was then generated using statistical software to determine the true positive rate (sensitivity) and false positive rate (100% - specificity). The area under the curve (AUC) for ClotChip Tpeak was 0.9185 for rivaroxaban, 0.8132 for apixaban and 1 for dabigatran (Fig 1D).Conclusions: There are currently no approved laboratory assays that reliably measure the anticoagulant effect of DOACs. ClotChip offers a portable POC platform for rapid, comprehensive assessment of hemostasis using a miniscule amount of whole blood (<10 µL). In this pilot study, ClotChip showed excellent sensitivity at detecting the anticoagulant effects of DOACs. A larger clinical trial is currently being planned to validate these results and help establish ClotChip as a reliable method to detect and monitor DOAC activity. DisclosuresMaji:Case Western Reserve University: Patents & Royalties: Licensed to XaTek, Inc. . Mohseni:Case Western Reserve University: Patents & Royalties: Licensed to XaTek, Inc.; XaTek, Inc.: Consultancy. Suster:XaTek, Inc.: Consultancy; Case Western Reserve University: Patents & Royalties: licensed to XaTek, Inc..

Detecting clinically relevant rivaroxaban or dabigatran levels by routine coagulation tests or thromboelastography in a cohort of patients with atrial fibrillation

BackgroundTraditional coagulation tests are included in emergency guidelines for management of patients on direct oral anticoagulants (DOACs) who experience acute bleeding or require surgery. We determined the ability of traditional coagulation tests and fast whole blood thromboelastography (ROTEM®) to screen for anticoagulation activity of dabigatran and rivaroxaban as low as 30 ng/mL.MethodsOne hundred eighty-four citrated blood samples (75 dabigatran, 109 rivaroxaban) were collected from patients with non-valvular atrial fibrillation (NVAF), to perform screening tests from different manufacturers, (diluted, D) PT, aPTT, TT and ROTEM®. The activity of DOACs was quantitatively determined by clot detection assays: Hemoclot DTT and DiXaI test (Biophen), on CS2100 (Siemens). The clotting time (CT) of INTEM and EXTEM ROTEM® (Werfen) were used as test parameters.ResultsDabigatran, ≥ 30 ng/mL, was accurately detected by five coagulation tests: APTT Actin FSL (93%), PT Neoplastin (93%), APTT Cephascreen, Thromboclotin, and Thrombin (all 100%), but not by PT Innovin (49%). CT-EXTEM (91%) was sufficiently sensitive, but not CT-INTEM (52%). APTT Cephascreen and Thrombin showed good linearity (R2 = 0.71,R2 = 0.72). For the other tests linearity was moderate to poor. Rivaroxaban was accurately detected by PT Neoplastin (98%) and less so by APTT Cephascreen (85%). In addition, rivaroxaban was also accurately detected by CT-INTEM (96%). PT Neoplastin showed good linearity (R2 = 0.81), all other tests had moderate to poor linearity.ConclusionIn patients with NVAF, the ability of routine coagulation tests to detect the presence of significant levels of DOACs is test and reagent dependent. CT-INTEM and CT-EXTEM may be fast whole blood alternatives.Trial registrationThe Institutional Review Board of the MUMC approved this study (December 2011, project number 114069).

Direct Oral Anticoagulants and their Clinical Management

Thrombotic disorders are treated mostly with Heparin and Warfarin in cases requiring anticoagulation drugs. Due to limitations to these drugs including risk of bleeding and need for regular laboratory monitoring several Direct oral anticoagulants (DOACs) have been developed and shown to have more advantages than Warfarin. Nevertheless, these drugs are limited to use in some circumstances. As other anticoagulants, these drugs need to be managed and controlled to cope their over activities. Subsequently, many antidotes have been developed to reverse these DOACs. However, these antidotes are still understudies and need to be fully approved. The assessment of DOACs is developing and several drug-specific calibrators have been developed to measure DOACs activity in plasma.

Does type 2 diabetes affect the on-treatment levels of direct oral anticoagulants in patients with atrial fibrillation?

AimsType 2 diabetes (T2D) is connected with several abnormalities in haemostasis; and with higher risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NV-AF). However, it is recently unknown whether T2D affects the activity of direct oral anticoagulants (DOACs). The aim of this study was to determine the impact of T2D on DOACs activity in patients with NV-AF. MethodsThis pilot prospective study enrolled totally 65 patients with NV-AF (20 dabigatran-treated, 110 mg/twice daily; 28 rivaroxaban-treated, 15 mg/daily; 17 apixaban-treated, 5 mg/twice daily). 25 patients had T2D (8 dabigatran-treated, 11 rivaroxaban-treated, and 6 apixaban-treated). DOAC activity was tested with Hemoclot® Thrombin Inhibitor assay in dabigatran-treated patients, and with factor Xa-calibrated anti-Xa chromogenic analysis in rivaroxaban- and apixaban-treated patients prior and two hours after drug administration. ResultsThere were no significant differences in dabigatran baseline (62.1 ± 8.0 vs. 51.8 ± 38.9 ng/ml, p = .76) and 2-h-post-drug-administration (91.7 ± 57.2 vs. 72.2 ± 33.2 ng/ml, p = .48) activity comparing T2D and non-diabetic patients. Similarly, no significant differences were found in rivaroxaban baseline (35.9 ± 22.5 vs. 55.3 ± 45.1 ng/ml, p = .19) and 2-h-post-drug-administration (145.7 ± 74.1 vs. 202.6 ± 135.0 ng/ml, p = .22) anti-Xa activity. In addition, no significant differences were present in apixaban baseline (96.0 ± 54.5 vs. 63.9 ± 36.8 ng/ml, p = .24) and 2-h-post-drug-administration (151.0 ± 78.3 vs. 151.7 ± 59.1 ng/ml, p = .98) anti-Xa activity between T2D and non-diabetic patients. ConclusionsThis pilot study did not detect differences in DOACs activity according to T2D status in patients with NV-AF.

Simultaneous quantification of direct oral anticoagulants currently used in anticoagulation therapy

Direct oral anticoagulants (DOACs) are among the most effective options to prevent serious thromboembolic events in patients with atrial fibrillation. Coagulation assays are used to assess DOAC activity, but lack the possibility to quantify drugs with concurrent pharmacodynamic effect. We developed a selective multi-drug assay to analyze apixaban, betrixaban, dabigatran, edoxaban, edoxaban M4, and rivaroxaban with ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) in plasma fulfilling all requirements of the FDA und EMA guidelines for bioanalytical method validation. Plasma samples were extracted using solid phase extraction in a 96-well micro volume format. Chromatographic separation was performed on a Waters BEH Phenyl 1.7μm column coupled to tandem mass spectrometry. Extraction recoveries exceeded 80 %. Concentrations of 1–1000 ng/ml can be precisely quantified (correlation coefficient of >0.99) using 100 μL plasma volume. Intra-day and inter-day accuracies ranged between 91.0 % and 116 %. Precisions at low and high concentrations were below 13.3 %. The method was applied within a clinical drug trial and eight short pharmacokinetic profiles of patients under DOAC therapy were analyzed. The assay allows for highly sensitive and selective simultaneous quantification of DOACs in patient plasma samples.

Determination of anticoagulant activity of direct oral anticoagulants: the current state of knowledge and a single center experience

Direct oral anticoagulants (DOACs) increasingly replace vitamin K antagonists in the treatment and prevention of venous thromboembolism, and stroke prevention in non-valvular atrial fibrillation. Due to their predictable pharmacological mechanism, no diet restrictions and lack of interaction with most drugs, DOACs do not require routine laboratory monitoring. However, in cases of suspected concentrations beyond the expected range or a need for an emergent surgical procedure, access to laboratory methods for reliable DOACs determination is necessary. In a routine laboratory, the anticoagulant effect of dabigatran can be determined using methods based on ecarin or the modified dilute thrombin time. Rivaroxaban, apixaban and edoxaban may be measured using the chromogenic anti-FXa assays. Our own experience (the longest in Poland) from the John Paul II Hospital laboratory with the 24-hour availability of DOACs measurement involves determinations of dabigatran (n = 136), rivaroxaban (n = 374) and apixaban (n = 22) in outpatients and hospitalized patients between May 2013 and April 2017. Determination of the level of DOACs should be available 24/7 in highly specialized medical centers, especially in those which perform emergency procedures for treating patients with acute stroke and hemorrhages.

Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS

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. 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. 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). 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.