Laboratory Assessment of the Anticoagulant Activity of Apixaban in Patients With Nonvalvular Atrial Fibrillation (2024)

Abstract

Our aim is to determine the most appropriate laboratory tests, besides anti-factor Xa(anti-FXa) chromogenic assays, to estimate the degree of anticoagulation with apixaban andcompare it with that of rivaroxaban in real-world patients. Twenty patients withnonvalvular atrial fibrillation treated with apixaban 5 mg twice daily and 20 patients onrivaroxaban 20 mg once daily were studied. Conventional coagulation tests, thrombingeneration assay (TGA), and thromboelastometry (nonactivated TEM [NATEM] assay) wereperformed in the 40 patients and 20 controls. The anti-FXa chromogenic assays were used tomeasure apixaban and rivaroxaban plasma levels. The NATEM measurements showed nosignificant difference between the 2 groups of patients. Concerning TGA, endogenousthrombin potential (ETP) was significantly decreased in patients on rivaroxaban ascompared to those treated with apixaban (P < .003). A statisticallysignificant, strong inverse correlation between apixaban plasma concentrations and ETP(P < .001) was observed. Apixaban significantly reduces ETP comparedto controls, but to a lesser extent than rivaroxaban. Thrombin generation assay mightprovide additional information on apixaban exposure, which is required in order toindividualize treatment especially for patients with a high bleeding risk. Our findingshave to be further investigated in studies with larger sample sizes, in the entire rangeof apixaban exposure, with other direct oral anticoagulants, and in relation to clinicaloutcomes.

Introduction

Atrial fibrillation (AF) is the most common chronic cardiac rhythm abnormality withworldwide prevalence ranged between 1% and 2%.¹ Direct oral anticoagulants (DOACs), which are inhibitors of both thrombin and factorXa (FXa), have been developed and demonstrated a favorable benefit–risk profile for primaryand secondary prevention of stroke and systemic embolism in patients with nonvalvular atrialfibrillation (NV-AF). Unlike vitamin K antagonists, DOACs (ie, apixaban, dabigatran,edoxaban, and rivaroxaban) are administered in fixed doses and do not usually requireroutine laboratory monitoring for dose adjustment, due to their more predictablepharmacokinetics and pharmacodynamics.²

However, measuring their anticoagulant activity and/or plasma levels may be helpful incertain clinical circumstances, such as bleeding or thrombosis during treatment,preoperative state, suspected overdose, and in certain populations including those withextremes in body weight, the elderly individuals, patients with renal insufficiency, andpatients with AF presenting with acute ischemic stroke prior to administration ofthrombolytic therapy.³

The lack of a readily available method to determine the degree of anticoagulation createschallenges to both clinicians and laboratory staff in terms of assessing the risk forbleeding of patients receiving these drugs in emergency. Anti-FXa chromogenic assays seem tobe the most appropriate assays for the quantitative measurement of FXa inhibitor plasma levels.⁴ Nevertheless, this type of assay is time-consuming and not commonly available. Morerecently, global coagulation assays such as viscoelastic tests (ROTEM and TEG) and thrombingeneration assays (TGAs) have also been recommended as potential tests for assessing theanticoagulant effect of DOACs. These assays have several advantages including providingglobal information on the coagulation process in short turnaround time.^5–7 The relatively limited published data on the laboratory testing of apixaban⁸ suggest that rotational thromboelastometry (ROTEM) and TGA may be useful forscreening, with ROTEM providing quick results in emergency situations.⁹ In most cases, these data were collected from in vitro studies using blood samplesspiked with apixaban.^6,9–11

Our research group has recently studied the anticoagulant activity of dabigatran andrivaroxaban, as measured by these 2 assays in real-life patients with NV-AF.^12,13 The aim of this study is to identify the most appropriate laboratory tests, besidesanti-FXa chromogenic assays, to measure the degree of anticoagulation with apixaban 5 mgtwice daily and to compare it with that of rivaroxaban 20 mg once daily in real-lifepatients with NV-AF.

Methods

The study population included patients with NV-AF who were on anticoagulation and wererecruited from the Second University Department of Cardiology and the Department ofHaemostasis in the “Attiko” University Hospital in Athens, Greece (Supplemental material).The group A consisted of patients on rivaroxaban 20 mg once daily, who were also included ina recently published study by our research team.¹³ Patients matched on age and sex with those in group A and taking the standard dose ofapixaban (5 mg orally twice daily) recommended for patients with high CHA2DS2-VASc score(congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke/transientischemic attack, vascular disease, age 65-75, and female sex) comprised group B. The samehemostatic parameters were also measured in age- and sex-matched, healthy participants.Individuals with platelet count <100 × 10⁹/L and active thrombosis orbleeding, patients on simultaneous anticoagulant and antiplatelet therapy, those with renaland/or hepatic and/or thyroid dysfunction, those with malignancy or with chronic infectiousor autoimmune diseases, and patients with active infection at the start of the study wereexcluded.

The study was performed in accordance with the Declaration of Helsinki and was approved bythe hospital’s institutional review board (approval number: 135, July 10, 2017). Informedconsents were obtained from all patients.

For the study participants, the CHA2DS2-VASc score was calculated, detailed personalmedical history (related to thrombotic and/or bleeding complications) was obtained, and athorough clinical examination was performed. At least 7 days after study enrollment, thefollowing laboratory examinations were undertaken: complete blood count, activated partialthromboplastin time (aPTT) and international normalized ratio (INR), fibrinogen, D-dimers,thrombin generation (TG), and ROTEM assay. Chromogenic assays for the in vitro quantitativemeasurement of FXa direct inhibitor (DiXaI, Biophen; Hyphen BioMed, Neuville-sur-Oise,France, and liquid anti-Xa assay; HemosIL Instrumentation Laboratory Company, Milan, Italy)were used to measure the plasma concentration of rivaroxaban and apixaban, respectively, inthe tested samples.

The last dose of antithrombotic medication was administered approximately 3 hours beforeblood sampling. The ROTEM analysis and conventional coagulation tests were done on the sameday, within 2 hours from sampling. Complete blood counts were performed on a Sysmex XE-2100analyzer (Elgin, Roche, Illinois). The aPTT, PT, and INR, fibrinogen, and d-dimerswere all measured on BCS XP System hemostasis analyzer (Siemens Healthcare Diagnostics,Marburg, Germany). Pathromtin* SL (Siemens Healthcare Diagnostics) was used to determineaPTT and the Thromborel S reagent (Siemens Healthcare Diagnostics) was used for PTdetermination. Plasma concentrations of fibrinogen were measured using a modification of theClauss method with the Fibrinogen Multifibren* U reagent (Siemens Healthcare Diagnostics).D-dimers were assessed by the Innovance D-dimer assay (Siemens Healthcare Diagnostics), aparticle-enhanced immunoturbidimetric method. Blood samples for analysis were anticoagulatedwith 0.109 mol/L trisodium citrate (9:1, vol/vol blood anticoagulant) and immediatelycentrifuged at 2500g for 20 minutes.

Results

The group A included 20 patients on rivaroxaban with a median age of 73 years (IQR:66.5-78) and half of them being females. The group B consists of 20 patients (70% females)on apixaban with a median age of 77 years (IQR: 63.5-79.5). Descriptive characteristics ofpatients, associated comorbidities, and their hematological and biochemical parameters areshown in Table 1. The 2 patientgroups were similar without statistically significant differences. The anti-FXa levelsbetween the 2 groups were comparable.

Table 2 presents the results ofcomparisons of coagulation parameters among the 3 groups (the 2 patient groups and thehealthy controls). In terms of the conventional coagulation tests, patients on rivaroxabanhad a statistically significant prolongation of aPTT and higher INR values than patientstreated with apixaban (P < .001 and P = .036,respectively). Viscoelastic measurements on the ROTEM analyzer showed a statisticallysignificant prolongation of CT and CFT and a decrease in a angle in bothgroups of patients as compared to controls (P < .05). However, the 2groups of patients on rivaroxaban and apixaban did not significantly differ, despite thegreater impact of rivaroxaban on CT in comparison to apixaban.

Concerning TGA, the procoagulant activity was significantly decreased in patients onrivaroxaban than those on apixaban (AUC: P < .003; Cmax:P < .009). Both tlag and tmax were also affected more by rivaroxabanthan apixaban, but this difference was not statistically significant.

The correlation of apixaban plasma values, as determined by the anti-FXa chromogenic assay,with all hemostatic parameters is presented in Table 3. There is statistically significant, stronginverse correlation between apixaban plasma concentrations and AUC (P <.001; Figure 1) or Cmax(P < .001). No correlation was found between ETP and rivaroxabanlevels. The plasma levels of apixaban and rivaroxaban were not significantly associated withNATEM variables.

Discussion

This study shows a stronger procoagulant effect of rivaroxaban than of apixaban andhighlights the potential role of TGA in assessing the anticoagulant activity of apixaban invivo in certain clinical contexts. To the best of our knowledge, a direct comparison of theanticoagulant activity of different DOACs using hemostatic variables measured in real-lifesettings has not been done.

Few studies have investigated the value of elastography or elastometry in the context ofapixaban therapy. In most cases, in vitro experiments were done that demonstrated asignificant impact of apixaban on CT or reaction time.^6,7,9,11,14 In our study, NATEM CT and CFT were also significantly prolonged in patients onapixaban or rivaroxaban compared to controls. However, there was not any correlation betweenapixaban/rivaroxaban plasma concentrations and NATEM parameters. A strong correlationbetween apixaban and rivaroxaban plasma concentrations and the LowTF-ROTEM CT in in vitroexperiments have been noted by Adelmann et al, but such a relation was not reported in exvivo samples.⁷ Several studies have been conducted to evaluate the role of thromboelastometry in thesetting of rivaroxaban therapy, yielding however inconsistent results.^7,15–17 In a recent study of our team, a significant prolongation of CT and CFT was observed,but no association was found between ROTEM variables and anti-Xa values in real-world patients.¹³ The available data insofar do not support the use of viscoelastic coagulation assayseither for excluding the presence of rivaroxaban or for determining rivaroxabanconcentration, due to the lack of adequate sensitivity. Thus, although viscoelasticcoagulation tests could play a role in detecting the presence of direct FXa inhibitors, moreevidence is needed before these tests are considered appropriate assays for detecting andmeasuring the anticoagulant effect of these inhibitors.¹⁸

Regarding TGA, few in vitro studies have assessed the results of this assay in relation toplasma levels of apixaban.^10,11,14,19 Apixaban influenced all parameters in a dose-dependent manner, but the sensitivityvaried. Thus, there is insufficient evidence to recommend the use of TGA for assessingapixaban effect in clinical practice.¹⁸ In this study, apixaban, compared to controls, reduced TG, with prolongations of lagtime and a decrease in peak thrombin level. Moreover, significant correlations betweenapixaban plasma levels and almost all parameters of the ETP assay were also observed. Theimpact of rivaroxaban on TG has been more extensively evaluated. Rivaroxaban produceddose-dependent effects on all TG parameters with variable sensitivity, but ETP (AUC)appeared to have lower responsiveness to rivaroxaban plasma levels than did the other TG parameters.^13,18,20 This does not seem to hold for apixaban. Unlike rivaroxaban, a strong, statisticallysignificant, inverse correlation between ETP and apixaban plasma concentrations was observedin our study. Moreover, apixaban resulted in a significantly lower reduction in ETP andCmax, compared to rivaroxaban, indicating a different effect of the 2 FXa inhibitors on TGcurve. The strong correlation between ETP and apixaban plasma levels in real-life patientssuggests that TGA may be useful in quantifying the anticoagulant activity of apixaban. It isnoteworthy that dabigatran plasma levels were also reported to be strongly inverselycorrelated with ETP in real-world patients.^12,21 This is in accordance with the findings of a recent study, which showed that TG canmeasure the anticoagulation effect of commonly used DOACs, with each drug having a unique TG profile.²²

Based on the effect of the 2 direct FXa inhibitors on ROTEM and TGA parameters, theanticoagulant effect of rivaroxaban 20 mg once daily is stronger than that of apixaban 5 mgtwice daily. Rivaroxaban use, compared to apixaban, resulted in significant prolongations oflag time, decreases in peak thrombin level and ETP, prolonged aPTT, and higher INRvalues.

Since the introduction of the DOACs, it has been attempted to compare and rank them interms of their safety and efficacy. In the absence of head-to-head randomized controlledtrials, alternative methods of comparisons using existing trial data have been used,including cross-study comparisons using meta-analyses and network meta-analyses.^23–26 Based on current evidence, the treatment with apixaban has the most favorable safetyprofile in terms of risk for bleeding among the DOACs and is considered to have the optimalbenefit to risk ratio. These findings are in line with current American HeartAssociation/American Stroke Association recommendations that classify apixaban as the DOACwith the highest level of evidence (class I; level of evidence A) for prevention ofrecurrent stroke in patients with a history of stroke or transient ischemic attack incomparison with dabigatran (class I; level of evidence B) and rivaroxaban (class IIa; levelof evidence B).²⁷

To the best of our knowledge, no direct comparisons among DOACs, regarding theiranticoagulant activity in real-life patients, are available. This is the first attempt todirectly compare anticoagulant effects in patients with NV-AF between the 2 standard, directFXa inhibitors. Based on TGA, laboratory findings are in accordance with clinical evidencethat suggests that apixaban has the best benefit/risk profile among the 3 NOACs. Apixabansignificantly reduces ETP compared to controls, but to a lesser extent than rivaroxaban anddabigatran, which both have a comparable effect on the amount of thrombin generated inreal-life patients.¹³ This might be the optimal level of anticoagulant activity in order to achieve thebest benefit to risk ratio for ischemic stroke prevention in patients with NV-AF. Ifapixaban is found to affect ETP in a dose-dependent manner, TGA could be used for assessingapixaban effect in clinical practice, by defining relative reference ranges in order toindividualize treatment in certain clinical settings, especially in patients with highbleeding risk.

Limitations of our study are the small sample size and the absence of long-term follow-upto evaluate the clinical relevance of the laboratory parameters that were tested. On theother hand, our analyses are based on real-life patients, and it is also important that thelaboratory findings based on TGA support current clinical evidence that classifies apixabanas the DOAC with the optimal benefit/risk profile.

In order to verify the potential impact of apixaban or other DOACs on specific coagulationassays, like TGA or the viscoelastic coagulation tests, in a valid and reliable way, severalcritical issues have to be addressed. The large number of specialized and modified assaysthat are available, the different reagents used by several laboratories, the limited use ofex vivo samples, and the lack of standardization probably cause delays in the developmentand establishment of new methods that can appropriately detect and quantify theanticoagulant activity of DOACs.

Conclusively, it seems that TGA can provide additional information on apixaban exposure andthe benefit–risk profile, which are needed to individualize treatment under certain clinicalcircumstances, especially in patients with high bleeding risk. Hypotheses based on ourfindings have to be further investigated in studies with larger sample sizes, in the entirerange of apixaban exposure, with other DOACs, and in relation to clinical outcomes.

Supplemental Material

Footnotes

References

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