# Fibrinography and thrombography
(Thrombodynics-4D) in atrial fibrillation ADAGE
patients aged 80 years and older receiving DOAC
therapy

Geoffrey Foulon-Pinto, Georges Jourdi, Maxime Delrue, Carmelo

Lafuente-Lafuente, Candice Cavalie, Isabelle Gouin-Thibault, Julien Le Guen,

Pascale Gaussem, Tristan Mirault, Etienne Puymirat, et al.

► To cite this version:

Geoffrey Foulon-Pinto, Georges Jourdi, Maxime Delrue, Carmelo Lafuente-Lafuente, Candice Cavalie, et al. Fibrinography and thrombography (Thrombodynics-4D) in atrial fibrillation ADAGE patients aged 80 years and older receiving DOAC therapy. Research and Practice in Thrombosis and Haemostasis, 2025, 9 (5), pp.102969. 10.1016/j.rpth.2025.102969. hal-05232507

HAL Id: hal-05232507

https://hal.science/hal-05232507v1

Submitted on 6 Nov 2025

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ORIGINAL ARTICLE

# Fibrinography and thrombography (thrombodynamics-4D) in atrial fibrillation assessment of direct oral anticoagulants in geriatrics patients aged 80 years and older receiving direct oral anticoagulant therapy

Geoffrey Foulon-Pinto<fnref n="1" />,<fnref n="2" />

Georges Jourdi<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Maxime Delrue<fnref n="1" />,<fnref n="3" />

Carmelo Lafuente-Lafuente<fnref n="4" />,<fnref n="5" />

Candice Cavalie<fnref n="1" />

Isabelle Gouin-Thibault<fnref n="6" />,<fnref n="7" />

Julien Le Guen<fnref n="8" />

Pascale Gaussem<fnref n="1" />,<fnref n="9" />

Tristan Mirault<fnref n="10" />

Etienne Puymirat<fnref n="11" />

Thomas Lecompte<fnref n="6" />,<fnref n="12" />

Eric Pautas<fnref n="1" />,<fnref n="13" />

Emmanuel Curis<fnref n="2" />,<fnref n="14" />

Virginie Siguret<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

<figure><img src="image_2.png" /></figure>

**Correspondence**

Virginie Siguret, INSERM UMR_S1144
(Optimisation en neuropharmacologie),
Université Paris Cité, 4 avenue de
l'Observatoire, FR-75270 Paris Cedex 06,
France.
Email: virginie siguret@aphp.fr

**Handling Editor:** Dr Henri Spronk

**Abstract**

**Background:** Scarce data are available on pharmacodynamic (PD) variability in very elderly patients receiving direct oral anticoagulants (DOACs) for atrial fibrillation (AF). Thrombodynamics-4D (TD-4D), which simultaneously assesses fibrin clot formation and thrombin generation, has not yet been tested in patients on rivaroxaban, apixaban, or dabigatran.

**Objectives:** To (i) evaluate TD-4D's ability to assess DOAC effect added to normal plasma; (ii) assess DOAC PD in very elderly patients with AF along with DOAC concentrations; (iii) identify factors associated with interindividual variability of DOAC PD at peak and trough levels.

<footnote n="1">¹INSERM UMR_S1140, Université Paris Cité, Paris, France</footnote>

<footnote n="2">²Hématologie Biologique, Hôpital Lariboisière, Assistance Publique-Hôpitaux de Paris, Paris, France</footnote>

<footnote n="3">³INSERM UMR_S1144, Université Paris Cité, Paris, France</footnote>

<footnote n="4">⁴GH Pitié Salpêtrière-Charles Foix-Gériatrie à orientation cardiologique et neurologique, Sorbonne Université, Ivry-sur-Seine, France</footnote>

<footnote n="5">⁵Clinical Epidemiology and Ageing Team, INSERM, IMRB, Université Paris-Est Créteil, Créteil, France</footnote>

<footnote n="6">⁶Service d'Hématologie Biologique, CHU de Rennes, Rennes, France</footnote>

<footnote n="7">⁷INSERM CIC 1414 (Centre d'Investigation Clinique de Rennes), CHU de Rennes, Université de Rennes, Rennes, France</footnote>

<footnote n="8">⁸Gériatrie aiguë, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France</footnote>

<footnote n="9">⁹Hématologie Biologique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France</footnote>

<footnote n="10">¹⁰Médecine vasculaire, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, INSERM U970 PARCC team 5, Université Paris Cité, Paris, France</footnote>

<footnote n="11">¹¹Cardiologie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France</footnote>

<footnote n="12">¹²Médecine vasculaire, CHRU Nancy, Université de Lorraine, Nancy, France</footnote>

<footnote n="13">¹³GH Pitié Salpêtrière-Charles Foix-Gériatrie aiguë polyvalente, Sorbonne Université, Ivry-sur-Seine, France</footnote>

<footnote n="14">¹⁴UR 7537 BioSTM, Faculté de Pharmacie, Université Paris Cité, Paris, France</footnote>
**Methods:** Assessment of Direct oral Anticoagulants in Geriatrics (NCT02464488) is a prospective, multicenter study including inpatients aged ≥80 years receiving DOACs for AF for at least 4 days. Fibrinography and thrombography parameters were measured using TD-4D along with plasma DOAC concentrations (antifactor [F]Xa or anti-FIIa activity) and fibrinogen.

**Results:** We analyzed pooled normal plasma samples spiked with DOACs and 345 samples from 187 Assessment of Direct oral Anticoagulants in Geriatrics patients (mean ± SD, age 87 ± 4 years; 69% females): 69 on rivaroxaban, 70 on apixaban, and 48 on dabigatran. All 3 DOACs prolonged fibrinography lag time and decreased initial rate of clot growth and clot size at 30 minutes in a concentration-dependent manner in spiking experiments and patients. DOACs prolonged temporal thrombography parameters while decreasing thrombin peak height and endogenous thrombin potential. At trough, apixaban and dabigatran concentrations were the only significant predictors of interindividual variability in both thrombin peak height (thrombography) and initial rate of clot growth (fibrinography). In rivaroxaban patients, cardiac failure significantly influenced thrombin peak height variability.

**Conclusion:** Fibrinography and thrombography, assessed simultaneously with TD-4D, provided consistent results for all 3 DOACs, including dabigatran. Substantial PD variability was observed, partly influenced by DOAC concentrations. The clinical relevance of such variability remains to be demonstrated.

KEYWORDS

atrial fibrillation, direct oral anticoagulant, elderly, fibrin formation, pharmacodynamics, thrombin generation

Essentials

* Data on PD variability in very elderly patients on DOACs are scarce.

* We assessed thrombography/fibrinography (Thrombodynamics) in patients ≥80 years old, along with DOAC levels.

* DOACs, including dabigatran, exerted a significant concentration-dependent effect on most thrombography/fibrinography parameters.

* Substantial PD variability was observed, partly influenced by DOAC concentrations.

1 | INTRODUCTION

The prevalence of nonvalvular atrial fibrillation (AF) increases with aging, reaching >10% in patients aged ≥80 years [1,2]. Particular attention must be paid to very elderly patients with AF as they frequently have numerous comorbid conditions and are polymedicated, leading to high risk for both thrombosis and bleeding. Current guidelines recommend the use of direct oral anticoagulants (DOACs), ie, inhibitors of factor (F)Xa (rivaroxaban, apixaban, and edoxaban) or thrombin (dabigatran), as first-line treatment in the prevention of systemic embolic events in patients with AF [3,4]. In the last decade, meta-analyses of randomized clinical trials and real-life studies have confirmed the favorable clinical benefit-risk ratio of these drugs compared with vitamin K antagonists in this fragile population [5-8]. Given their large therapeutic margin and

predictable pharmacokinetics (PK) and pharmacodynamics (PD), DOACs can be administered at fixed dose without laboratory monitoring, except in specific situations. However, PK studies have documented substantial interindividual variability in DOAC plasma concentrations, regardless of the regimen (full or reduced dose), which was greater in octogenarians and nonagenarians than that observed in younger patients [9-18]. In the Assessment of Direct oral Anticoagulants in Geriatrics (ADAGE) study conducted by our group in patients aged ≥80 years with AF receiving rivaroxaban or apixaban, we evidenced important (>50%) coefficients of variation (CVs) in both maximal (Cmax) and minimal plasma concentrations of both anti-FXa inhibitors [17]. Regarding the assessment of DOAC PD, the study of thrombin generation (TG) has been shown to be a suitable and reliable assay to assess *in vitro* or *ex vivo* anticoagulant effect of direct FXa inhibitors, in contrast to prothrombin time [17,19-31]. In
ADAGE study, substantial variability in thrombin peak heights was noticed, especially at low concentration intervals for both rivaroxaban and apixaban, suggesting an impact of the underlying coagulation status of elderly inpatients [17]. While thrombography has been widely evaluated in patients on DOACs in different settings, the effect of DOACs on fibrinography, ie, fibrin clot formation and growth (taking into account not only fibrinogen level), and the potential relationships between thrombography and fibrinography have been poorly investigated. It was therefore of particular interest to further evaluate DOAC PD in ADAGE patients, in whom hyper-fibrinogenemia is common, using the Thrombodynamics-4D (TD-4D) analyzer. Indeed, this unique system has the advantage of enabling complete phenotyping of both TG and fibrin clot growth [32,33].

To date, TD-4D has already been shown to be suitable for exploring hypercoagulability states in different settings [34–39], as well as hypocoagulability states. It is particularly used in clinical practice to monitor patients receiving heparin therapy in some countries [40–43]. However, very few studies with a limited number of patients (<10 for each DOAC) have assessed DOAC PD using TD-4D [44].

In the present study, we first evaluated the ability of TD-4D to assess the effect of DOACs (rivaroxaban, apixaban, and dabigatran) in spiked pooled normal plasma samples. Then, in plasma samples from ADAGE patients, we sought to (i) characterize the PD profile of DOACs using TD-4D, along with DOAC concentrations; (ii) identify factors associated with interindividual variability in DOAC PD parameters at peak and trough concentrations.

## 2 | METHODS

### 2.1 | Materials

#### 2.1.1 | Pooled normal plasma samples spiked with rivaroxaban, apixaban, or dabigatran

Frozen pooled normal plasma samples (Cryocheck-Cryopep) were spiked with increasing concentrations of DOACs (0-450 ng/mL), as previously described [45]. Rivaroxaban, apixaban, and dabigatran were kindly provided by Bristol Myers Squibb/Pfizer, Bayer Health-care, and Boehringer Ingelheim, respectively.

#### 2.1.2 | ADAGE patients and sampling

ADAGE is an academic, prospective, observational multicenter study (NCT 02464488; for details, see Foulon-Pinto et al. [17]). We recruited hospitalized patients from the university hospital's geriatric department (Assistance Publique-Hôpitaux de Paris, Paris, France). Briefly, patients were eligible if they were aged ≥80 years and received rivaroxaban, apixaban, or dabigatran for AF for at least 4 days. The DOAC dose regimen was at the discretion of the physician. Exclusion criteria included any acute, unstable comorbid condition or an estimated life expectancy of a few weeks. Sampling (see below) and last DOAC intake times were

recorded on a standardized request form. Sampling time points were at the discretion of the physicians over a period of 20 days following inclusion. Time points were classified according to the time elapsed from the last oral intake (T, hours) for patients receiving rivaroxaban. T1 to T4 corresponded to peak time (Tmax), and T20 to T24 corresponded to trough (Tmin). For patients receiving apixaban or dabigatran (twice daily), T1 and T4 corresponded to Tmax, and T10 to T12 corresponded to Tmin. Physicians prospectively collected demographic, clinical, and laboratory data (including creatinine clearance [CrCl] calculated with the Cockcroft-Gault formula). The study was approved by the institutionel Ethics Committee (Comité consultatif de protection des personnes pour la recherche biomédicale-Île-de-France-6-number 15.074bis). All participants gave written informed consent to participate in the study. Only patients for whom remaining plasma aliquots were available were included in the present study.

### 2.2 | Methods

#### 2.2.1 | Blood collection and processing

Blood samples in patients and controls were collected by venepuncture into a citrate tube (buffered trisodium citrate 0.109 M, 9:1 v/v). Tubes were sent to each centre's laboratory within 2 hours after sampling and immediately double-centrifuged for 15 minutes at 2000 × g at 15 to 22 °C to obtain platelet-poor plasma. Plasma samples (500 µL aliquots) were stored at -80 °C prior to shipment on dry ice for central analysis (Haematology Laboratory, Hôpital Lariboisière). Plasma samples were thawed for 3 to 5 minutes in a 37 °C water bath just before testing.

#### 2.2.2 | Measurement of plasma DOAC and fibrinogen levels

We measured DOAC concentrations (expressed in nanograms per milliliter [ng/mL]) in plasma samples with the STA-R analyzer (Stago) using chromogenic anti-FXa assays (STA-Liquid Anti-FXa, Stago) for rivaroxaban and apixaban, and anti-FIIa activity using Hemoclot Direct Thrombin Inhibitors (Hyphen-Biomed) for dabigatran. Dedicated calibrations and controls were used. The lower limit of quantification was 20 ng/mL for the 3 assays.

Plasma fibrinogen levels were centrally measured in each sample using Clauss method (Dade Thrombin Reagent, Siemens).

#### 2.2.3 | TD-4D assay

The TD-4D assay was performed centrally using the Thrombodynamics analyzer system (Hemacore, Diapharma) with the TD-4D PLS dedicated kit (Hemacore, Diapharma). The system simultaneously measures spatiotemporal parameters of fibrin clot formation (fibrinography) and TG process (thrombography) in a thin layer of
unstirred plasma [32,41,42,46]. Briefly, 120 µL of platelet-poor plasma is added to a microcuvette containing corn trypsin inhibitor to prevent contribution of contact phase activation, then 5 µL of phospholipid solution (manufacturer's reagent) is added. After incubation for 15 minutes at 37 °C in the Thrombodynamics device, the mixture is transferred to a second microcuvette containing calcium ions, and then immediately transferred to the measurement cuvette. Finally, the activator insert, whose surface is coated with immobilized tissue factor (100 pmol/m²), is then transferred to the cuvette. The process of fibrin clot formation is recorded in a time-lapse video-microscopy mode using a dark-field light scattering method. Simultaneously, the system monitors TG by fluorimetry, based on the ability of thrombin to cleave a fluorogenic substrate, 7-amino-4-methylcoumarin (AMC) bound to a short amino acid sequence (Arg-Gly-Gly). The rate of AMC formation is proportional to local thrombin concentration. The microcuvette is maintained at 37 °C and is illuminated with red (625 nm) or ultra-violet (365 nm) light emitting diodes. Clot growth is detected with red light scattering, while AMC fluorescence is detected with UV. Scattered red light and fluorescence passing through the macro lens are recorded by a charge-coupled device system. Images under red and blue light are acquired once per minute. The whole analysis lasts 1 hour. The Thrombodynamics software (Hemacore, Diapharma) calculates parameters of spatiotemporal dynamics of fibrin clot formation and TG [40,46].

The following fibrinography parameters were recorded (see Supplementary Table 1): (i) lag time (Tlag, minutes); (ii) initial rate of clot growth (Vi, µm/min); (iii) rate of clot growth (V, µm/min); (iv) clot size (CS, µm) measured at 30 minutes; (v) clot density (D, arbitrary units); (vi) time to spontaneous clotting (Tsp, minutes). The following thrombography parameters (at the near-activator area) were recorded (see Supplementary Table 1): (i) Tlag (minutes); (ii) time to thrombin peak (minutes); (iii) maximal thrombin concentration (ie, thrombin peak height, Cmax_ATG, AU/L); (iv) endogenous thrombin potential (ETP_ATG, AU·min/L); (v) stationary amplitude of moving peak of thrombin (Ast, AU/L); (vi) rate of thrombin peak propagation (µm/min; see Supplementary Videos 1–3).

### 2.2.4 | TD-4D assay performances—reference intervals

Interassay CVs for all parameters measured in pooled normal plasma (Cryocheck) were <8.0%, except for Ast and Tsp (Supplementary Table 2). Mean values of fibrinography and thrombography parameters were within the manufacturer's reference intervals [35,40], except for D. The lower D we observed is thought to result from the additives present in Cryocheck, leading to hypodense clots. We locally tested frozen plasma samples from 30 DOAC-free volunteers, with a mean (SD) age of 31 ± 8 years, enrolled in the Dabigatran etexilate and Rivaroxaban Influence of Genetics factors in healthy

volunteers (DRIVING) study [25] (Supplementary Table 2). Reference intervals were established as mean ± 2 SD and were of the same order of magnitude as those of the manufacturer [40].

In addition, we tested samples from randomly selected 35 elderly subjects, referred to the Haematology Laboratory of Lariboisière University Hospital (Paris, France) for coagulation testing, aged ≥80 years, without any anticoagulant treatment, and having normal full blood count and routine coagulation assay results (including prothrombin time ratio, activated partial thromboplastin time, and fibrinogen level). In accordance with French regulations, they all approved the use of their leftover plasma samples via a nonopposition form. Their mean (SD) age was 85 ± 5 years (minimum-maximum [min-max], 80-95 years), with 66% being females; half had mild to normal renal function, and half had moderate renal failure. Their mean fibrinography and thrombography parameters were within the manufacturer's reference intervals, except for Vi and CS, which were slightly higher [35,40] (Supplementary Table 2).

### 2.2.5 | Statistical analysis

Quantitative variables were expressed as mean ± SD, CV, median (IQR [25th-75th percentiles]), or min-max. The associations between DOAC concentrations, fibrinography, and thrombography parameters were tested using Spearman's rank correlation. Comparisons between ADAGE patients and DOAC-free elderly subjects were performed using Mann-Whitney U-test for each DOAC.

We prespecified that the Vi (fibrinography) and Cmax_ATG (thrombography) would be chosen as the 2 Thrombodynamics parameters for statistical analysis, given their sensitivity to a wide range of DOAC concentrations. Potential predictors of Vi (fibrinography) and Cmax_ATG (thrombography), including intake of amiodarone, any CYP450 inhibitor, any P-glycoprotein inhibitor or substrate, CrCl, and the presence of heart failure, at Tmin and Tmax, were tested using either Mann-Whitney U-test (categorical predictor) or Spearman tests (quantitative predictor, univariate analysis). To account for confounding effects of DOAC or fibrinogen concentrations (for Vi) on the parameters, multivariate analysis was also performed using a linear model with both predictors and confounders. The P value for the predictor was computed using type I sums of squares, with the predictor being the last term in the model.

Statistical analyses were conducted using R (version 4.0.2) [47] and Python (version 3.9.12) [48], with the latter in a Jupyter Notebook environment (version 6.4.8). Python libraries, including pandas [49], NumPy [50], matplotlib [51], seabin [52], and SciPy [53], were used for additional data processing and analysis. A P value < .05 was considered statistically significant after multiplicity correction; all results are given as raw P values.
# 3 | RESULTS

## 3.1 | TD-4D results in normal plasma spiked with DOACs

We first evaluated fibrinography parameters using TD-4D as a function of DOAC concentrations, using pooled normal plasma

samples spiked with rivaroxaban, apixaban, or dabigatran (Figure 1). The 3 DOACs prolonged Tlag and decreased rate of growth and CS at 30 minutes in a concentration-dependent manner ($P < 10^{-4}$). Early Tsp ($<30$ minutes) occurred only in 2.8% of the samples.

Regarding thrombography, increasing DOAC plasma concentrations were significantly associated with prolonged temporal

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Fibrinography parameters (Thrombodynamics-4D) as a function of direct oral anticoagulant (DOAC) concentrations in spiking experiments. (A) Lag time (Tlag), (B) initial rate of clot growth (Vi), (C) rate of clot growth (V), (D) clot size (CS) at 30 minutes, (E) clot density (D), and (F) time to spontaneous clotting (Tsp). Pooled normal plasma (Cryocheck) samples were spiked with rivaroxaban (R), apixaban (A), or dabigatran (D) to obtain final concentrations ranging from 0 to 1000 nM. DOAC concentrations were measured in the spiked samples. Concentrations are expressed in both nanograms per milliliter (ng/mL) and nanomolar (nM) to facilitate comparisons between R (red dots), A (blue dots), and D (green dots). Spontaneous clotting occurred in $n = 5$ (14%), $n = 5$ (14%), and $n = 12$ (33%) samples spiked with R, A, and D, respectively, mainly at low DOAC concentrations.</figcaption></figure>
parameters (Lag_ATG and Tmax_ATG), while Cmax_ATG, ETP_ATG, and rate of thrombin peak propagation decreased (P < 10⁻⁴) with each DOAC (Figure 2). Ast also decreased with increasing concentrations of apixaban (P = .0039) and dabigatran (P < 10⁻⁴), but not rivaroxaban (P = .269; Figure 2). Dabigatran had a more pronounced effect on Cmax_ATG, ETP_ATG, and Ast than rivaroxaban or apixaban (Figure 2).

## 3.2 DOAC PD in ADAGE patient samples using TD-4D

### 3.2.1 | Patient characteristics

A total of 345 plasma samples from 187 ADAGE patients were analyzed with TD-4D. Patient characteristics are summarized in

<figure><img src="image_4.png" /></figure>

**FIGURE 2** Thrombography parameters (Thrombodynamics) as a function of direct oral anticoagulant (DOAC) concentrations in spiking experiments. (A) Lag time (Lag_ATG), (B) time to peak (Tmax_ATG), (C) maximal thrombin concentration (peak height, Cmax_ATG), (D) endogenous thrombin potential (ETP_ATG), (E) stationary amplitude of thrombin moving peak (Ast), and (F) rate of thrombin peak propagation (Vt). Pooled normal plasma (Cryocheck) samples were spiked with rivaroxaban (R), apixaban (A), or dabigatran (D; see Figure 1 legend). Concentrations of DOACs are expressed in both nanograms per milliliter (ng/mL) and nanomolar (nM) to facilitate comparisons between R (red dots), A (blue dots), and D (green dots). ETP, endogenous thrombin potential.
**Table 1.** The mean (SD) age was 87 ± 4 years (min-max, 80-98 years), with 69% of the patients being female. Sixty-nine patients received rivaroxaban, 70 received apixaban, and 48 received dabigatran; most patients received a reduced dose (Table 1).

The average Cumulative Illness Rating Scale-Geriatrics score of 10.5 showed substantial comorbid conditions. The mean CrCl (Cockcroft-Gault) was 51.0 mL/min. Polymedication was frequent, with an average of 6.1 drugs per patient in addition to DOACs (Table 1).

Of the 345 samples, 148 were collected from patients on rivaroxaban (with an average of 2.1 time points per patient), 129 from patients on apixaban (1.8 time points per patient), and 68 from patients on dabigatran (1.4 time points per patient).

### 3.2.2 | Association of DOAC concentrations with fibrinography parameters in ADAGE patients

In ADAGE patients, DOAC plasma concentrations ranged from < 20 ng/mL to 676 ng/mL, 547 ng/mL, and 665 ng/mL for rivaroxaban, apixaban, and dabigatran, respectively. Tlag was prolonged, and the Vi measured in the 2- to 6-minute interval decreased with increasing DOAC concentration (P < 10⁻⁴), irrespective of the DOAC (Figure 3). The CS decreased with increasing rivaroxaban, apixaban, and dabigatran concentrations (P = .0002, P = .002, P < 10⁻⁴, respectively). Tlag, Vi, and CS values presented substantial variability in the 3 DOACs at a given concentration interval (eg, 100-200 ng/mL; Figure 3).

<table><caption>**TABLE 1** Characteristics of the Assessment of Direct oral Anticoagulants in Geriatrics patients with Thrombodynamics-4D results at inclusion.</caption><thead><tr><th>Patient characteristics</th><th>Rivaroxaban<br>n = 69</th><th>Apixaban<br>n = 70</th><th>Dabigatran<br>n = 48</th><th>Whole cohort<br>N = 187</th></tr></thead><tbody><tr><td><strong>Demographic characteristics</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Age (y), mean ± SD</td><td>86.4 ± 4.2</td><td>87.0 ± 4.1</td><td>86.1 ± 4.3</td><td>86.6 ± 4.2</td></tr><tr><td>Females, %</td><td>69.6</td><td>72.9</td><td>62.5</td><td>69.0</td></tr><tr><td>Body weight (kg), mean ± SD</td><td>65.2 ± 13.0</td><td>67.4 ± 16.1</td><td>67.0 ± 16.0</td><td>66.5 ± 14.9</td></tr><tr><td><strong>Clinical characteristics</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>CHA<sub>2</sub>DS<sub>2</sub>VASC score, mean ± SD</td><td>4.9 ± 1.4</td><td>5.1 ± 1.3</td><td>4.2 ± 1.6</td><td>4.9 ± 1.4</td></tr><tr><td>HEMORR<sub>2</sub>HAGES score, mean ± SD</td><td>2.0 ± 1.0</td><td>2.6 ± 1.1</td><td>2.1 ± 0.8</td><td>2.3 ± 1.0</td></tr><tr><td>CIRS-G score, mean ± SD</td><td>9.5 ± 3.9</td><td>11.7 ± 5.2</td><td>9.1 ± 3.4</td><td>10.4 ± 4.6</td></tr><tr><td>Previous stroke or TIA, %</td><td>26.5</td><td>30.4</td><td>27.7</td><td>28.3</td></tr><tr><td>Diabetes mellitus, %</td><td>10.6</td><td>27.5</td><td>20.0</td><td>19.4</td></tr><tr><td>Heart failure, %</td><td>54.4</td><td>53.6</td><td>23.4</td><td>46.2</td></tr><tr><td><strong>Laboratory data, mean ± SD</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>CrCl<sup>a</sup> (mL/min)</td><td>50.8 ± 18.0</td><td>49.2 ± 15.5</td><td>54.5 ± 22.1</td><td>50.9 ± 18.1</td></tr><tr><td>Albumin (g/L)</td><td>33.1 ± 3.9</td><td>32.9 ± 6.1</td><td>32.3 ± 5.9</td><td>32.9 ± 5.2</td></tr><tr><td>Hemoglobin (g/dL)</td><td>12.5 ± 1.4</td><td>11.7 ± 1.8</td><td>12.4 ± 1.5</td><td>12.2 ± 1.6</td></tr><tr><td>Fibrinogen (g/L)</td><td>5.0 ± 1.8</td><td>5.2 ± 1.3</td><td>4.4 ± 1.7</td><td>4.9 ± 1.6</td></tr><tr><td><strong>Therapeutic data, %</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Reduced dose DOAC<sup>b</sup></td><td>88.4</td><td>77.1</td><td>100.0</td><td>87.2</td></tr><tr><td>No. of medications, mean ± SD</td><td>6.1 ± 2.7</td><td>6.6 ± 2.9</td><td>5.4 ± 2.2</td><td>6.1 ± 2.7</td></tr><tr><td>≥1 CYP3A4/5 inhibitor</td><td>14.9</td><td>27.5</td><td>22.7</td><td>21.7</td></tr><tr><td>≥1 P-glycoprotein inhibitor</td><td>37.3</td><td>47.8</td><td>43.2</td><td>42.8</td></tr><tr><td>Amiodarone</td><td>14.9</td><td>27.5</td><td>18.2</td><td>20.6</td></tr><tr><td>Antiplatelet therapy</td><td>10.6</td><td>24.6</td><td>2.3</td><td>14.0</td></tr></tbody></table>

CHA<sub>2</sub>DS<sub>2</sub>VASC, congestive heart failure, hypertension, age ≥75 (doubled), diabetes mellitus, prior stroke or transient ischemic attack (doubled), vascular disease, age 65-74, female; CIRS-G, Cumulative Illness Rating Scale-Geriatrics; CrCl, creatinine clearance; DOAC, direct oral anticoagulant; HEMORR<sub>2</sub>HAGES, hepatic or renal disease, ethanol abuse, malignancy, older age, reduced platelet count or function, re-bleeding, hypertension, anemia, genetic factors, excessive fall risk and stroke; TIA, transient ischemic attack.

<footnote><sup>a</sup>Calculated using Cockcroft-Gault formula.</footnote>

<footnote><sup>b</sup>Rivaroxaban 15 mg once daily, apixaban 2.5 mg twice daily, and dabigatran 110 mg twice daily.</footnote>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

DOAC:

<figure><img src="image_10.png" /></figure>

**FIG URE 3** Fibrinography parameters (Thrombodynamics) as a function of direct oral anticoagulant (DOAC) concentrations in all Assessment of Direct oral Anticoagulants in Geriatrics (ADAGE) patients. (A) Lag time (Tlag), (B) initial rate of clot growth (V), (C) clot size (CS) at 30 minutes, (D) clot density (D), and (E) time to spontaneous clotting (Tsp). Concentrations of DOACs are expressed in both nanograms per milliliter (ng/mL) and nanomolar (nM) to facilitate comparisons between rivaroxaban (R; red dots), apixaban (A; blue dots), and dabigatran (D; green dots). Dashed lines indicate the locally determined reference intervals in healthy subjects. Of note, 1 patient on R (676 ng/mL) is represented by an open black diamond (beyond the limits of quantification), a.u., arbitrary units.

D was significantly associated with dabigatran concentration (P = .0048), but not with rivaroxaban or apixaban concentrations. D was strongly associated with plasma fibrinogen level of the 3 DOACs (P < 10⁻⁴ for rivaroxaban and apixaban; P = .032 for dabigatran; Supplementary Figure 1). Remarkably, Tsp occurred in 94% of ADAGE patient samples, with an early median time of 18 minutes (IQR, 10-27), which hampered the recording of data for V (measured within the 15-25-minute interval) and CS (measured at 30 minutes) in a substantial number of samples. There was no relationship between time to spontaneous clot formation and

rivaroxaban or apixaban levels, but a significant association with dabigatran levels (P = .0001; Figure 3).

### 3.2.3 | Association of DOAC concentrations with thrombography parameters in ADAGE patients

Tlag and time to peak were prolonged with increasing DOAC plasma concentrations (P < 10⁻⁴), while Cmax_ATG (P < 10⁻⁴) and ETP_ATG decreased (P = .0009, P = .0001, P < 10⁻⁴ for rivaroxaban, apixaban,
dabigatran, respectively; **Figure 4**). Cmax_ATG and ETP_ATG values presented marked interindividual variability in the 3 DOACs (**Figure 4**).

**Figure 2)** as well as in spiked samples (not shown). These results demonstrate that the delay in clot formation and the decrease in clot growth reflect the decrease in TG induced by DOACs.

### 3.2.4 | Relationships between thrombography and fibrinography parameters

Strong associations ($P < 10^{-4}$ for most parameters) were found between thrombography and fibrinography parameters, especially for temporal parameters in ADAGE patients (Supplementary

### 3.2.5 | PK and PD variability in ADAGE patients at Tmax and Tmin

We then focused on the subset of ADAGE patients who had a time point at Tmax or Tmin (Tables 2 and 3). In the subset of patients receiving a reduced dose, ie, 15 mg o.d. for rivaroxaban, 2.5 mg twice

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

DOAC:

Rivaroxaban
Uncoagulable sample

Apixaban

Dabigatran

**FIGURE 4** Thrombography parameters (Thrombodynamics-4D) as a function of direct oral anticoagulant (DOAC) concentrations in all Assessment of Direct oral Anticoagulants in Geriatrics (ADAGE) patients. (A) Lag time (Lag_ATG), (B) time to peak (Tmax_ATG), (C) maximal thrombin concentration (Cmax_ATG), (D) endogenous thrombin potential (ETP_ATG), and (E) stationary amplitude of thrombin moving peak (Ast). Concentrations of DOACs are expressed in both nanograms per milliliter (ng/mL) and nanomolar (nM) to facilitate comparisons between rivaroxaban (R; red dots), apixaban (A; blue dots), and dabigatran (D; green dots). Dashed lines indicate the locally determined reference intervals in healthy subjects. Of note, 1 patient on R (676 ng/mL) is represented by an open black diamond (beyond the limits of quantification). AU, arbitrary units; ETP, endogenous thrombin potential.
daily for apixaban, and 110 mg twice daily for dabigatran, we observed a wide interindividual variability in DOAC concentrations at both Tmax and Tmin, with CVs varying from 47% to 67% (Table 2). In contrast, regarding PD, CVs were <30% for fibrinography parameters, regardless of the DOAC, except for Tlag at Tmax in rivaroxaban (CV = 36%) and dabigatran (CV = 51%), and for time to Tsp of the 3 DOACs (Figure 5). Regarding thrombography, a greater variability was observed in Tlag (CV > 50%) of the 3 DOACs. For Cmax_ATG and ETP_ATG, the variability was remarkably low in patients receiving apixaban (CVs < 34%) at both time points (Table 3). The highest variability was observed in Cmax_ATG at Tmax in patients receiving rivaroxaban or dabigatran (CV = 41% and 63%,

respectively), and at Tmax for ETP_ATG in patients receiving dabigatran (CV = 56%; Figure 6).

We sought to identify covariates, namely DOAC plasma concentrations together with individual characteristics, which are potentially associated with Vi and Cmax_ATG at Tmin and Tmax (Supplementary Table 3). In multivariate analysis, at trough level (Tmin), apixaban and dabigatran concentrations were the only predictors of the interindividual variability of both Cmax_ATG and Vi. In patients on rivaroxaban at Tmin, cardiac failure was the only predictor of Cmax_ATG. At peak level, apixaban and dabigatran concentrations were predictors of the interindividual variability of Cmax_ATG; in patients on rivaroxaban, no predictors were found.

<figure><img src="image_16.png" /><figcaption>**FIGURE 5** Fibrinography parameters (Thrombodynamics-4D) in a subset of Assessment of Direct oral Anticoagulants in Geriatrics (ADAGE) patients on rivaroxaban, apixaban, or dabigatran (reduced dose) at peak time (Tmax) and trough (Tmin) compared with pooled normal plasma (Normal Pool; gray) and elderly direct oral anticoagulant (DOAC)-free subjects (Elderly control; yellow). (A) Lag time (Tlag), (B) initial rate of clot growth (Vi), (C) clot size (CS) at 30 minutes, and (D) time of spontaneous clotting (Tsp). Fibrinography parameters are presented as scattered dot plots. ADAGE patients on rivaroxaban o.d. 15 mg (pink) or 20 mg (red); ADAGE patients on apixaban twice daily (b. i.d.) 2.5 mg (light blue) or 5.0 mg (dark blue); ADAGE patients on 110 mg dabigatran twice daily (green). Dashed lines indicate the locally determined reference intervals in healthy subjects. Comparisons between the mean values of each parameter in the Elderly control group and ADAGE patients are shown as ***P < 10⁻³, **P < 10⁻², *P < .05 (Mann-Whitney U-test). Of note, 1 patient on rivaroxaban (676 ng/mL) is represented by an open black diamond (beyond the limits of quantification). b.i.d., twice-daily; o.d., once daily.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>FIGURE 6 Thrombography parameters in a subset of Assessment of Direct oral Anticoagulants in Geriatrics (ADAGE) patients on rivaroxaban, apixaban, or dabigatran (reduced dose) at peak time (Tmax) and time to trough (Tmin) compared with pooled normal plasma (Normal Pool; gray) and direct oral anticoagulant (DOAC)-free elderly subjects (Elderly control; yellow). (A) Lag time (Lag_ATG), (B) time to peak (Tmax_ATG), (C) maximal thrombin concentration (Cmax_ATG), (D) endogenous thrombin potential (ETP_ATG), and (E) stationary amplitude of thrombin moving peak (Ast). Thrombography parameters are presented as scattered dot plots. ADAGE patients on rivaroxaban o.d. 15 mg (pink) or 20 mg (red); ADAGE patients on apixaban 2.5 mg (light blue) or 5.0 mg (dark blue) twice daily; ADAGE patients on 110 mg dabigatran (green) twice daily. Dashed lines indicate the locally determined reference intervals in healthy subjects. Comparisons between the mean values of each parameter in the elderly control group and ADAGE patients are shown as ***P < 10⁻³, **P < 10⁻², *P < .05 (Mann-Whitney U-test). Of note, 1 patient on rivaroxaban (676 ng/mL) is represented by an open black diamond (beyond the limits of quantification). AU, arbitrary units; b.i.d., twice-daily; ETP, endogenous thrombin potential; o.d., once daily.</figcaption></figure>

4 | DISCUSSION

In the present large-scale prospective study, we simultaneously
evaluated fibrinography and thrombography using TD-4D system in

plasma samples from very elderly patients receiving direct oral FXa
or thrombin inhibitors for AF, along with spiked pooled normal
plasma samples. For the first time, we demonstrated the ability of
TD-4D system to assess the *in vitro* and *ex vivo* effects of DOACs,
<table><caption>TABLE 2 Direct oral anticoagulant concentrations and fibrinography parameters (Thrombodynamics-4D) at peak time (Tmax) and trough (Tmin) in the Assessment of Direct oral Anticoagulants in Geriatrics patients with reduced direct oral anticoagulant dose.</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="2">Rivaroxaban 15 mg once daily</th><th colspan="2">Apixaban 2.5 mg twice daily</th><th colspan="2">Dabigatran 110 mg twice daily</th></tr><tr><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th></tr></thead><tbody><tr><td colspan="7"><strong>DOAC concentration (ng/mL)</strong></td></tr><tr><td>Samples, n</td><td>24</td><td>43</td><td>25</td><td>49</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>248 (181-397)</td><td>42 (27-69)</td><td>170 (133-210)</td><td>74 (49-117)</td><td>171 (123-280)</td><td>39 (20-70)</td></tr><tr><td>Range</td><td>64-676</td><td>20-149</td><td>69-442</td><td>20-240</td><td>79-665</td><td>20-106</td></tr><tr><td>CV, %</td><td>49.5</td><td>66.0</td><td>46.8</td><td>59.5</td><td>66.7</td><td>58.9</td></tr><tr><td colspan="7"><strong>Tlag (min)</strong></td></tr><tr><td>Samples, n</td><td>22</td><td>32</td><td>24</td><td>47</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>2.0 (1.7-2.6)</td><td>1.2 (1.0-1.4)</td><td>1.3 (1.2-1.4)</td><td>1.1 (1.0-1.3)</td><td>1.3 (1.2-2.0)</td><td>1.0 (0.9-1.1)</td></tr><tr><td>Range</td><td>1.3-4.7</td><td>0.8-2.1</td><td>0.9-2.3</td><td>0.7-1.8</td><td>1.0-3.9</td><td>0.8-1.3</td></tr><tr><td>CV, %</td><td>36.0</td><td>23.0</td><td>21.9</td><td>21.6</td><td>51.0</td><td>15.3</td></tr><tr><td colspan="7"><strong>Initial rate of clot growth (µm/min)</strong></td></tr><tr><td>Samples, n</td><td>22</td><td>32</td><td>24</td><td>47</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>60.6 (56.2-65.3)</td><td>70.4 (64.9-75.8)</td><td>64.9 (60.9-74.2)</td><td>68.3 (64.8-76.2)</td><td>60.9 (57.8-65.6)</td><td>70.3 (66.9-75.1)</td></tr><tr><td>Range</td><td>45.0-84.6</td><td>56.5-87.1</td><td>56.1-95.5</td><td>54.6-88.8</td><td>54.5-74.4</td><td>57.5-79.5</td></tr><tr><td>CV, %</td><td>16.4</td><td>11.5</td><td>14.2</td><td>10.5</td><td>9.6</td><td>7.6</td></tr><tr><td colspan="7"><strong>CS (µm)</strong></td></tr><tr><td>Samples, n</td><td>9</td><td>11</td><td>10</td><td>17</td><td>9</td><td>9</td></tr><tr><td>Median (IQR)</td><td>1236 (1112-1393)</td><td>1691 (1632-1734)</td><td>1362 (1317-1698)</td><td>1655 (1395-1780)</td><td>1436 (1332-1583)</td><td>1707 (1634-1904)</td></tr><tr><td>Range</td><td>1019-1759</td><td>1276-2029</td><td>1134-1857</td><td>1170-2147</td><td>1273-2125</td><td>1612-2205</td></tr><tr><td>CV, %</td><td>18.0</td><td>12.5</td><td>15.0</td><td>15.6</td><td>16.8</td><td>11.1</td></tr><tr><td colspan="7"><strong>D (a.u.)</strong></td></tr><tr><td>Samples, n</td><td>23</td><td>32</td><td>24</td><td>47</td><td>5</td><td>13</td></tr><tr><td>Median (IQR)</td><td>23,230 (20,060-28,420)</td><td>23,940 (20,550-30,160)</td><td>25,340 (22,600-27,790)</td><td>25,470 (21,190-29,910)</td><td>28,810 (27,570-29,170)</td><td>32,220 (30,160-36,290)</td></tr><tr><td>Range</td><td>14,690-33,710</td><td>18,160-37,370</td><td>14,090-34,880</td><td>15,530-37,550</td><td>22,740-29,360</td><td>26,190-39,810</td></tr><tr><td>CV (%)</td><td>21.1</td><td>21.8</td><td>19.0</td><td>22.1</td><td>9.0</td><td>11.4</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="2">Rivaroxaban 15 mg once daily</th><th colspan="2">Apixaban 2.5 mg twice daily</th><th colspan="2">Dabigatran 110 mg twice daily</th></tr><tr><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th></tr></thead><tbody><tr><td>Time to Tsp (min)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Samples, n</td><td>21</td><td>42</td><td>23</td><td>44</td><td>13</td><td>21</td></tr><tr><td>Median (IQR)</td><td>18.5 (11.4-31.6)</td><td>10.9 (7.1-23.1)</td><td>22.9 (12.1-31.1)</td><td>23.0 (12.8-28.1)</td><td>27-2 (24.7-33.2)</td><td>13.7 (11.0-22.2)</td></tr><tr><td>Range</td><td>7.7-58.1</td><td>3.2-42.3</td><td>6.8-48.2</td><td>6.8-46.7</td><td>9.8-58.5</td><td>7.4-54.6</td></tr><tr><td>CV, %</td><td>62.9</td><td>69.7</td><td>49.8</td><td>49.2</td><td>39.7</td><td>63.0</td></tr></tbody></table>

au., arbitrary units; CS, clot size; CV, coefficient of variation; D, clot density; DOAC, direct oral anticoagulant; Tlag, lag time; Tmax, peak time; Tmin, trough; Tsp, spontaneous clotting.

which reduced TG and subsequently delayed and slowed fibrin clot formation.

We first showed that rivaroxaban, apixaban, and dabigatran exerted a significant concentration-dependent effect on most thrombography and fibrinography parameters, both in spiked normal pooled plasma samples and in ADAGE patient samples. Thrombography results, ie, the prolongation of temporal parameters and the decrease in Cmax_ATG and ETP_ATG, were consistent with those obtained from other thrombography systems, such as calibrated automated thrombogram (CAT), using tissue-factor micelles in suspension [17,19-31]. However, unlike CAT, we did not observe a paradoxical increase in TG at low dabigatran concentrations, either in spiked or patient samples. Indeed, an apparent enhancement of TG has been observed with CAT system, most probably due to different calculation algorithms [44,54-56]. In contrast, TD-4D was able to assess both direct FXa and thrombin inhibitor effects on TG. Another advantage of the TD-4D analyzer is that it requires only 120 µL of plasma samples, making it particularly suitable for geriatric and pediatric patients. One disadvantage is that thrombin is quantified in arbitrary units, not in nanomolar, making comparisons with other thrombography systems difficult.

We further evidenced that DOACs affected fibrinography parameters in a concentration-dependent manner, increasing the lag time and decreasing the rates of growth at initiation and at 30 minutes (when measurable), as well as the CS. Moreover, we evidenced strong associations between thrombography and fibrinography parameters, especially for temporal parameters. Our results demonstrate that the delay in clot formation and decrease in clot growth reflect the decrease in TG induced by DOACs. In other words, the TD-4D system mimics the common coagulation pathway until final substrate (fibrinogen) transformation. Besides, we observed that D was found to be strongly correlated with fibrinogen concentration measured using the Clauss method, especially in ADAGE patients, most of whom had hyperfibrinogenemia. Interestingly, we found that the measured D was not sensitive to anti-FXa DOACs, regardless of the drug concentration. In contrast, it was sensitive to dabigatran concentration, probably due to its direct, reversible, competitive inhibitory effect on thrombin, whether free or bound to a fibrin clot, thus probably reflecting some analytical interference.

In addition, we evidenced substantial interindividual variability in thrombography and fibrinography parameters with increasing concentrations of DOACs in patients. We highlighted an underlying hypercoagulable state in some ADAGE patients, for whom shorter temporal parameters, combined with higher peak heights and endogenous thrombin potential, were observed compared with spiked normal plasma. It is well known that advanced age and chronic comorbid conditions lead to an imbalance between procoagulant factors and natural coagulation inhibitors, potentially resulting in hypercoagulability [57]. This variability deserves further studies in a geriatric setting.

Besides the relationship between PD parameters and DOAC concentrations in ADAGE patients, we also assessed the interindividual variability of DOAC PK/PD in a subset of patients who were
<table><caption>TABLE 3 Thrombography parameters (Thrombodynamics-4D) at peak time and trough in the Assessment of Direct oral Anticoagulants in Geriatrics patients with reduced direct oral anticoagulant dose.</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="2">Rivaroxaban 15 mg once daily</th><th colspan="2">Apixaban 2.5 mg twice daily</th><th colspan="2">Dabigatran 110 mg twice daily</th></tr><tr><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th><th>Tmax</th><th>Tmin</th></tr></thead><tbody><tr><td colspan="7"><strong>Tlag (min)</strong></td></tr><tr><td>Samples, n</td><td>23</td><td>43</td><td>25</td><td>49</td><td>11</td><td>22</td></tr><tr><td>Median (IQR)</td><td>1.1 (0.8-1.5)</td><td>0.2 (0.1-0.4)</td><td>0.3 (0.2-0.6)</td><td>0.2 (0.2-0.6)</td><td>0.6 (0.4-0.8)</td><td>0.2 (0.1-0.2)</td></tr><tr><td>Range</td><td>0.6-3.9</td><td>0.1-0.9</td><td>0.1-1.3</td><td>0.1-1.0</td><td>0.2-1.5</td><td>0.1-0.5</td></tr><tr><td>CV, %</td><td>56.9</td><td>79.5</td><td>71.0</td><td>69.9</td><td>55.8</td><td>54.1</td></tr><tr><td colspan="7"><strong>Time to peak (min)</strong></td></tr><tr><td>Samples, n</td><td>23</td><td>43</td><td>25</td><td>49</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>3.1 (2.6-3.8)</td><td>2.1 (1.6-2.1)</td><td>2.1 (2.1-2.6)</td><td>2.1 (2.1-2.1)</td><td>2.6 (2.1-3.1)</td><td>1.85 (1.6-2.1)</td></tr><tr><td>Range</td><td>2.6-7.1</td><td>1.6-3.1</td><td>1.2-4.6</td><td>1.6-3.6</td><td>1.6-4.6</td><td>1.6-2.1</td></tr><tr><td>CV, %</td><td>35.8</td><td>18.4</td><td>26.9</td><td>18.4</td><td>33.0</td><td>13.5</td></tr><tr><td colspan="7"><strong>Peak height (AU/L)</strong></td></tr><tr><td>Samples, n</td><td>23</td><td>43</td><td>25</td><td>49</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>203 (140-268)</td><td>289 (254-422)</td><td>309 (240-395)</td><td>325 (292-379)</td><td>193 (98-243)</td><td>349 (279-380)</td></tr><tr><td>Range</td><td>64-371</td><td>132-544</td><td>206-507</td><td>158-511</td><td>8-354</td><td>168-499</td></tr><tr><td>CV, %</td><td>40.7</td><td>33.0</td><td>29.2</td><td>22.9</td><td>63.0</td><td>23.8</td></tr><tr><td colspan="7"><strong>ETP (AU·min/L)</strong></td></tr><tr><td>Samples, n</td><td>23</td><td>43</td><td>25</td><td>49</td><td>14</td><td>22</td></tr><tr><td>Median (IQR)</td><td>1391 (1176-1774)</td><td>1832 (1322-2480)</td><td>1698 (1280-1961)</td><td>1711 (1436-1978)</td><td>1036 (724-1501)</td><td>1741 (1602-2290)</td></tr><tr><td>Range</td><td>601-2371</td><td>557-3572</td><td>971-3095</td><td>924-2958</td><td>80-2078</td><td>1111-2715</td></tr><tr><td>CV, %</td><td>30.5</td><td>38.3</td><td>34.1</td><td>25.5</td><td>56.1</td><td>24.9</td></tr></tbody></table>

AU, arbitrary unit; CV, coefficient of variation; ETP, endogenous thrombin potential; Tlag, lag time; Tmax, peak time; Tmin, trough;

sampled at peak and/or trough levels. PK variability was high regardless of the DOAC; all CVs of plasma concentration at Tmax and Tmin were around ≥50%, as consistently reported by different groups, including ours, in patients in this age group [9-12,14,15,17]. As a reminder, ADAGE patients, with a mean age of 87 years and 69% being women, had substantial comorbid conditions (eg, renal impairment) and frequent polymedication, which contribute to the PK variability in this fragile population [17]. Using TD-4D, we evidenced that the variability of thrombography parameters, eg, peak height, was elevated in patients receiving rivaroxaban 15 mg o.d. and dabigatran 110 mg twice daily at Tmax (>40%), but less important in patients receiving 2.5 mg twice daily apixaban at both Tmax and Tmin (<30%). Finally, even though the sample size was limited, apixaban or dabigatran concentrations at Tmax and Tmin were the main contributors to Cmax_ATG and subsequently to the initial rate of growth, whereas cardiac failure in patients on rivaroxaban was the main determinant of Cmax_ATG at Tmin. These findings, obtained with TD-4D based on a coagulation process initiated with immobilized TF, remarkably confirm our previous results, which were obtained using another thrombography system (ST Genesia, Stago, soluble TF) for analyzing rivaroxaban and apixaban ADAGE samples [17], thus

reinforcing the relevance of TD-4D in assessing DOAC PD. Although rivaroxaban has an impact on TD-4D parameters, rivaroxaban concentration was not found predictors when considering only concentrations at Tmin or Tmax. This is explained by the lower span of concentration ranges, the smaller sample size, and the high variability of parameters, all of which lower power.

Another interest of TD-4D was the evaluation of spatial propagation of thrombin and clot growth. However, an early Tsp (before 30 minutes) hampered the recording of some parameters (namely V and stationary amplitude of thrombin peak) in a substantial number of plasma samples, either from DOAC-free elderly subjects or ADAGE patients on DOACs. Some authors have proposed centrifuging samples at 10,000 × g to decrease Tsp [46,58], although this centrifugation condition is not recommended for performing TG tests. Lipets et al. [58] hypothesized that the combination of circulating active factors, circulating procoagulants, and contact pathway-activating extracellular vesicles is the predominant mechanism causing Tsp in patient plasma. Nevertheless, whether there is clinical relevance to Tsp or not needs to be further investigated [58,59].

Our study has several limitations. First, this study was conducted in a subset of ADAGE patients, depending on the availability
of plasma samples, and not in the whole cohort. Moreover, the
recruitment of patients with dabigatran was difficult, with fewer
patients included than expected. Second, frequent early Tsp has
limited the collection of the temporal-spatial parameters in a number
of samples. Evaluating PD using TD-4D on fresh plasma
or centrifuging plasma at high speed may solve this problem and
overcome these drawbacks, yet this cannot be verified in ADAGE
patients, as aliquots had already been prepared. Third, DOAC-free
elderly individuals were not matched with ADAGE patient charac-
teristics. A study including a larger number of matched controls
(based on kidney/liver function) and a greater number of patients in
the subgroups is of interest to better understand PD variability in the
very elderly. Finally, the present study was not designed to evaluate
the potential relationship between PK/PD data and clinical events, as
it was underpowered to answer this question.

In conclusion, in this large-scale prospective study, fibrinography and thrombography simultaneously assessed with TD-4D provided consistent PD results for all 3 DOACs, including dabigatran. Substantial PD variability was observed. Apixaban and dabigatran concentrations at Tmin were the only significant predictors of interindividual variability in both Cmax_ATG (thrombography) and Vi (fibrinography). In rivaroxaban patients, cardiac failure was significantly associated with Cmax_ATG variability. The clinical relevance of such variability remains to be demonstrated.

FUNDING

We acknowledge the CONNY-MAEVA Charitable Foundation and the Société Française de Cardiologie for the purchase of reagents. The funding sources had no role in the design and conduct of the study, collection, management, analysis, and interpretation of the data.

AUTHOR CONTRIBUTIONS

G.F.-P., V.S., G.J., and E.C. wrote the manuscript; C.L.-L., V.S., E. Pautas, and P.G. designed the research; C.L.-L., J.L.G., E. Puymirat, T. M., E. Pautas, and M.D. were clinical investigators; G.F.-P., V.S., G.J., and C.C. performed the research; G.F.-P., V.S., G.J., T.L., and E.C. analyzed the data; all authors reviewed the manuscript.

RELATIONSHIP DISCLOSURE

V.S., I.G.-T., E. Pautas, E. Puymirat, M.D., and P.G. have received honoraria for participating in expert meetings on dabigatran (from Boehringer Ingelheim), rivaroxaban (from Bayer Healthcare AG), or apixaban (Bristol Myers Squibb-Pfizer). The other authors declare no conflicts of interest. T.M. has received honoraria from Novartis, Amarin, and Novo Nordisk and nonfinancial support from Amgen, Bristol Myers Squibb-Pfizer, and MSD.

ORCID

Virginia Siguret ID https://orcid.org/0000-0002-6509-8964

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SUPPLEMENTARY MATERIAL

The online version contains supplementary material available at
https://doi.org/10.1016/j.rpth.2025.102969