In vitro evaluation of anticoagulant therapy management when urgent percutaneous coronary intervention is required in rivaroxaban-treated patients

We investigated in vitro the management of intraprocedural anticoagulation in patients requiring immediate percutaneous coronary intervention (PCI) while using regular direct oral anticoagulants (DOACs). Twenty-five patients taking 20 mg of rivaroxaban once daily comprised the study group, while five healthy volunteers included the control group. In the study group, a beginning (24 h after the last rivaroxaban dose) examination was performed. Then, the effects of basal and four different anticoagulant doses (50 IU/kg unfractionated heparin (UFH), 100 IU/kg UFH, 0.5 mg/kg enoxaparin, and 1 mg/kg enoxaparin) on coagulation parameters were investigated at the 4th and 12th h following rivaroxaban intake. The effects of four different anticoagulant doses were evaluated in the control group. The anticoagulant activity was assessed mainly by anti-factor Xa (anti-Xa) levels. Beginning anti-Xa levels were significantly higher in the study group than in the control group (0.69 ± 0.77 IU/mL vs. 0.20 ± 0.14 IU/mL; p < 0.05). The study group’s 4th and 12th-h anti-Xa levels were significantly higher than the beginning level (1.96 ± 1.35 IU/mL vs. 0.69 ± 0.77 IU/mL; p < 0.001 and 0.94 ± 1.21 IU/mL vs. 0.69 ± 0.77 IU/mL; p < 0.05, respectively). Anti-Xa levels increased significantly in the study group with the addition of UFH and enoxaparin doses at the 4th and 12th h than the beginning (p < 0.001 at all doses). The safest anti-Xa level (from 0.94 ± 1.21 to 2.00 ± 1.02 IU/mL) was achieved 12 h after rivaroxaban with 0.5 mg/kg enoxaparin. Anticoagulant activity was sufficient for urgent PCI at the 4th h after rivaroxaban treatment, and additional anticoagulant administration may not be required at this time. Twelve hours after taking rivaroxaban, administering 0.5 mg/kg of enoxaparin may provide adequate and safe anticoagulant activity for immediate PCI. This experimental study result should confirm with clinical trials (NCT05541757).

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in the world and is associated with significant symptoms, impaired quality of life, and cardiovascular morbidity and mortality (Hindricks et al. 2021;Benjamin et al. 1994;Rodríguez-Mañero et al. 2019). Stroke prevention is among the primary therapeutic goals in managing AF. Clinical guidelines for AF recommend using long-term oral anticoagulant therapy driven by individually predicted stroke risk (Hindricks et al. 2021;January et al. 2019). In pivotal clinical trials, direct oral anticoagulants (DOACs) were non-inferior to vitamin K antagonists (VKAs) in preventing thromboembolic stroke and had a lower risk of intracranial bleeding (Ruff et al. 2014). Therefore, DOACs were suggested as the first-choice therapy for stroke prevention in patients with nonvalvular AF (Hindricks et al. 2021;January et al. 2019).
Approximately 30% of patients with AF have concomitant coronary artery disease (CAD) (Kralev et al. 2011;Lip et al. 2014). In addition, 7 to 10% of myocardial infarction patients and 10 to 15% of patients undergoing percutaneous coronary intervention (PCI) have a history of AF (Sutton et al. 2016;Lopes et al. 2012). The prevalence of prior AF has been reported to be between 3.0 and 4.9% in ST-segment elevation myocardial infarction (STEMI) patients treated with primary PCI. Also, AF recognized during AMI was associated with increased in-hospital and long-term mortality compared to patients with sinus rhythm (Batra et al. 2016;Topaz et al. 2017;Braga et al. 2015). Primary PCI refers to the strategy of directly transporting a patient with STEMI to a cardiac catheterization laboratory to undergo mechanical revascularization, and it is the best reperfusion modality for these patients (Ibanez et al. 2018). Dual antiplatelet therapy and intravenous (IV) anticoagulation are the standard of care and first-line treatment for patients presenting with STEMI (Ibanez et al. 2018). Anticoagulant therapy aims to control ongoing coronary thrombosis and facilitate primary PCI. The treatment of STEMI patients with primary PCI while taking DOACs is a challenging clinical situation for cardiologists to manage. ESC guidelines recommend the addition of intraprocedural low-dose anticoagulation (e.g., IV enoxaparin 0.5 mg/kg or UFH 60 IU/kg) during PCI in acute coronary syndrome (ACS) patients using DOACs, regardless of the time of the last DOAC dose (Lip et al. 2019;Collet et al. 2021). Nevertheless, no prospective randomized study has specifically examined the management of intraprocedural anticoagulation in STEMI/NonSTEMI patients undergoing PCI while receiving continuous DOAC therapy.
It is still unclear whether additional anticoagulation is required intraprocedural in patients who use regular DOACs and need PCI. Other questions to be clarified include which anticoagulant is suitable, the anticoagulant's dose, and whether the time between the last DOAC dose and the index procedure is significant. In this study, we investigated the answers to these questions in vitro environment.

Study population
Twenty-five patients using the direct oral factor Xa inhibitor rivaroxaban were included in the study group. These patients with persistent or permanent AF had been using rivaroxaban 20 mg daily for at least 1 month. A control group of five healthy adults of similar ages was formed. Patients with the following criteria were excluded from the study: malignancy, coagulopathy, heart valve disease, active infection, chronic systemic or inflammatory disease, recent ACS or elective PCI, uncontrolled hypertension and diabetes, cerebrovascular accident, thyroid disorder, left ventricular systolic dysfunction (ejection fraction <50%), glomerular filtration rate <50 mL/min/1.73 m 2 , body mass index <18.5 and >30, age >75 years, hyperbilirubinemia, and hypertriglyceridemia (>350 mg/dL). The study was approved by the local institution's ethical committee, and written informed consent was obtained from all participants.

Study group measurements
Rivaroxaban is a reversible, direct-acting oral inhibitor of factor Xa. Bioavailability after oral administration is dosedependent (66% after a 20 mg dose), with plasma concentration peaking between 2 and 4 h after ingestion and a half-life ranging from 6 to 13 h (11-13 h in elderly subjects), depending on dose and age (Samama et al. 2013).
Blood samples were drawn from the patients in the rivaroxaban group three times: beginning (24 h after the last rivaroxaban dose), 4 h after rivaroxaban, and 12 h after rivaroxaban (Fig. 1). Firstly, 7 mL of venous blood was drawn from patients for beginning values before rivaroxaban intake. This blood was divided into three tubes: the first tube (9NC sodium citrate, 3.2%, Ayset Medical Products, Adana, Turkey) for prothrombin time (PT), activated partial thromboplastin time (aPTT), and international normalized ratio (INR), the second tube (BlacT-ACT Celite ACT tubes, Hemonart LTD, Istanbul, Turkey) for activated clotting time (ACT), and the third tube (9NC Sodium citrate, 3.2%, Ayset Medical Products, Adana, Turkey) for anti-factor Xa (anti-Xa).
Each participant's total blood volume was estimated using Nadler's equation (Nadler et al. 1962). Nadler's equation is as follows: male blood volume = [0.3669 × (height, m) 3 + 0.03219 × (weight, kg) + 0.6041] and female blood volume = [0.3561 × (height, m) 3 + 0.03308 × (weight, kg) + 0.1833]. For each participant, 500 mL of isotonic saline (sodium chloride 0.9% solution for IV infusion) solutions were prepared to which anticoagulants were added at doses calculated using this estimated blood volume. For instance, suppose it is planned to administer UFH at a 50 IU/kg dose to a woman standing 160 cm tall and weighing 70 kg. Using Nadler's equation, the total blood volume of this woman is approximately 3950 mL. After removing the amount of fluid planned to be added, 3100 IU of UFH was put into 500 mL of isotonic saline solution. Thus, a solution containing 6.2 IU of UFH was prepared in 1 mL. By adding 1 mL of this solution to 6 mL of venous blood drawn from the woman described in the example, 7 mL of blood is obtained as if this woman had been administered 50 IU/kg UFH (this is the equivalent of 3500 IU IV UFH).
Five syringes of blood were drawn from the patients using a branule 4 and 12 h after taking 20 mg of rivaroxaban ( Fig. 1). Patients were never given UFH or enoxaparin. Four different doses of anticoagulants (50 IU/ kg unfractionated heparin (UFH) (Koparin vial, Koçak Pharmaceuticals, Istanbul, Turkey), 100 IU/kg UFH, 0.5 mg/kg enoxaparin (Clexane, Sanofi Winthrop Industrie, Maisons-Alfort, France), and 1 mg/kg enoxaparin) were added to the syringes containing the patients' blood. A calculated amount of anticoagulant was added to the blood in the syringes according to the patient's estimated blood volume, so an in vitro environment was created that simulated IV anticoagulant administration to the patient. These syringes were prepared as follows: 1. injector: 7 mL of venous blood (for basal values at that moment without the addition of anticoagulant); 2. injector: 6 mL of venous blood plus 1 mL of isotonic saline solution containing 50 U/kg UFH (according to patient's estimated blood volume); 3. injector: 6 mL of venous blood plus 1 mL of isotonic saline solution containing 100 U/kg UFH (according to patient's estimated blood volume); 4. injector: 6 mL of venous blood plus 1 mL of isotonic saline solution containing 0.5 mg/kg enoxaparin (according to patient's estimated blood volume); and 5. injector: 6 mL of venous blood plus 1 mL of isotonic saline solution containing 1 mg/kg enoxaparin (according to patient's estimated blood volume). The blood in these syringes was also divided into three tubes, and the parameters PT, aPTT, INR, ACT, and anti-Xa were measured (Fig. 1). A fresh UFH or an isotonic saline solution containing enoxaparin was prepared each time (4th and 12th h) to prevent the anticoagulant activity from changing over time.

Control group measurements
Five syringes of blood were drawn at any one time from volunteers in the control group. Then, the same procedure was applied to the blood of the control group as the rivaroxaban group (Fig. 1).

Measurement of the anti-factor Xa level and coagulation parameters
The 2 mL blood samples were centrifuged immediately at 5000 RCF (relative centrifugal force) for 10 min at room temperature in the biochemistry laboratory. The plasma samples were put into one Eppendorf tube using a plastic pipe and stored in a deep freezer at −20 °C until analysis. Anti-Xa levels were measured in plasma samples obtained in the biochemistry laboratory using a Berichrom Heparin kit (Berichrom Heparin, Siemens Healthineers, Marburg, Germany) and a Sysmex CS-5100 System (Siemens Healthcare Diagnostics, Erlangen, Germany) device. The Berichrom Heparin kit is a chromogenic test and contains an antithrombin III reagent. The Berichrom Heparin LMW Calibrator was used to calibrate the Berichrom Heparin kit before the anti-Xa measurement. Other coagulation parameters measured were INR, PT, and aPTT. Venous blood samples in coagulation tubes were centrifuged at 5000 RCF for 10 min, and the INR, PT, and aPTT levels were measured in the biochemistry laboratory using a Sysmex CS 5100 device, Dade Actin FS, Activated PTT reagent, and Thromborel S reagent.

Statistical analysis
Continuous variables are presented as the mean value ± standard deviation. Categorical variables are presented as absolute numbers and percentages. The equality of the data to the normal distribution was assessed with the Shapiro-Wilk test, skewness, and kurtosis. Categorical parameters were compared by the chi-squared test or Fisher's exact test. The Student's t-test was used to compare normally distributed variables, and the Mann-Whitney U test was used for nonnormally distributed variables. The analysis of the repeated measurements was performed with the paired t-test and Wilcoxon signed-rank test. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 21 (IBM Corporation, Armonk, New York, USA). All statistical tests were two-sided, and p values of less than 0.05 were considered significant.

Results
Twenty-five patients (15 men and 10 women; mean age 58.44 ± 10.85 years) were included in the study group who used rivaroxaban 20 mg daily due to persistent or permanent AF. Hypertension was present in 68% (17) of the patients, 36% (9) of the patients had diabetes mellitus, and 20% (5) had CAD. The average CHA 2 DS 2 VASc score was 2.6 ± 1.2. A control group of five healthy adults (two men and three women; mean age of 57.20 ± 8.17 years) was established. The demographic characteristics and laboratory parameters of the groups are shown in Table 1. There was no statistically significant difference between the two groups.

Effect of anticoagulants on coagulation parameters
The anti-Xa level of the study group before rivaroxaban was higher than that of the control group (0.69 ± 0.77 IU/mL vs. 0.20 ± 0.14 IU/mL; p < 0.05). Before taking rivaroxaban, anti-Xa levels in the study group ranged from 0.14 to 3.6 IU/ mL. The ACT, PT, aPTT, and INR parameters were similar between the two groups ( Table 2). The basal values of both groups and coagulation parameters were obtained as a result of adding 50 IU/kg UFH, 100 IU/kg UFH, 0.5 mg/kg enoxaparin, and 1 mg/kg enoxaparin, as shown in Tables 3  and 4. Anti-Xa levels were higher in patients receiving rivaroxaban than in the control group in all measurements with added anticoagulants. However, this difference did not attain statistical significance except adding 0.5 mg/kg enoxaparin at the 4th h. In the rivaroxaban group, the ACT level was statistically significantly higher than that in the control group in all anticoagulant-added measurements (p < 0.001 in all). PT, aPTT, and INR levels were also more elevated in the rivaroxaban group with the addition of anticoagulants, but statistically significant differences were heterogeneous (Tables 3 and 4).
In the study group, anti-Xa, ACT, PT, aPTT, and INR values were significantly higher than beginning at the 4th h after 20 mg of rivaroxaban, as expected (p < 0.001, p < 0.01, p < 0.01, p < 0.01, p < 0.01, respectively) ( Table 3). The anti-Xa levels obtained in the study group are shown in Figs. 2 and 3. All coagulation parameters increased significantly after adding 50 IU/kg and 100 IU/kg UFH at the 4th h (Table 3). After adding 0.5 mg/kg and 1 mg/kg of enoxaparin at the 4th h, there was a statistically significant increase in anti-Xa, ACT, and aPTT values (Table 3). When the effects of 50 IU/kg UFH and 0.5 mg/kg enoxaparin on coagulation parameters were compared at the 4th h, a more significant increase was found in anti-Xa, PT, and INR values with 50 IU/kg UFH (p < 0.05, p < 0.001, p < 0.001, respectively). When high-dose anticoagulants (100 IU/kg UFH and 1 mg/kg enoxaparin) were compared at 4 h, anti-Xa, ACT, PT, and INR values significantly increased with 100 IU/kg UFH (p < 0.001, p < 0.05, p < 0.001, p < 0.001, respectively) (Table 3).

Discussion
Acute coronary syndrome guidelines recommend adding intraprocedural low-dose anticoagulation during the urgent PCI in patients using regular DOACs, regardless of the time of the last DOAC dose (Lip et al. 2019;Collet et al. 2021). However, no prospective randomized study has specifically examined the management of intraprocedural anticoagulation in this situation. The purpose of this study was to determine whether an additional anticoagulant is needed, which anticoagulant should be used, and at what dose in patients taking DOACs who require urgent PCI. The anti-Xa level of the study group before rivaroxaban was higher than that of the control group. As expected, anti-Xa, ACT, PT, aPTT, and INR values in the study group were significantly higher than beginning at the 4th h after 20 mg of rivaroxaban. All coagulation parameters significantly increased with the addition of 50 IU/kg UHF, 100 IU/kg UFH, 0.5 mg/kg enoxaparin, and 1 mg/kg enoxaparin 4 h after rivaroxaban intake. Twelve hours after taking rivaroxaban, all anticoagulation parameters were still significantly higher than before the drug. The mean anti-Xa level at 12 h was 0.94 ± 1.21 IU/mL but less than 0.5 IU/mL in 24% (6/25) of patients. Among the anticoagulants added 12 h after rivaroxaban, the best results were obtained with 0.5 mg/kg of enoxaparin (from 0.94 ± 1.21 to 2.00 ± 1.02 IU/mL). The enoxaparin doses are given at 4 and 12 h had a softer and more well-distributed anticoagulant effect than the UFH doses. The American College of Chest Physicians' consensus guideline states that the therapeutic anti-Xa activity for treating venous thromboembolism is between 0.3 and 0.7 IU/ mL for UFH and 0.6 to 1.0 IU/mL using twice-daily lowmolecular-weight heparins (LMWHs) (Hirsh et al. 2008). Although the target for LMWH anticoagulant activity in PCI has not been clearly established, anti-Xa activity levels ranging from 0.5 to 1.8 IU/mL have been targeted in studies involving the administration of IV enoxaparin for PCI (Kereiakes et al. 2001;Moliterno et al. 2003;Montalescot et al. 2006). In the STEEPLE (Safety and Efficacy of Enoxaparin in Percutaneous Coronary Intervention Patients, an International Randomized Evaluation) trial, 3528 patients undergoing elective PCI were randomized to receive 0.50 mg/kg IV enoxaparin, 0.75 mg/kg IV enoxaparin, or IV UFH (Montalescot et al. 2006). Anti-Xa levels of 0.5-1.8 IU/mL were targeted in the enoxaparin arms, while an ACT level of 300-350 s was targeted in the UFH arm (if glycoprotein IIb/IIIa inhibitors are also used, this value is 200-300 s). This study achieved target anticoagulation levels in 86% of patients receiving enoxaparin without monitoring, compared with 20% of patients receiving UFH with ACT monitoring. The STEEPLE study showed that both doses of enoxaparin (0.5 and 0.75 mg/kg) were highly effective, and the incidence of major bleeding was significantly lower in both enoxaparin groups than in the UFH group. The ATOLL (The Acute Myocardial Infarction Treated with Primary Angioplasty and Intravenous Enoxaparin or Unfractionated Heparin to Lower Ischemic and Bleeding Events at Short-and Long-term Follow-up) trial compared an IV bolus of 0.5 mg/ kg enoxaparin with UFH in 910 STEMI patients undergoing primary PCI. In the per-protocol analysis of the ATOLL trial, comprising more than 87% of the study population (795 patients), enoxaparin showed superiority over UFH in reducing the primary endpoint, mortality, and major bleeding and improved net clinical benefit (Collet et al. 2013). Compared to UFH, enoxaparin has been demonstrated to have a more stable and predictable anticoagulant effect, with almost all patients achieving an optimal level of anticoagulation during the procedure.
Routine monitoring is not required for DOAC therapy. If necessary, the anticoagulant effect of oral factor Xa inhibitors is evaluated by anti-Xa activity. The anti-Xa assay can be used to measure DOAC activity via chromogenic reagents and special DOAC calibrators (Asmis et al. 2012;Willekens et al. 2021). Studies have demonstrated a linear, dose-dependent, and strong correlation (R 2 = 0.95 to 1.00) between plasma rivaroxaban concentration and anti-Xa activity over a wide concentration range (20 to 660 ng/mL) when chromogenic reagents and rivaroxaban calibrators are used (Asmis et al. 2012;Willekens et al. 2021;Douxfils et al. 2013). A steady-state peak and trough anti-Xa activity range from 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL   Table 4 Basal values and the effect of anticoagulants on coagulation parameters in the control group, basal values at 12 h after rivaroxaban, and anticoagulants' effect in the study group 1 3 were calculated for rivaroxaban based on published drug concentrations in previous pharmacokinetic studies by Beyer et al. (Beyer et al. 2016). For apixaban 5 mg twice daily, these ranges are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. Our study group's mean anti-Xa level before rivaroxaban was 0.63 ± 0.73 IU/mL. In this situation, it can be said that the coagulation parameters obtained 24 h after 20 mg daily rivaroxaban taking in our study group are compatible with previous studies, and adequate anticoagulation continues even 24 h after taking the drug. However, it should be noted that seven patients had anti-Xa levels less than 0.5 IU/mL in our study. The anti-Xa level 24 h after rivaroxaban administration may be sufficient for prophylaxis of deep vein thrombosis or nonvalvular AF but not for elective or primary PCI. All coagulation parameters were significantly higher at 4 h after taking 20 mg of rivaroxaban than before the drug (Table 3). Anti-Xa activity was measured on average at 1.96 ± 1.35 IU/mL at the 4th h after rivaroxaban intake. This value is slightly higher than the mean anti-Xa levels  obtained in the elective PCI studies NICE-4 (The initial National Investigators Collaborating on Enoxaparin) and ELECT (Evaluating Enoxaparin Clotting Times) (1.7 ± 0.9 IU/mL and 1.6 ± 0.8 IU/mL, respectively) (Kereiakes et al. 2001;Moliterno et al. 2003). The X-PLORER (Evaluating Optimal Concomitant Anticoagulation in Rivaroxaban Treated Patients, an Oral Direct Factor Xa Inhibitor, During Percutaneous Coronary Revascularization) study has shown that rivaroxaban (with or without UFH) effectively inhibits coagulation activity during elective PCI in patients with chronic coronary syndrome compared with UFH alone (Vranckx et al. 2015). Periprocedural rivaroxaban given 2 to 4 h prior to elective PCI appeared to provide adequate anticoagulation without an increased risk of bleeding. As a result, it is possible to achieve adequate anticoagulation for primary or elective PCI 4 h after drug ingestion in patients using regular rivaroxaban.
The latest guidelines on coronary revascularization and ACS focus on reducing bleeding complications in PCI and antithrombotic therapies (Collet et al. 2021;Neumann et al. 2019). Guidelines for anticoagulated patients undergoing primary PCI recommend some strategies to reduce the risk of bleeding: preferring the radial approach as the access site, performing primary PCI without interruption of oral anticoagulant, not applying UFH if INR > 2.5 in patients using VKA, and adding low-dose parenteral anticoagulation (e.g., enoxaparin 0.5 mg/kg or UFH 60 IU/kg) in patients using DOACs, regardless of the timing of the last DOAC administration (Lip et al. 2019;Collet et al. 2021). In our study, anti-Xa levels were significantly elevated by the addition of 50 IU/kg UHF, 100 IU/kg UFH, 0.5 mg/kg enoxaparin, and 1 mg/kg enoxaparin 4 h after rivaroxaban intake (3.58 ± 1.44 IU/mL, 5.79 ± 1.21 IU/mL, 2.73 ± 1.18 IU/mL, and 3.95 ± 1.20 IU/mL, respectively) ( Table 3 and Fig. 2). These values are higher than the targeted and safe anti-Xa levels in previous primary or elective PCI studies with enoxaparin. A patient who regularly uses rivaroxaban 20 mg daily may not need additional intraprocedural anticoagulation 4 h after taking the drug. Our findings contradict the guidelines' recommendation to give low-dose parenteral anticoagulation to patients who use DOACs, no matter when the last dose of DOACs was taken. Adding an anticoagulant 4 h after rivaroxaban, even at a low dose, may shift the balance of major bleeding and thrombotic events in favor of bleeding in a STEMI patient who underwent primary PCI.
Twelve hours after rivaroxaban ingestion, all anticoagulation parameters remained significantly higher than before the drug. The mean anti-Xa level was 0.94 ± 1.21 IU/mL at 12 h after rivaroxaban. In patients receiving enoxaparin in the STEEPLE study, the median anti-Xa at the start and end of PCI was 0.94 and 0.82 IU/mL, respectively, and an anti-Xa level of approximately 0.9 IU/mL was associated with an optimal risk-benefit ratio (Montalescot et al. 2008).
According to the STEEPLE study's findings, the mean anti-Xa level at the 12th h of our patient group can be considered optimum. However, the distribution of the mean anti-Xa measurements at 12 h in our study group was wide. Based on the target values of the STEEPLE study, the anti-Xa value of 6 patients (24%) was measured below 0.5 IU/mL, and the value of 2 patients (8%) was above 1.8 IU/mL. Additionally, primary PCI in patients with STEMI is performed in a more thrombotic environment than elective PCI. Inadequate anticoagulation, which occurs in approximately one-quarter of patients, may increase thrombotic risk. The addition of 50 IU/kg UHF, 100 IU/kg UFH, 0.5 mg/kg enoxaparin, and 1 mg/kg enoxaparin 12 h after rivaroxaban administration significantly increased anti-Xa levels to 2.79 ± 1.16 IU/mL, 5.62 ± 1.53 IU/mL, 2.00 ± 1.02 IU/mL, and 3.60 ± 1.09 IU/ mL, respectively (Table 4). When anti-Xa and other coagulation parameters are investigated, as shown in Table 4 and Fig. 3, it is evident that the safest anticoagulant addition is 0.5 mg/kg enoxaparin. Also, previous randomized studies have shown that 0.5 mg/kg IV enoxaparin is superior to UFH in elective or primary PCI (Montalescot et al. 2006;Collet et al. 2013).

Study limitations
First, our study was carried out experimentally, and answers were sought to questions about clinical practices based on an in vitro study. However, the importance of such research prior to clinical trials is evident. Our study may guide future clinical studies. Second, we preferred rivaroxaban, an oral factor Xa inhibitor, taken once daily in our research. We do not know if our findings apply to apixaban, taken twice daily, and dabigatran, a direct thrombin inhibitor. Third, all DOACs currently present reach a peak plasma level within 1 to 4 h after ingestion and reach their trough level between 12 and 24 h. We examined the effects of anticoagulants added 4 and 12 h after taking rivaroxaban. It is also possible to investigate different times, such as the 2nd, 8th, and 18th h. Last, the control group consisting of five healthy individuals is a small sample size. However, the purpose of establishing a control group was to evaluate anticoagulants' efficacy and laboratory data's reliability rather than to compare the two groups. Consequently, the small sample size of the control group has no bearing on the study results.

Conclusions
In patients using rivaroxaban and requiring emergency PCI, additional anticoagulants may not be needed at or around the 4th h after the drug, given the balance between major bleeding and thrombotic events. Twelve hours after taking rivaroxaban, adding 0.5 mg/kg of enoxaparin may provide more effective and safe anticoagulation. The use of UFH could result in excessive and unpredictable anticoagulation. This experimental study will guide future clinical trials, and this experimental study result should confirm with clinical trials. The authors declare that all data were generated in-house and that no paper mill was used.

Authors contributions
Data Availability This trial data will be shared if the editor wants to evaluate.

Competing interests
The authors declare no competing interests.