{'fraction_non_alphanumeric': 0.03206122660047575, 'fraction_numerical': 0.022391146964525804, 'mean_word_length': 5.35733070348455, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Impact of Total Antithrombotic Effect on Bleeding Complications in Patients Receiving Multiple Antithrombotic Agents

Background: Few reports have evaluated the total antithrombotic effect of multiple antithrombotic agents. Methods and Results: Thrombus formation was evaluated with the Total Thrombus-formation Analysis System (T-TAS ® ) using 2 types of microchips in 145 patients with stable coronary artery disease receiving oral anticoagulants plus single- or dual-antiplatelet therapy. The PL-chip coated with collagen is designed for analysis of the platelet thrombus formation process under shear stress condition (18 µ L/min). The AR-chip coated with collagen and tissue thromboplastin is designed for analysis of the fibrin-rich platelet thrombus formation process under shear stress condition (4 µ L/min). The results were expressed as an area under the flow pressure curve (PL 18 -AUC 10 and AR 4 -AUC 30 , respectively). Bleeding events occurred in 43 patients during a 22-month follow-up. AR 4 -AUC 30 was significantly lower in patients with bleeding events than in those without (584 [96–993] vs. 1,028 [756–1,252], P=0.0003). Multivariate logistic regression analysis identified AR 4 -AUC 30 (odds ratio 3.18) as a significant predictor of bleeding events, in addition to baseline anemia and usage of the standard dose of direct oral anticoagulants. However, PL 18 -AUC 10 was not significantly related to bleeding events. Conclusions: A lower AR 4 -AUC 30 level was associated with increasing risk of subsequent bleeding complications in patients with stable coronary artery disease who received multiple antithrombotic agents.

expected benefit of thrombotic event reduction. The conventional therapy has been to combine all 3 drugs in a strategy known as triple therapy. However, this approach has resulted in excessive major bleeding, with rates of 2.2% within the first month and 4-12% within the first year of treatment. 2 The WOEST (What Is the Optimal Antiplatelet and Anticoagulant Therapy in Patients with Oral Anticoagulation and Coronary Stenting) trial demonstrated that single-antiplatelet therapy with clopidogrel plus oral anticoagulation was associated with a significantly lower risk of bleeding complications than was triple therapy with aspirin, clopidogrel, and oral anticoagulation in patients undergoing PCI who required anticoagulation treatment. 3 In addition, the PIONEER AF-PCI (Open-Label, Randomized, Controlled, Multicenter Study Exploring Two Treatment Strategies of Rivaroxaban and a Dose-L ong-term treatment with oral anticoagulants is necessary in patients with mechanical heart valves, and in most with atrial fibrillation (AF) or venous thromboembolism. Approximately 20-30% of these patients have concomitant coronary artery disease (CAD) that requires percutaneous coronary intervention (PCI) with stenting. 1 Antiplatelet therapy has been a cornerstone of medical therapy in patients with CAD, but the combination of oral anticoagulants and antiplatelet therapy is associated with an increased risk of bleeding complications. Therefore, the optimum treatment in patients who have coexisting CAD and mechanical heart valves, AF, or venous thromboembolism is unclear when thrombotic and bleeding risks are both taken into account. The treatment strategy for patients receiving multiple antithrombotic agents, oral anticoagulants plus single-or dual-antiplatelet therapy must balance the risk of a bleeding event with the in 145 patients with stable CAD (125 men; mean age, 74 years) who were undergoing oral anticoagulation treatment in addition to single-or dual-antiplatelet therapy. In this retrospective and observational study, all study patients were on oral anticoagulation treatment with direct oral anticoagulants (DOACs) or vitamin K antagonists plus antiplatelet therapy with 100 mg/day of aspirin and/or a Japanese standard dose of a P2Y12 receptor inhibitor (75 mg/day clopidogrel or 3.75 mg/day prasugrel) for at least 7 days. Oral anticoagulants and antiplatelet agents were prescribed at each doctor's discretion. Exclusion criteria included any bleeding events within 30 days before enrollment, a hemoglobin level <7 g/dL or >18 g/dL, a platelet count <50,000/µL or >500,000/µL, hematologic or malignant disease, hemodialysis, severe liver dysfunction, and administration of a loading dose of P2Y12 receptor inhibitors, or fibrinolytic agents within 7 days before enrollment. The study protocol was approved by the Ethics Committee of Yokohama City University. Written, comprehensive, informed consent was given by all patients.

Collection of Blood Samples
Blood samples were collected as described in detail previously. 8 Briefly, blood samples were collected from the antecubital vein with a 21-gauge needle into the following tubes: hirudin-containing blood sampling tube (MP0600 [Verum Diagnostica]; final concentration of hirudin 25 μg/mL); blood collection tubes (VP-CA050K70, Venoject II; Terumo), and syringe containing 0.11 mL of 3.8% sodium citrate solution. Whole-blood samples were kept at room temperature for 1 h, and thereafter thrombus formation was measured with T-TAS.
All blood samples of patients receiving DOACs were obtained 2-4 h after taking DOACs, and those of patients receiving vitamin K antagonists were obtained after warfarin doses were fixed in the steady state.

Measurement of Thrombogenicity Using T-TAS
T-TAS is an automated, microchip-based flow chamber Adjusted Oral Vitamin K Antagonist Treatment Strategy in Subjects with Atrial Fibrillation who Undergo Percutaneous Coronary Intervention) trial demonstrated that lowdose rivaroxaban plus single-antiplatelet therapy and very-low-dose rivaroxaban plus dual-antiplatelet therapy could significantly lower the risk of bleeding complications compared with conventional triple therapy with warfarin and dual-antiplatelet therapy. 4 However, both trials showed that about 14-15% of patients receiving singleantiplatelet therapy plus anticoagulant agents had bleeding complications requiring medical attention. There may be an inter-individual variability in the response to antithrombotic agents, so assessment of antithrombotic effects is desirable to avoid bleeding complications, especially in patients receiving multiple antithrombotic agents. However, it is difficult to monitor the total antithrombotic effect using conventional methods, such as prothrombin time-international normalized ratio (PT-INR), activated partial thromboplastin time (aPTT), and platelet function tests in patients receiving multiple antithrombotic agents.
Recently, the Total Thrombus-formation Analysis System (T-TAS; Fujimori Kogyo Co Ltd., Japan), a microchip-based flow chamber system that can evaluate whole-blood thrombogenicity, was developed as an easyto-use system for quantitative analysis of thrombus formation. 5,6 A previous study reported that quantitative analysis of thrombus formation with T-TAS using the AR-chip was potentially useful for predicting bleeding events in AF patients undergoing catheter ablation. 7 Thus, the aim of the present study was to evaluate the relationship between quantitative analysis of antithrombotic effects with T-TAS and subsequent bleeding complications in patients receiving multiple antithrombotic agents.

Subjects
Between September 2013 and May 2017, quantitative analysis of antithrombotic effects, using T-TAS, was performed Inside the microchip, platelets adhere and aggregate on the surface of the collagen, and microchip capillaries are occluded. The AR-chip contains a single capillary channel (width, 300 µm; depth, 80 µm) coated with type I collagen plus tissue thromboplastin. Inside the microchip, activation of the platelets and the coagulation system is triggered simultaneously by collagen system developed for easy and quick assessment of thrombus formation under shear stress conditions as previously described. 5,8,9 Briefly, this system is equipped with a rectangular capillary, a pneumatic pump, and a flow pressure sensor. Thrombus formation is analyzed by continuous monitoring of the flow pressure change resulting from capillary occlusion. Thrombus formation is evaluated with 2 types of microchips: a PL-chip and an AR-chip. The PL-

Distribution of T-TAS Parameters
De-escalation from triple therapy to dual therapy was and tissue thromboplastin, respectively. The area under the flow pressure curve (AUC) was analyzed to assess thrombogenicity inside the microchip. The AUC for the first 10 min for the PL-chip at a flow rate of 18 µL/min is described as PL18-AUC10, and the AUC for the first 30 min for the AR-chip at a flow rate of 4 µL/min was described as AR4-AUC30. These pressure-time integrals were used to quantify the overall stability of thrombus. Lower PL18-AUC10 or AR4-AUC30 values presumably reflect slow or reduced thrombus growth, or alternatively a rapid breakdown of thrombus. 9

Clinical Outcomes
Prognostic information was obtained using electronic medical records or telephone interviews. Bleeding complications were defined as type 1, 2, 3, or 5 bleeding, according to the Bleeding Academic Research Consortium (BARC) definitions. 10 Major adverse cardiovascular events (MACE) were defined as death from cardiac cause, myocardial infarction, or ischemic stroke.  Table 1. Total Effect of Multiple Antithrombotic Agents found in 37 patients during the follow-up periods (median time of de-escalation was 85 days). However, escalation from dual therapy to triple therapy was not observed. Bleeding events occurred in 43 patients during an average 22-month follow-up. The definition of BARC 1, 2, and 3 was met 16 (37%), 18 (42%), and 9 (21%), respectively, of those patients with bleeding events. Figure 1 shows the distribution of AR4-AUC30 (median, 967; IQR, 491-1,215) and PL18-AUC10 (median, 131; IQR, 51-188) in all study patients. The effects of antithrombotic agents assessed using T-TAS widely varied among individuals. There was a weak correlation between AR4-AUC30 and PL18-AUC10 (r=0.17, P=0.043).

Discussion
To our knowledge, this is the first study to assess whether low total thrombogenicity increases the risk of bleeding complications in patients with CAD who receive multiple antithrombotic agents (combination of oral anticoagulants and antiplatelet drugs). In the present study, whole-blood thrombogenicity was assessed using T-TAS; results were expressed as AR4-AUC30. The effects of antithrombotic agents assessed using the AR-chip varied widely among individuals, and patients with bleeding complications had low on-treatment thrombogenicity compared with those without. In addition, AR4-AUC30 was an independent predictor of subsequent bleeding events. However, PL18-AUC10, conventional coagulation tests, including aPTT and PT-INR, P2Y12 receptor inhibitors use, and triple therapy were not significant predictors of bleeding events. Currently, available coagulation assays (e.g., rotational thromboelastometry, thromboelastography) are mostly performed under static conditions, so the complex interactions among erythrocytes, platelets, white blood cells, and coagulation factors under various shear rates cannot be appreciated. Other systems with high shear rates, including the Platelet Function Analyzer and the Cone and Plate analyzer are available to assess platelet adhesion and aggregation; however, their results do not provide information beyond primary hemostasis. 11 In addition, the present study showed that PL18-AUC10 as the result of platelet adhesion and aggregation tests was not related to clinical outcomes. In contrast, the AR-chip, which is novel and easy to use for quantitative monitoring of the levels of thrombogenicity under a whole-blood flow state, is a potential tool for the assessment of platelet activation and

Predictors of Bleeding Events
Univariate logistic regression analysis demonstrated that baseline anemia, prasugrel use, usage of DOACs, usage of the standard dose of DOACs, and AR4-AUC30 were related to bleeding events ( Table 2). However, aPTT, PT-INR, PL18-AUC10, and triple therapy (combination of dualantiplatelet therapy and oral anticoagulants) were not significantly related to bleeding events. Multivariate logistic regression analysis identified AR4-AUC30, usage of the standard dose of DOACs, and baseline anemia as significant predictors of bleeding events ( Table 3).

Impact of Thrombogenicity Measured With T-TAS on Clinical Outcomes
AR4-AUC30 was significantly lower in patients with bleeding events than in those without (584 [96-993] vs. 1,028 [756-1,252], P=0.0003) (Figure 2). Figure 3 shows the incidence of patients with bleeding events across AR4-AUC30 quartiles. Approximately three-quarters (73%) of patients with bleeding events were in the first and second AR4-AUC30 quartiles.
Study patients were also classified into 2 groups according to median AR4-AUC30 level: low thrombogenicity group (Gr-L, n=72) and high thrombogenicity group (Gr-H, n=73). Patients' characteristics and blood examinations in the 2 groups are shown in Supplementary Table. At 22 months, the incidence of bleeding events was significantly higher in Gr-L than in Gr-H (38% vs. 16%, P=0.0007) (Figure 4A). MACE occurred in 8 patients during follow-up; however, there were no significant differences in the incidence of MACE between the 2 groups (3% vs. 5%, P=0.23) (Figure 4B). According to the median PL18-AUC10, there were no significant differences in the incidence of bleeding events (32% vs. 22%, P=0.18) and MACE (3% vs. 5%, P=0.37) between the lower PL18-AUC10 relatively small number of patients. Thus, the sample size was too small to evaluate ischemic outcomes. Further, large population studies are needed to evaluate the relationship between AR4-AUC30 measured with T-TAS and rates of ischemic complications. Second, we did not obtain baseline AR4-AUC30 values before the administration of any antithrombotic agents in our study. However, AR4-AUC30 levels in the present study were lower than those in healthy volunteers in previous reports. 5 In addition, we did not evaluate serial measurements of peak and trough values in patients receiving DOACs. However, all blood samples from patients receiving DOACs were obtained 2-4 h after taking DOACs, which were estimated as the peak values, and those of patients receiving vitamin K antagonists were obtained after warfarin doses were fixed in the steady state. It remains unclear whether the peak or trough point of blood sampling is more strongly related to bleeding complications in patients receiving DOACs. 27-30 Moreover, we did not assess the plasma concentrations of DOACs, anti-Xa activity, or the relationship between T-TAS parameters and the concentrations of DOACs or anti-Xa activity. Furthermore, there was no significant difference in the frequency of triple therapy between patients with and without bleeding complications. This finding might be related to the low intensity of vitamin K antagonist therapy and frequent use of underdosing of DOACs in patients treated with triple therapy. As shown in the present study, there is inter-individual variability in the intensity of oral anticoagulants plus antiplatelet agents. Another explanation is that some patients with triple therapy were switched to dual therapy. Finally, 37% of patients with bleeding complications had minimal bleeding. Despite these limitations, the results of the present study are expected to contribute to bleeding risk stratification in CAD patients receiving multiple antithrombotic agents.

Conclusions
Total effects of antithrombotic agents measured with T-TAS widely varied among individuals. The AR4-AUC30 level was an independent and significant marker for predicting subsequent bleeding complications in stable CAD patients treated with a combination of oral anticoagulants and antiplatelet agents.
the coagulation system in patients receiving various types of antithrombotic therapy. 12,13 It has been demonstrated that patients receiving multiple antithrombotic agents have higher rates of bleeding complications, and patients with bleeding complications are associated with poor clinical outcomes compared with those without bleeding complications. 14 Therefore, guidelines recommend that duration of triple therapy should be minimized according to bleeding and ischemic risks. 15 Although approximately 6-8% of patients undergoing PCI have an indication for long-term oral anticoagulants for various conditions such as AF, mechanical heart valves, or venous thromboembolism, these patients should be considered at high risk of bleeding, and the indication for oral anticoagulants or antiplatelet therapy should be reassessed and treatment continued only if a compelling indication exists (e.g., high CHADS2 score, presence of mechanical heart valves, a history of recurrent venous thromboembolism, complexity of treated CAD, or a history of stent thrombosis). 15 However, the optimal strategy to balance prevention of thrombosis with the risk of bleeding is unclear. 16, 17 The dose intensity of vitamin K antagonist should be carefully monitored, with the PT-INR in the lower part of the recommended target range. In patients receiving DOACs, it is difficult to monitor the antithrombotic effect. 18-21 Our study showed that usage of the standard dose of DOACs as part of multiple antithrombotic therapy was a significant predictor of bleeding events. Some studies suggest a low dose of DOACs as part of a combination of antiplatelet therapy has a favorable outcome, although those studies were underpowered for the assessment of meaningful differences in the incidence of relevant ischemic events. 4,15, 22 In addition, the use of prasugrel or ticagrelor as part of triple therapy should be avoided. 23 Recent studies have prompted efforts to seek new therapeutic strategies. 24-26 Three new promising approaches have emerged to reduce the risk of bleeding among patients for whom oral anticoagulants and antiplatelet therapy are indicated. 3,4, 22 These studies demonstrated that a combination of oral anticoagulants and single-antiplatelet therapy was associated with a lower rate of clinically significant bleeding than was standard therapy with oral anticoagulants plus dual-antiplatelet therapy in patients with an indication for oral anticoagulants who underwent PCI. However, clinically significant bleeding complications occurred in approximately 15% of study patients even in the dual-therapy groups (oral anticoagulants+single-antiplatelet therapy) within the first year of treatment. 3,4,22 Therefore, a useful methodology for assessing the effects of multiple antithrombotic agents is desirable to avoid bleeding complications.
Ito et al report that thrombogenicity measured with T-TAS using the AR-chip is useful for assessing the efficacy of vitamin K antagonists and DOACs in AF patients who have undergone catheter ablation and that this marker could be useful for predicting periprocedural bleeding complications. 7 In the present study, AR4-AUC30 was a potentially useful marker of future bleeding events in patients with CAD receiving oral anticoagulants plus single-or dual-antiplatelet therapy, although conventional laboratory data such as aPTT and PT-INR were not.

Study Limitations
First, this single-center, observational study included a