Utility of the Total Thrombus-Formation Analysis System as a Tool for Evaluating Thrombogenicity and Monitoring Antithrombotic Therapy in Pediatric Fontan Patients

There is no consensus regarding thromboprophylaxis after Fontan procedure, and novel tools to assess thrombogenicity are needed to establish optimal thromboprophylaxis. The Total Thrombus-formation Analysis System (T-TAS) was developed for the quantitative analysis of thrombus formation using microchips with thrombogenic surfaces. This prospective study evaluated the utility of T-TAS in the assessment of thrombogenicity in pediatric Fontan patients. The participants included 20 consecutive Fontan patients who underwent cardiac catheterization and 30 healthy controls. Blood samples collected without and with antithrombotic therapy (aspirin or aspirin and warfarin) were used for T-TAS to compute the area under the curve (AUC) in the atheroma (AR10-AUC30) and platelet (PL18-AUC10) chips. A higher AUC indicates higher thrombogenicity. T-TAS values showed that patients in the Fontan group without antithrombotic therapy had lower thrombogenicity than those in the control group [PL18-AUC10, median (interquartile range) 356 (313–394) vs. 408 (392–424); AR10-AUC30, median (interquartile range) 1270 (1178–1351) vs. 1382 (1338–1421)]. Aspirin and warfarin therapies significantly decreased PL18-AUC10 and AR10-AUC30, respectively, compared with those of patients without antithrombotic therapy (P < 0.001 for each comparison). Subgroup analysis divided by low (< 9 mmHg) or high (≥ 9 mmHg) central venous pressure (CVP) showed that CVP affects the reduction in AR10-AUC30 with antithrombotic therapy. T-TAS may be a useful tool for monitoring thrombogenicity and antithrombotic therapy in Fontan patients.


Introduction
The Fontan procedure is the final palliative operation commonly performed in children with single ventricle physiology. Thromboembolism is one of the most important complications in children after this procedure. The prevalence of thromboembolism after the Fontan surgery ranges from 17 to 33%, with a mortality rate of 25% [1,2].
The current clinical guidelines suggest that thromboprophylaxis after the Fontan procedure is adequate to reduce the risk of thromboembolism. However, these guidelines emphasize a need for further studies to gather sufficient evidence to determine optimal antithrombotic therapy [3,4]. In children, thromboprophylaxis after the Fontan procedure is typically performed with antiplatelet (aspirin) and anticoagulation (warfarin) therapies. A previous randomized controlled trial found no significant difference between these two drugs in the Fontan postoperative group [5]. The use of direct oral anticoagulant (DOAC) therapy after the Fontan procedure may be a promising alternative for thromboprophylaxis; however, there are insufficient data to recommend DOACs. Moreover, in Japan, bleeding-related complications are common with antithrombotic therapy, especially anticoagulation with warfarin [6].
The risk of thromboembolism after Fontan surgery increases over time and is expected to be lower in children than in adults. Abnormalities of various coagulation and fibrinolytic factors have been reported after Fontan surgery, making it difficult to assess the thrombogenicity in individual patients using conventional tests [7][8][9][10]. Hence, novel tools for monitoring the thrombogenicity and efficacy of antithrombotic therapy are needed to establish optimal antithrombotic therapy.
The Total Thrombus-formation Analysis System (T-TAS; Fujimori Kogyo Co Ltd., Japan) is an automated flow chamber system using microchips for simple analysis of the thrombus formation process in whole blood samples under blood flow mimicking in vivo conditions [11]. Several studies have shown that T-TAS is a useful diagnostic tool for monitoring therapy or predicting bleeding events [12][13][14][15].
Thus, we hypothesized that T-TAS would be a useful index for assessing thrombogenicity and the efficacy of antithrombotic therapy in patients after the Fontan procedure. This study investigated whether T-TAS is useful in assessing thrombogenicity and antithrombotic therapy in pediatric Fontan patients.

Study Population and Protocol
This observational study enrolled 21 consecutive Fontan patients who underwent cardiac catheterization at Kumamoto University Hospital from October 2020 to September 2021. All patients had received two-drug antithrombotic therapy with aspirin (4 mg/kg) and warfarin. Warfarin was controlled with a target prothrombin time international normalized ratio (PT-INR) of 1.5-2.0.
We excluded patients with postoperative periods of < 1 and > 10 years, a history of thrombosis or arrhythmias, and moderate or severe atrioventricular valve regurgitation. We excluded one patient with a postoperative period of > 10 years, and the remaining 20 patients were included in the present study (details of the Fontan group are shown in Table 1). Since there were no pediatric T-TAS reference values, we recruited a control group of 30 children with no organic abnormalities and no family history of thrombosis. The children in the control group had visited our hospital after an abnormal ECG or heart murmur was noted during school or medical examinations (Fig. 1a).
All procedures were conducted in accordance with the Declaration of Helsinki and its amendments. The study protocol was approved by the human ethics committee of Kumamoto University, and written informed consent was obtained from each patient's family.

Hemodynamics Evaluation by Cardiac Catheterization
Intracardiac and great vessel pressures, including central venous pressure (CVP), were measured. Oxygen consumption was estimated using age, sex, and heart rate; cardiac index (CI; l/min/m 2 ) was measured using Fick's principle. Systemic arterial resistance (Rs) and pulmonary arterial resistance (Rp) were also calculated.

Anticoagulation Regimen and Collection of Blood Samples
According to the regular clinical protocol, aspirin and warfarin were discontinued for 14 days prior to cardiac catheterization in the Fontan group. Aspirin was resumed the day after cardiac catheterization. Warfarin was resumed 3 weeks after cardiac catheterization. Blood samples were obtained at the time of catheterization (drug-free point), 3 weeks after the resumption of aspirin (on aspirin), and 1 month after the resumption of warfarin (on aspirin and warfarin) (Fig. 1b).
Blood samples were collected from the antecubital vein using a 21-gauge butterfly needle. Whole blood samples for the T-TAS assay were kept at room temperature for 1-4 h before the T-TAS assay. The blood samples for the biomarker assay were prepared immediately after collection: plasma samples were centrifuged at 2000 g for 10 min at 4 °C and serum samples were centrifuged at 3000 rpm for 5 min at room temperature.

Measurement of Thrombogenicity Using T-TAS
T-TAS is an automated microchip-based flow chamber system developed for easy and rapid assessment of platelet thrombus formation under flow conditions [11][12][13][14].
Briefly, using a simple procedure, this system analyzes different thrombus-forming processes using two disposable microchips with different thrombogenic surfaces.
A whole blood sample (330 μL) anticoagulated with BAPA was applied to one of the chips [a platelet chip (PL-chip) coated with type I collagen] at a flow rate of 18 μL min −1 , corresponding to an initial wall shear rate of 1500 s −1 . Inside the microchip, platelets adhere and aggregate on the surface of collagen, occluding the microchip capillaries. The other chip, the atheroma chip (AR-chip), is coated with type I collagen plus tissue thromboplastin. A whole blood sample collected into 3.2% sodium citrate was mixed with CaCl 2 and corn trypsin inhibitor immediately before the assay. The sample was then applied into the AR-chip at a flow rate of 10 μL min −1 , corresponding to an initial wall shear rate of 600 s −1 . Inside the microchip, activation of both the platelets and coagulation system is triggered simultaneously by collagen and tissue thromboplastin, respectively. The area under the flow pressure curve (AUC) was computed to assess platelet thrombogenicity inside the microchips, and a higher AUC indicates higher thrombogenicity. The PL 18 -AUC 10 parameter represents the AUC for the first 10 min for the PL-chip, tested at a flow rate of 18 μL min −1 . AR 10 -AUC 30 represents the AUC for the first 30 min for the AR-chip, tested at a flow rate of 10 μL min −1 .

Statistical Analysis
No information was missing in the dataset of all participants who met the inclusion criteria and not the exclusion criteria. To describe the characteristics of the enrolled participants of the current study, data were summarized as median and interquartile range (IQR) for continuous variables and number and percentage for categorical variables. Correlation of parameters was evaluated using Spearman's rank correlation coefficient. Next, to evaluate the treatment effect of aspirin and/or warfarin on parameters of blood coagulation, linear mixed effect models were applied using random effects by participant. In this model, the variance-covariance structure of the random effects was set as unstructured. In the subgroup analysis, interactions were evaluated using likelihood ratio tests. Fitting for each mixed model was visually examined using a standardized normal probability plot. All statistical analyses were performed using Stata 15.0 (Stata-Corp, College Station, TX, USA). All P values were twotailed, and P < 0.05 was considered statistically significant.

T-TAS Parameters and Baseline Characteristics
We compared the control group with the Fontan group at the time of catheterization (without treatment with aspirin or warfarin). The baseline characteristics of the control and Fontan groups are shown in Table 2. Age and sex were not

Laboratory Data and Hemodynamic Parameters Affecting T-TAS Parameters in Fontan Patients
Further, we analyzed factors affecting the T-TAS parameters in Fontan patients not treated with antithrombotic therapy. The platelet count was significantly correlated with PL 18 -AUC 10 and AR 10 -AUC 30 . Other CBC and biomarkers tested were irrelevant. Regarding hemodynamic parameters, PL 18 -AUC 10 was negatively correlated with CVP and positively correlated with Rs (Table. 4).

Effects of Aspirin and Warfarin on T-TAS Parameters, APTT, and PT-INR
To evaluate the utility of PL 18 -AUC 10 and AR 10 -AUC 30 measured by T-TAS, we compared the parameters with and without antithrombotic therapy in Fontan patients. Serial changes in T-TAS parameters (PL 18 -AUC 10 and AR 10 -AUC 30 ) and APTT and PT-INR with no antithrombotic therapy, aspirin therapy, and aspirin and warfarin combination therapy are shown in Fig. 3a

CVP Impacts the Reduction of AR 10 -AUC 30 with Antithrombotic Therapy
Subsequently, we next examined the association between T-TAS parameters and CVP and CI, considering the possibility that the effects of elevated CVP and decreased cardiac output, which are characteristics of the Fontan circulation, on the coagulation system are associated with a risk of hemorrhagic complications due to antithrombotic therapy. Negative correlations were found between the reduction in AR 10 -AUC 30 and CVP (rho − 0.7686, P < 0.001). Therefore, we focused on AR 10 -AUC 30 (Fig. 3f). However, there were no significant differences in the reduction in PL 18 -AUC 10 (Fig. 3e) , APTT (Fig. 3g), and PT-INR (Fig. 3h).

Discussion
The main feature of the present study was the assessment of whole blood thrombogenicity using T-TAS in pediatric Fontan patients. The main findings were as follows: (1) pediatric Fontan patients had less propensity for thrombogenicity than controls, as assessed by T-TAS; (2) PL 18 -AUC 10 and AR 10 -AUC 30 could be useful markers for monitoring the antithrombotic effects of aspirin and warfarin; and (3) the degree of reduction in AR 10 -AUC 30 with antithrombotic therapy may reflect the hemodynamics of Fontan patients, i.e., the impact of elevated CVP on the coagulation system. To the best of our knowledge, this is the first report to describe the utility of T-TAS as a tool for evaluating thrombogenicity and monitoring antithrombotic therapy in Fontan patients.
The Fontan circulation presents a thrombotic environment and associated complex pathophysiology affecting all three elements of Virchow's triad: endothelial cell dysfunction, abnormal blood flow, and increased coagulability [6,7].  10 , area under the curve for the first 10 min for the platelet chip tested at a flow rate of 18 µL/min; AR 10 -AUC 30 , area under the curve for the first 30 min for the atheroma chip tested at a flow rate of 10 µL/min WBC white blood cell; Hb hemoglobin; Hct hematocrit; PLT platelet count; T-bil total-bilirubin; AST aspartate aminotransferase; ALT alanine aminotransferase; γGTP γ-glutamyl transpeptidase; BUN urea nitrogen; Crea creatinine; BNP brain natriuretic peptide; APTT activated partial thromboplastin time; PT-INR PT international normalized ratio; FIB fibrinogen; ATIII antithrombin III; TAT thrombinantithrombin complex; PIC α2-plasmin inhibitor complex; F factor; vWF von Willebrand factor; TM thrombomodulin; CI cardiac index: CVP central venous pressure; Rs systemic artery resistance: Rp pulmonary artery resistance; SaO 2 arterial oxygen saturation PL 18  Several studies have reported hypercoagulability caused by coagulation factor abnormalities and platelet activation after Fontan surgery. The risk of thromboembolism is also expected to increase as endothelial and multiorgan damage associated with congestion progresses over time [1,16,17]. Few pediatric cohort studies have investigated thromboembolic phenomena in Fontan circulation, and our knowledge of coagulopathy in pediatric Fontan patients is limited.
The main advantage of the T-TAS system is that it assesses thrombus formation under flow conditions using whole blood samples. Thus, to some extent, T-TAS reflects physiological conditions [11,18]. Some studies have also demonstrated the usefulness of T-TAS in detecting abnormal coagulation and platelet function [13,[19][20][21]. Therefore, we believe that T-TAS is suitable for evaluating thrombogenicity in Fontan patients with abnormal blood flow, various coagulation factor abnormalities, and platelet activation.
The present study found that pediatric Fontan patients showed a low propensity for thrombus formation. This result is supported by reports of a decreased risk of thromboembolism within 1 year and after 10 years of Fontan surgery, as well as reports of more bleeding events with antithrombotic therapy in children [1,6].
Using thromboelastography, Leslie et al. also did not find hypercoagulability in pediatric Fontan patients [22]. T-TAS can quantitatively measure the thrombogenic process under physiological flow conditions and is sensitive enough to detect abnormalities in platelet and coagulation function. This suggests that T-TAS may be more suitable for assessing thrombogenic potential in specific Fontan circulation.
In this study, we examined factors affecting T-TAS parameters in pediatric Fontan patients and found a positive correlation between platelet counts and T-TAS parameters. In previous reports, the platelet count was significantly related to the AUC of T-TAS in healthy participants [23,24]. Since Fontan patients tend to have low platelet counts due to factors such as splenomegaly caused by elevated portal pressure, the AUC may be low simply due to low platelet counts. Therefore, careful attention should be paid to the evaluation of thrombogenicity by T-TAS when platelet counts are extremely low.
Reportedly, in a good Fontan circulation, CVP decreases and Rs increases during childhood, and perfusion pressure is maintained [25]. In the current study, increasing PL 18 -AUC 10 was positively correlated with lower CVP and increased Rs, suggesting that adequate primary hemostatic performance may be maintained in these patients with preserved good perfusion pressure of the Fontan circulation.
This study shows that the antiplatelet effects of aspirin and anticoagulant effects of warfarin can be monitored by T-TAS, as reported in adult patients with cardiovascular diseases. These results suggest that T-TAS can be used to evaluate aspirin resistance, which is often a problem in thromboprophylaxis in postoperative Fontan patients [26,27]. Moreover, T-TAS is reportedly a useful monitoring tool for DOACs [14,15]. DOACs have recently been considered for thromboprophylaxis in Fontan patients [28]. We believe that T-TAS could be useful for monitoring this patient population.
In addition, we found a significant association between a reduction in AR 10 -AUC 30 and CVP due to anticoagulation therapy. The Fontan circulation results in a low flow rate condition due to elevated venous pressure driving pulmonary arterial blood flow. A reduced flow rate propagates the initiation of the coagulation system and increases the likelihood of thrombus formation. Hepatic dysfunction and protein-losing enteropathy due to elevated CVP may further compound the coagulation abnormalities and provide an additional mechanism for the dysregulation of hemostasis [29][30][31]. AR 10 -AUC 30 can evaluate the reduction in thrombogenicity by anticoagulation reflecting Fontan hemodynamics, which is not observed by PT-INR or APTT. This suggests that assessment by T-TAS may be suitable for risk stratification of bleeding complications associated with thromboprophylaxis, which may reflect the hemodynamic impact on the coagulation system in Fontan patients.
This study had some limitations. First, this single-center observational study had a small sample size which could lead to an overestimation of the results. Second, the clinical outcomes (thrombotic and bleeding events) associated with antithrombotic therapy have not been assessed. Finally, as previously reported, there are individual differences in T-TAS parameters [23]. To better assess thrombogenicity by T-TAS, multiple evaluations in the same individual are desirable. Further large population studies are needed to examine the relationship between T-TAS parameters and clinical outcomes in Fontan patients and to establish optimal antithrombotic therapy.
In conclusion, the present study demonstrated that T-TAS may be a useful tool for monitoring thrombogenicity in Fontan patients. AR 10 -AUC 30 and PL 18 -AUC 10 measured by T-TAS are potentially suitable indices for the assessment of antithrombotic therapy in Fontan patients. Fig. 3 Effect of aspirin and warfarin therapy on parameters related to blood coagulation in pediatric Fontan patients (a-d) Time-course changes of a PL 18 -AUC 10 , b AR 10 -AUC 30 , c APTT (sec), and d PT-INR in children with aspirin therapy and subsequent warfarin therapy compared with those of drug-free children. P values were calculated for comparisons between aspirin/warfarin therapy and the drug-free state. e-h Subgroup analysis divided by low (< 9 mmHg) or high (≥ 9 mmHg) CVP. Time-course changes of e PL 18 -AUC 10 , f AR 10 -AUC 30 , g APTT (sec), and h PT-INR in the pediatric Fontan patients treated with aspirin and subsequent warfarin therapy. P values were evaluated in comparisons between the low and high CVP groups in each therapy condition. The red and blue lines indicate the high and low CVP in pediatric Fontan patients, respectively. PL 18 -AUC 10 is defined as the area under the curve (AUC) for the first 10 min for the platelet chip tested at a flow rate of 18 μL/min. AR10-AUC30 is defined as the AUC for the first 30 min for the platelet chip tested at a flow rate of 10 μL/min. APTT, activated partial thromboplastin time; PT-INR, PT international normalized ratio; CVP, central venous pressure ◂