Study design

This prospective study was performed at 1st Chair and Department of Cardiology, Medical University of Warsaw, Poland in collaboration with Department of Coronary Artery Disease and Heart Failure, Institute of Cardiology, Jagiellonian University Medical College at St. John Paul II Hospital, Kraków, Poland. A complete assessment of thrombin generation and clot lysis were conducted in the Krakow Center for Medical Research and Technologies at the St. John Paul II Hospital, Kraków.

The design and protocol of the study was compliant with Declaration of Helsinki and was approved by Ethics Committee of the Medical University of Warsaw (Approval Number: KB/128/2018, KB/4/A2021). Informed consent was obtained from all study participants.

Study participants

We enrolled 135 patients with advanced symptomatic AS who were qualified for elective TAVI as per local Heart Team recommendation. Severe AS was defined as aortic valve area (AVA) < 1.0 cm2 or an indexed AVA < 0.6 cm2/m2, calculated using the continuity equation on transthoracic echocardiography (TTE) [1, 4]. The indication for TAVI was determined in accordance with the current guidelines of the European Society of Cardiology (ESC) [4]. It was determined that patients qualified for TAVI had an elevated surgical risk, defined as at least 4% in EuroScore II or STS calculator [4]. To distinguish between severe and pseudo-severe AS, the following tests were additionally performed if needed: (1) dobutamine stress echocardiography in low-flow, low-gradient, reduced left ventricular ejection fraction (LVEF) patients and (2) computed tomography with aortic valve calcium score assessment in low-flow, low-gradient, preserved LVEF patients. Inclusion and exclusion criteria are listed in Table 1.

Table 1 Inclusion and exclusion criteria in the studyTAVI procedure

A multidetector computed tomography angiography (CTA) was used to select the size and approach for transcatheter aortic valves. TAVI procedures were performed using femoral access in a hybrid operating room. The procedure was performed by an interventional cardiologist in collaboration with a cardiac surgeon. Following TAVI, single antiplatelet therapy (mostly acetylsalicylic acid) was administered in patients with no indication for oral anticoagulation (OAC) [1]. If the patient had indications before TAVI to take dual antiplatelet therapy (DAPT), such therapy was maintained as recommended.

Sample collection and routine laboratory tests

During hospitalization, blood samples were collected at two time points: (1) one day before the TAVI procedure, and (2) 5–7 days after (at hospital discharge). A fasting blood sample of 25 ml was collected from the antecubital vein and immediately preserved in tubes containing 3.2 trisodium citrate. Sample processing took place within 60 min of blood collection.

The Cobas 8000 CC/c702 + c502 Roche and Dimension EXL Siemens was used to analyze serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, glucose, glycated hemoglobin (HbA1c), and high sensitivity C-reactive protein (hsCRP). Creatinine levels were determined and estimated glomerular filtration rate (eGFR) calculated using the chronic kidney disease epidemiology collaboration (CKD-EPI) formula. A complete blood count (CBC), including red and white blood cell count, hemoglobin, hematocrit, red cell distribution width, platelet distribution width, and platelet count, was assessed with Yumizen H 2500 Horiba and XN2000 Sysmex.

Laboratory investigations of coagulation parameters

Venous blood was obtained from the antecubital vein between 7:00 and 09:00 AM using citrated tubes (9:1 ratio of 0.106 M sodium citrate; Monovette, Sarstedt, Nümbrecht, Germany). Subsequently, the collected blood underwent centrifugation at 2500 g and 20 °C for 20 min to obtain platelet-poor plasma. This plasma was rapidly snap-frozen within 30 min of collection and stored in aliquots at − 80 °C until analysis.

Thrombin generation

Thrombin generation kinetics were assessed using the Calibrated Automated Thrombogram (CAT, Thrombinoscope BV, Maastricht, the Netherlands) following the manufacturer’s instructions. The measurements were conducted in a 96-well plate fluorometer (Ascent Reader, Thermolabsystems OY, Helsinki, Finland) equipped with the 390/460 filter set at a temperature of 37 °C. In summary, 80 µL of platelet-poor plasma were diluted with 20 µL of a reagent containing 5 pM recombinant tissue factor (TF), 4 micro-molar phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine vesicles, and 20 µL of FluCa solution (HEPES, pH 7.35, 100 nM CaCl2, 60 mg/mL bovine albumin, and 2.5 mM Z-Gly-Gly-Arg-7-amino-4-methylcoumarin). The Peak thrombin represents the maximum concentration of thrombin formed during the recording time, while the area under the curve corresponds to the endogenous thrombin potential (ETP). Lag time demonstrates the initiation phase of coagulation, and time to peak (ttPeak) signifies the propagation phase of thrombin generation. Each plasma sample underwent duplicate analysis, and the intraassay variability was determined to be 7%.

Plasma clot lysis assay

The fibrinolysis capacity was assessed utilizing a clot lysis time (CLT) assay recommended by the International Society on Thrombosis and Haemostasis (ISTH) Subcommittee, as detailed previously [20]. In brief, citrated plasma was combined with 15 mM calcium chloride, 0.5 U/mL human thrombin (Merck), 15 µM phospholipid vesicles (Rossix, Mölndal, Sweden), and 18 ng/mL recombinant tissue plasminogen activator (rtPA, Boehringer Ingelheim, Germany). Subsequently, the mixture was transferred to a microtiter plate, and its turbidity was measured at 405 nm at a temperature of 37 °C. CLT is defined as the time interval between the midpoints of the clear-to-maximum-turbid transition and the midpoints of the maximum-turbid-to-50% clear transition (50% lysis). Lysis variables had intraassay coefficients below 8%.

Echocardiographic evaluation

Full echocardiographic evaluation was performed in all studied subjects. Precise measurements of aortic valve including maximal velocity through aortic valve (Vmax), aortic valve area (AVA), aortic valve area indexed for body surface area (AVAi), maximal and mean aortic gradient, left ventricular ejection fraction. The examination was performed be an experienced cardiologist. All scans were performed with a Philips Sparq ultrasound machine (Philips Healthcare, Andover, MA) using a phased-array transducer probe (2–4 MHz), with additional analysis using Philips Q-Station 3.3.2.

Endpoints

The primary endpoint was the change in (1) fibrin clot properties, and (2) thrombin generation before vs. after TAVI. The secondary endpoint was the predictive value of baseline (1) thrombin generation and (2) clot lysis time for changes in aortic valve flow parameters, namely: AVA, AVAi, maximal or mean aortic gradient.

Statistical analysis

Since there are no data regarding the differences in thrombin generation, fibrin clot properties and clot susceptibility to lysis before and after TAVI, power calculation for the primary endpoint was based on the differences in fibrin clot properties in patients scheduled for TAVI, compared to those undergoing SAVR [21]. Patients scheduled for TAVI had, on average, a 0.1-fold higher clot density, compared to SAVR patients. The required sample size was calculated by a 2-sided t-test at a significance level of 0.05 with the following assumptions: (i) mean difference between the groups = 1.0, (ii) standard deviation (SD) ± 2.0, and (iii) nominal test power = 0.8. It was estimated that a total of 64 patients should be enrolled in the study to observe a difference in clotting parameters before and after TAVI.

Categorical variables are presented as number and percent. The Shapiro–Wilk test was used to assess normal distribution of continuous variables. Continuous variables were presented as mean and SD or median with interquartile range (IQR). Spearman rank correlation coefficients were calculated for associations between continuous variables with non-normal distributions. Univariable linear regression analyses demonstrated relationships between clinical variables and blood coagulation variables. Paired t-test of Wilcoxon signed rank test was used for repeated measures analysis, between two analysed timepoints. A 2-sided p-value below 0.05 was considered significant. Statistical analysis was conducted using GraphPad Prism version 8.0.1 (San Diego, CA, USA) and IBM SPSS ver. 28.0 (Armonk, NY, USA).