In the current study we assessed 79 non-cancer and hemodynamically stable PE patients recruited from December 2016 to March 2021 and described in detail previously [9, 18]. PE was diagnosed based on the occurrence of typical clinical symptoms confirmed by computed tomography angiography (angio-CT). The simplified PE severity index (sPESI) was assessed initially in all patients [19]. Invasive evaluation of pulmonary pressure was not performed in any subject due to the low probability of chronic thromboembolic pulmonary hypertension, (CTEPH) on transthoracic echocardiography (TTE) [20]. DVT was diagnosed within the first 48 h since enrolment based on a positive finding of color duplex sonography. Provoked VTE was diagnosed if a patient had surgery requiring general anesthesia, major trauma, plaster cast or hospitalization in the past month, pregnancy or delivery in the past 3 months. RV dysfunction and comorbidities were defined as described previously [18].

RPVO was defined as residual perfusion defects on control computed tomography angiography performed after 3–6 months of anticoagulation [1]. Post-PE syndrome, diagnosed at 6 months since the index PE event, was defined by persistent dyspnea reported at 3 and 6 months since the event (New York Heart Association [NYHA] class II or more) and impaired exercise capacity using the respective reference values [9]. The Jagiellonian University Medical College Ethical Committee approved the study, and participants provided written informed consent in accordance with the Declaration of Helsinki.

All subjects were evaluated on admission before initiation of anticoagulant therapy and after 5–7 days. Blood samples were drawn from an antecubital vein with minimal stasis. Blood cell count, glucose, fibrinogen, high-sensitivity C-reactive protein (hsCRP), lipid profile, D-dimer, and factor (F)VIII activity were assayed by routine laboratory techniques in the hospital laboratory. N-terminal B-type natriuretic propeptide (NT-proBNP), high-sensitivity troponin T (TnT) were assessed by routine laboratory techniques in the hospital laboratory, while E-selectin, interleukin-6 (IL-6), L-lactate and 8-isoprostane were assayed by the immunoenzymatic tests (ELISA; R&D Systems, Abingdon, United Kingdom; Quantikine, R&D Systems, Minneapolis, USA; Abcam, Cambridge, United Kingdom; Cayman Chemical, Ann Arbor, MI, USA). Positive TnT was defined as a value > 14 pg/mL [21].

At 3 months of anticoagulant therapy blood samples were drawn 24–28 h since the administration of the last dose of direct oral anticoagulants (DOACs) and samples were evaluated if the drug concentration was below 30 ng/ml [22]. A chromogenic assay was used to measure anti-factor X (FXa) activity (BIOPHEN, Hyphen-Biomed, Neuville-Sur-Oise, France) in patients who received rivaroxaban or apixaban. In patients treated with warfarin, blood samples were drawn 24 h after the last dose of low-molecular-weight heparin. To evaluate efficiency of fibrinolysis, PAI-1 antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity (both from Hyphen-Biomed, Neuville-Sur-Oise, France), α2-antiplasmin, and plasminogen activity were measured (both Berichrom, Siemens Healthcare Diagnostics, Marburg, Germany).

The endogenous thrombin potential (ETP) was measured using calibrated automated thrombography (Thrombinoscope BV, Maastricht, the Netherlands). For fibrin clot analysis, blood samples (vol/vol, 9:1 of 3.2% trisodium citrate) were spun at 2500 g for 20 min and the supernatant was aliquoted and stored at -80 °C. All measurements were performed by technicians blinded to the origin of the samples. Intra-assay and inter-assay coefficients of variation were 5–7%. Fibrin clot permeation (Ks), reflecting the average pore size in the fibrin network was determined using a pressure-driven system as described previously [23]. Briefly, 20 mM CaCl2 and 1 U/mL human thrombin (Merck, Darmstadt, Germany ) were added to

citrated plasma. Volume of the buffer flowing through the clots was measured within 60 min. Fibrinolysis capacity (clot lysis time, CLT) was measured according to Pieters et al. [24]. Briefly, citrated plasma was mixed with 15 mM calcium chloride, human thrombin (Merck) at a final concentration of 0.5 U/ml, 10 µM phospholipid vesicles, and 18 ng/ml recombinant tPA (Boehringer Ingelheim, Ingelheim, Germany). A turbidity of the mixture was measured at 405 nm. Intra-assay and interassay coefficients of variation for the two fibrin variables were < 5% and < 8%, respectively.

Variables were presented as numbers and percentages or median and interquartile range (IQR), as appropriate. Normality was assessed by Shapiro-Wilk test. Differences between the groups were compared using the Student’s t-test for normally distributed variables. In turn, the Mann-Whitney U-test was used for non-normally distributed variables. Categorical variables were compared by chi-squared test or Fisher’s exact test. Associations between parametric variables were assessed by the Pearson’s correlation test while between nonparametric by Spearman’s rank correlation coefficient. All independent variables potentially associated with both the exposure and outcome were included in the multivariable logistic regression to find parameters independently associated with RPVO. The best cut-off value that maximizes sensitivity and specificity of Ks and CLT for RPVO prediction was calculated by using the Receiver Operating Characteristics (ROC) curves. A two-sided P < 0.05 was considered statistically significant. All statistical analyses were performed using STATISTICA software Version 13.3 (StatSoft, Krakow, Poland) or IBM SPSS Statistics Version 26.0 (IBM Corp, Armonk, NY, USA).