Zsuzsanna Bereczky

Beta-antithrombin (β-AT), the isoform of antithrombin (AT) with a higher affinity for heparin, constitutes 5–10% of total AT in plasma. There are limited data regarding β-AT activity levels in thrombotic disorders. In our study, we analyzed samples from 200 non-AT-deficient patients who had experienced venous thromboembolism (VTE) compared to 200 healthy controls. Total AT activity was measured using a chromogenic anti-factor Xa assay. To measure β-AT, we used elevated NaCl (1.1 M) in the reagent to inhibit the heparin binding of α-AT. There were no significant differences in total AT activity (median (IQR)) levels between the control and VTE groups (100 (93–109)% and 99 (94–109)%, respectively; p = 0.955). However, the β-AT activity levels (median (IQR)) and the ratio of β-AT to total AT (mean ± SD) were significantly higher in the VTE group compared to the control group (93.3 (90.3–97.3)% vs. 89.3 (84.0–95.0)% and 9.34 ± 0.68% vs. 8.86 ± 0.88%; p < 0.001). β-AT activity levels and the ratios in the upper third were strongly associated with a higher risk of VTE (OR (95% CI): 5.78 (3.08–10.87) and 6.15 (3.36–11.24), respectively). Our study demonstrated an elevation of plasma levels of β-AT in patients with VTE. Further research is necessary to clarify the pathophysiological significance of this finding.

Background: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular bleeding disorder. The most common symptoms are recurrent, severe nosebleeds that occasionally necessitate intervention by an ENT (Ear, Nose, and Throat) specialist, as well as iron-deficiency anemia. Telangiectasia is typically located in the nasal cavity, lips, tongue, fingertips, and the gastrointestinal mucosa. Arteriovenous malformations (AVMs) are located in internal organs (brain, lungs, liver, etc.). The family history is positive for HHT. The diagnosis is based on the Curacao criteria. The endoglin and activin receptor-like kinase 1 genes (ENG and ACVRL1) are the most common mutation sites, leading to elevated endothelial growth factor (VEGF) levels. Methods: We conducted a retrospective analysis in the Department of Internal Medicine, Division of Hematology, and Center of Expertise for Rare Diseases at the University of Debrecen, spanning the period from 2010 to 2025. Records of patients referred with HHT were reviewed concerning demographic data, clinical presentations, laboratory findings, and treatment approaches. To evaluate management options, epistaxis severity was assessed using the Epistaxis Severity Score (ESS). Results: 48 HHT patients (21 male and 27 female) were included in this retrospective study. Genetic testing was positive in each case, showing mutations in the ENG (HHT1 subgroup) or ACVRL1 (HHT2 subgroup) genes. Most of the patients are followed-up with in our department. ESS was calculated at baseline and 6 months after antiangiogenic treatment by two independent physicians. Detailed computed tomography (CT) was performed in all patients. Seven patients were administered desmopressin, a synthetic analog of antidiuretic hormone (ADH), based on our previous experience in reducing bleeding in von Willebrand disease. Antiangiogenic therapy with thalidomide (50 mg oral tablets) was used in 24 patients, while bevacizumab was administered to 5 patients. Most patients experienced a remarkable decrease in epistaxis severity and a reduction in the need for transfusions (ESS before treatment: HHT1 patients, 4.15 ± 1.91 vs. ESS after treatment, 2.62 ± 0.99; HHT2 patients, 3.79 ± 3.19 vs. 2.02 ± 1.91). Subgroup analysis using paired ESS data showed a significant reduction in ESS in both HHT1 and HHT2 patients (p = 0.003 and p = 0.043, respectively). Bevacizumab further reduced the ESS, but the few cases were not suitable for statistical analysis. Serum iron levels significantly increased after antiangiogenic treatment in the HHT2 group (p = 0.01). Conclusions: HHT is a rare vascular bleeding disorder. Daily nosebleeds impair the patients’ quality of life and sometimes lead to severe transfusion-dependent iron-deficient anemia. Frequent hospitalization places a significant burden on the healthcare system. Thus, we have used treatment options for HHT patients that primarily act by inhibiting VEGF, and these treatment modalities have yielded successful results in our hands.

Background/Objectives: Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease, in which multiple genetic and environmental factors may contribute. This study aimed to identify potential genetic determinants in patients with CTEPH and to compare their occurrence to a control group, which included patients with pulmonary embolism who had not developed CTEPH. Methods: Tier 1 and 2 genes related to coagulation, fibrinolysis and platelet disorders—as recommended by the International Society on Thrombosis and Haemostasis—and genes associated with vascular conditions were analyzed in n = 15 patients with CTEPH and n = 17 controls using next-generation sequencing. Non-synonymous, rare variants were collected and interpreted. Results: As expected, no single gene or variant was consistently present among CTEPH patients. Instead, individuals carried different mutations and combinations of variants. We identified several variants that were not found in the control group. Candidate variants were detected in F12, F13A1, F13B, F5, KNG1, SERPIND1, THBD, ADAMTS13, VWF, STIM1, ETV6, THPO, MPL, SERPINA1, ENG, RASA1, ACVRL1, GDF2, NFE2, SOX17 and RNF213. We did not detect exclusive variants in FGA, CPB2, and BMPR2 although they were suggested as candidates in previous studies. Elevated factor VIII and von Willebrand factor in CTEPH could not be explained by mutations in VWF and F8. Conclusions: Our study supports the hypothesis of heterogeneous genetic background in CTEPH, involving multiple pathways such as coagulation, altered fibrinolysis and impaired angiogenesis. These results provide a basis for more detailed investigations into specific genes and variants potentially associated with CTEPH in larger cohorts.

Carotid artery intima–media thickness (cIMT), a pre-clinical vascular change that accompanies atherosclerosis is considered as a cardiovascular risk factor. Coagulation factor XIII (FXIII) stabilizes the fibrin clot and increases its resistance to fibrinolysis. Regarding FXIII Val34Leu polymorphism, the protective effect of the Leu34 allele in the presence of elevated fibrinogen levels against myocardial infarction was demonstrated. Our aim was to investigate the effect of FXIII polymorphisms on cIMT. Patients with obesity (n = 69), type 2 diabetes mellitus (T2DM) (n = 104), and age- and sex-matched healthy controls (n = 82) were enrolled. FXIII polymorphisms (Val34Leu, His95Arg, Intron-K C>G) were determined by RT-PCR with FRET detection and melting curve analysis. cIMT was determined by B-mode ultrasound. Differences in cIMT between control (median: 0.5965, IQR: 0.5115–0.6580 mm) and T2DM (median: 0.7105, IQR: 0.5948–0.7568 mm), as well as between obese (median: 0.6105, IQR: 0.5455–0.6780 mm) and diabetic groups, were found (p < 0.0001 and p = 0.003, respectively). Genotype and allele frequencies of the studied polymorphisms did not differ between subgroups. In the study group (n = 255) after adjustment for age and sex, the presence of Intron-K G allele showed a significant and independent protective effect against cIMT progression in a separate model (p = 0.005) and after adjusting for other parameters associated with cIMT (p = 0.015). FXIII Intron-K G allele provides a protective effect against subclinical vascular disease in the studied population, and this effect is independent of the presence of obesity, as well as T2DM, Leu34 allele, and fibrinogen levels.

The inactivation of thrombin by antithrombin is highly enhanced by the presence of heparin chains forming “bridges” between the two proteins. X-ray structures for such ternary complexes have been published, but the molecular background of the lower efficiency of smaller heparinoids on thrombin inhibition remains poorly understood. Antithrombin-resistant prothrombin mutants (mutations affecting Arg596 in prothrombin) have been reported that cause severe thrombophilia. Our aim was to study the interactions in the antithrombin–thrombin Michaelis complex both in the presence and the absence of a heparinoid chain and in the presence of pentasaccharide by using molecular dynamics. We also intended to study the complexes of thrombin mutants as well as a known alternative antithrombin conformation at the “hinge” region built using docking. The binding between the proteins was investigated by Gaussian Accelerated Molecular Dynamics (GaMD). We compared the contribution of several amino acids at the binding “exosites” between AT and the wild type and mutant thrombins and between systems containing or not containing a heparinoid. In the docking-based simulations, several of the analyzed amino acid pairs no longer contributed to the interaction, suggesting that the open “hinge” conformation has limited biological relevance. We could identify multiple conformational types using clustering, revealing high flexibility in mutants and systems without heparinoid, probably indicating lower stability. We were also able to detect the allosteric effects of the ligands on the bound thrombin. In summary, we were able to obtain conformations using GaMD that can explain the better protein–protein interactions in the ternary complexes and the impaired AT binding of the thrombin Arg596 mutants at an atomic level.

Background/Objectives:: Right ventricular (RV) pressure assessment is crucial in both the diagnosis and follow-up of patients with chronic thromboembolic pulmonary hypertension (CTEPH). While right heart catheterization (RHC) and pulmonary angiography (PA) are gold-standard invasive methods, transthoracic echocardiography (TTE) offers a safer and more accessible alternative. This study aimed to evaluate the reliability of echocardiographic RV pressure estimation compared to invasive techniques and to identify clinical predictors of response to balloon pulmonary angioplasty (BPA). Methods: In this prospective study, 17 patients with confirmed CTEPH underwent RV pressure assessment via TTE, RHC (Swan-Ganz catheterization), and PA within the same hospitalization period. BPA responders were defined based on clinical improvement and were compared to poor responders using pre- and post-treatment parameters. Results: A strong correlation was found between Swan-Ganz and PA-derived pressures (r = 0.96), with a slightly lower correlation between TTE and PA (r = 0.84), and the lowest between TTE and Swan-Ganz (r = 0.78). In the well-responding group, the 6 min walk distance (6MWD) increased by 60 ± 18 m, compared to 12 ± 10 m in poor responders (p < 0.01). NT-proBNP levels decreased by 40% in responders versus 10% in non-responders (p < 0.01). TAPSE improved significantly in responders (from 16.0 ± 2.0 mm to 19.5 ± 2.5 mm, p < 0.01), while RV basal diameter decreased by 15% (p < 0.05). No significant echocardiographic improvement was observed in poor responders. Conclusions: TTE provides a reliable estimate of RV pressure in CTEPH when standardized protocols are followed. NT-proBNP levels and RV size may serve as useful predictors of BPA response.

Alpha2-plasmin inhibitor (α2PI) has a heterogeneous structure due to proteolytic cleavages in the circulation. The C-terminally cleaved form loses the plasminogen binding site and is, therefore, a slow plasmin inhibitor (NPB-α2PI). As FXIII primarily crosslinks the plasminogen-binding intact form (PB-α2PI) to fibrin, the effect of NPB-α2PI on fibrinolysis has been less studied. Herein, we investigated the effect of C-terminal truncation. Total-, PB-, and NPB-α2PI antigen levels and α2PI incorporation were measured by ELISAs from samples of 80 healthy individuals. Clot lysis parameters of the same subjects were investigated using an in vitro clot lysis assay. α2PI incorporation into the clot was demonstrated by Western blotting. Clot lysis and clot structure were also analyzed using an α2PI-deficient plasma substituted with recombinant PB- and NPB-α2PI. Both plasma and clot-bound levels of total- and NPB-α2PI showed a significant positive correlation with clot lysis parameters. NPB-α2PI was detected in the clot due to non-covalent binding. Regardless of the type of binding, both forms affected the clot structure by increasing the thickness of the fibrin fibers and reducing the pore size. In conclusion, we found that NPB-α2PI can bind non-covalently to fibrin, and this binding contributes to changes in clot structure and inhibition of fibrinolysis.

Background: Laboratory monitoring of the effect of low-molecular-weight heparins (LMWHs) is generally not necessary. However, prompt evaluation of heparin inhibitory effects (i.e., anti-Xa activity) is important in cases of life-threatening bleeding, need for urgent surgery or acute thromboembolism under LMWH treatment. We aimed to establish a simple and reliable point-of-care method for the detection of enoxaparin. Methods: Eighty patients under enoxaparin therapy and ten healthy volunteers without any anticoagulant treatment were enrolled. Simultaneous measurements of anti-Xa activity using the chromogenic method and clotting times in the absence and presence of polybrene using viscoelastometric assays containing Russell’s viper venom (RVV-test) were performed on the ClotPro device. Results: Among the measured and derived RVV-test parameters, the ratio of the RVV clotting times (RVV CT) detected in the absence and presence of polybrene showed the best statistically significant correlation with anti-Xa activity (r = 0.774, p < 0.001). Based on ROC analysis, we designated RVV CT ratios of 1.02, 1.23 and 1.6 as the best cut-off values for separating anti-Xa ranges below and above 0.3 and 0.6 IU/mL, respectively. If the RVV CT ratio is below or above 1.23, the anti-Xa activity is suggested to be below 0.6 IU/mL or above 0.3 IU/mL with high certainty, respectively. Further differentiation is possible if the RVV CT ratio is measured below 1.02 or above 1.6. In these cases, the measured anti-Xa values are below 0.3 IU/mL or above 0.6 IU/mL, respectively, with high probability and good predictive values. Conclusions: Our method can provide semiquantitative information on the effect of enoxaparin and the expected anti-Xa activity within 10 min in real clinical situations.

The structure of aspirated coronary thrombus in ST-segment elevation myocardial infarction (STEMI) is still being studied. Our aims were to characterize coronary thrombi of different ages, focusing on the appearance of activated protein C (APC/PC) and its relation to the elements of neutrophil extracellular traps (NETs), and the factors closely related to fibrin as factor XIII (FXIII) and α2 plasmin inhibitor (α2-PI). The thrombi of n = 24 male patients with atherosclerotic coronary plaque rupture related to native coronary artery occlusion were selected for histopathology analysis. Thrombus age was distinguished as fresh, lytic, and organized, and then analyzed by immunofluorescent staining and confocal microscopy. FXIII was present at a high level and showed a high degree of co-localization with fibrin in all stages of thrombus evolution. The amount of α2-PI was low in the fresh thrombi, which increased significantly to the lytic phase. It was evenly distributed and consistently associated with fibrin. APC/PC appeared in the fresh thrombus and remained constant during its evolution. The presence of NET marker and CD66b was most dominant in the lytic phase. APC/PC co-localization with the elements of NET formation shows its role in NET degradation. These observations suggest the importance of searching for further targeted therapeutic strategies in STEMI patients.

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.