Search Results (204)

Background/Objectives: Direct oral anticoagulants (DOACs) are now the guideline-recommended alternative to vitamin K antagonists (VKAs) for long-term anticoagulation in patients with non-valvular atrial fibrillation. However, accurately assessing their impact on ischemic stroke outcomes remains challenging, primarily due to uncertainty regarding anticoagulation status at the time of hospital admission. This preliminary study addresses this gap by using point-of-care testing (POCT) to confirm DOAC activity at bedside, allowing for a more accurate comparison of 90-day functional outcomes between anticoagulated and non-anticoagulated stroke patients. Methods: We conducted a retrospective cohort study of 786 ischemic stroke patients admitted to the University of Pécs between February 2023 and February 2025. Active DOAC therapy was confirmed using the ClotPro^® viscoelastic testing platform, with ecarin Clotting Time (ECT) employed for thrombin inhibitors and Russell’s Viper Venom (RVV) assays for factor Xa inhibitors. Patients were categorized as non-anticoagulated (n = 767) or DOAC-treated with confirmed activity (n = 19). Mahalanobis distance-based matching was applied to account for confounding variables including age, sex, pre-stroke modified Rankin Scale (mRS), and National Institutes of Health Stroke Scale (NIHSS) scores at admission and 72 h post-stroke. The primary outcome was the change in mRS from baseline to 90 days. Statistical analysis included ordinary least squares (OLS) regression and principal component analysis (PCA). Results: After matching, 90-day functional outcomes were comparable between groups (mean mRS-shift: 2.00 in DOAC-treated vs. 1.78 in non-anticoagulated; p = 0.745). OLS regression showed no significant association between DOAC status and recovery (p = 0.599). In contrast, NIHSS score at 72 h (p = 0.004) and age (p = 0.015) were significant predictors of outcome. PCA supported these findings, identifying stroke severity as the primary driver of outcome. Conclusions: This preliminary analysis suggests that ischemic stroke patients with confirmed active DOAC therapy at admission may achieve 90-day functional outcomes comparable to those of non-anticoagulated patients. The integration of bedside POCT enhances the reliability of anticoagulation assessment and underscores its clinical value for real-time management in acute stroke care. Larger prospective studies are needed to validate these findings and to further refine treatment strategies. Full article

The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the “protein of interest” (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout this publication as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors than the respective preparations of both E. coli strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies. Full article

Background/Objectives: We define severe postpartum hemorrhage (PPH) with macroscopic hematuria as clinical disseminated intravascular coagulation (DIC), a life-threatening condition. We also report a methodology using machine learning, a subtype of artificial intelligence, for developing the boundary criterion for predicting hematuria on the fibrinogen–fibrin/fibrinogen degradation product (FDP) plane. A positive FDP–fibrinogen/3–60 (mg/dL) value indicates hematuria; otherwise, non-hematuria is observed. We aimed to validate this criterion using severe placental abruption (PA), and to examine the activation of the coagulation–fibrinolytic system in clinical DIC. Methods: Of 17,285 deliveries across nine perinatal centers in Japan between 2020 and 2024, 13 had severe PA without hematuria, 18 had severe PPH without hematuria, and 3 had severe PPH with hematuria, i.e., clinical DIC. We calculated the values of the criterion formula for 13 cases of severe PA to validate the boundary criterion and compared the laboratory tests for coagulation–fibrinolytic activation among the three groups. Results: The calculated values using the criterion for the 13 PA without hematuria ranged from −108.91 to −5.87 and all were negative. In cases of clinical DIC, fibrinogen levels (median, 62 mg/dL) were lower (p < 0.05), while levels of FDP (96 mg/dL), the thrombin–antithrombin complex (120 ng/mL), and the plasmin-α₂–plasmin inhibitor complex (28.4 μg/mL) were significantly higher than in the other two groups. Conclusions: This study demonstrated the validity of the boundary criterion for predicting hematuria using severe PA. The coagulation–fibrinolytic test results suggested that PPH cases with hematuria were assumed to have clinical DIC, indicating that this criterion may be considered for diagnosing DIC during delivery. However, further additional patient data are needed to confirm the usefulness of this criterion because of the very low number of hematuria cases. Full article

Platelets play critical roles in haemostasis and thrombosis. The platelet activation process is driven by agonist-induced rises in cytosolic [Ca²⁺]_i, where the patterns of Ca²⁺ responses are still incompletely understood. In this study, we developed a number of techniques to model the [Ca²⁺]_i curves of platelets from a single blood donor. Fura-2-loaded platelets were quasi-simultaneously stimulated with various agonists, i.e., thrombin, collagen, or CRP, in the presence or absence of extracellular Ca²⁺ entry, secondary mediator effects, or Ca²⁺ reuptake into intracellular stores. To understand the calibrated time curves of [Ca²⁺]_i rises, we developed two non-linear models, a multilayer perceptron (MLP) network and an autoregressive network with exogenous inputs (NARX). The trained networks accurately predicted the [Ca²⁺]_i curves for combinations of agonists and inhibitors, with the NARX model achieving an R² of 0.64 for the trend prediction of unforeseen data. In addition, we used the same dataset for the construction of a partial least square (PLS) linear regression model, which estimated the explained variance of each input. The NARX model demonstrated that good fits could be obtained for the nanomolar [Ca²⁺]_i curves modelled, whereas the PLS model gave useful interpretable information on the importance of each variable. These modelling results can be used for the development of novel platelet [Ca²⁺]_i-inhibiting drugs, such as the drug 2-aminomethyl diphenylborinate, blocking Ca²⁺ entry in platelets, or for the evaluation of general platelet signalling defects in patients with a bleeding disorder. Full article

Naturally occurring plant-based compounds are increasingly being explored for their therapeutic potential in treating a wide range of conditions, particularly those related to vascular health. The compound 3-deoxysappanchalcone (3-DSC), derived from Caesalpinia sappan L., has been proven to exhibit anti-inflammatory, anti-influenza, and anti-allergic properties, though its role in thrombosis and haemostasis remains unexplored. This study aimed to evaluate the anti-thrombotic potential of 3-DSC in both in vitro and in vivo models. The anticoagulant activities of 3-DSC were assessed using activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin (FIIa) and activated factor X (FXa) activity assays, as well as fibrin polymerization and platelet aggregation tests. Its effects on plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) expression were evaluated in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). The results demonstrated that 3-DSC extended aPTT and PT, suppressed thrombin and FXa activities, reduced their production in HUVECs, inhibited thrombin-induced fibrin polymerization and platelet aggregation, and exerted anticoagulant effects in mice. Furthermore, 3-DSC significantly decreased the PAI-1 to t-PA ratio. These findings suggest that 3-DSC possesses potent anti-thrombotic properties by modulating coagulation pathways and fibrinolysis. Its therapeutic potential warrants further investigation for the development of novel anticoagulant agents. Full article

Background/Objectives: Among other substrates, the a disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS7) protease degrades thrombospondin-5 (the cartilage oligomeric protein, COMP), thrombospondin-1 (TSP-1) and the tissue inhibitor of metalloproteinases-1 (TIMP-1) indicating a potential role of ADAMTS7 expression on coagulation cascade, tissue remodeling and wound healing. We analyzed the potential effect of direct oral anticoagulant (DOAC) treatment on ADAMTS7 promoter methylation and followed it over time to assess whether DOACs epigenetically modulate ADAMTS7 and induce pathways associated with coagulation or endothelium repair machinery. Methods: Eighty-four DOAC-treated atrial fibrillation (AF) patients followed-up from baseline (t0) to 7 days (t1, n = 70) and 28 days of treatment (t2, n = 62) and 19 non-AF controls were included in the study. Genomic DNA was extracted from blood at all timepoints and was bisulfite-converted prior to methylation analysis. ADAMTS7 promoter DNA methylation was analyzed with MIP-qMSP-PCR. Results: A total of 16 minor bleeding events occurred. The baseline percentage of ADAMTS7 methylation did not differ between AF patients and controls (15.8% vs. 16.1%, p = 0.908). In the patient cohort, DOAC therapy marginally decreased ADAMTS7 methylation from t0 to t2 (15.2% vs. 14.0%, p = 0.044). This ADAMTS7 demethylation from t0 to t2 was statistically significant only in patients experiencing bleeding (17.1%. vs. 13.4%, p = 0.010 in bleedings, 14.5% vs. 14.2%, p = 0.561 in non-bleedings). No other differences were observed. Conclusions: ADAMTS7 is demethylated during DOAC-related bleedings, a mechanism potentially leading to COMP degradation and thus thrombin-induced platelet aggregation, as well as the induction of endothelium repair through different ADAMTS7-dependent pathways. Full article

This study aimed to characterize and evaluate the fibrinolytic, thrombolytic, hematological, and toxicological aspects of a serine protease (AsKSP) from Aspergillus tamarii Kita UCP 1279. The enzyme was purified using a two-phase aqueous system and assessed for optimal pH (7.0) and temperature (50 °C), stability, and effects of metal ions, inhibitors, and surfactants. AsKSP exhibited stability for up to 120 min at 50 °C and 36 h at pH 7.0. Enzymatic activity was enhanced by Na⁺ and Zn²⁺ and non-ionic surfactants (Tween-80) but inhibited by Cu²⁺, Fe³⁺, Triton X-100, and SDS, reducing activity by up to 62.35%. The highest amidolytic activity was observed for the substrate N-succinyl-Gly–Gly–Phe-p-nitroanilide. SDS-PAGE analysis indicated an approximate molecular mass of 90 kDa. The enzyme showed fibrinolytic activity, degrading 38.81% of fibrin clots in vitro after 90 min, without affecting fibrinogen. Cytotoxicity assays indicated no toxicity (cell viability > 80%). Coagulation assays showed slight prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT), with no effect on thrombin time. No red blood cell lysis was observed, and albumin increased enzymatic activity by 31.70%. These findings demonstrate that Aspergillus tamarii Kita UCP 1279 produces a fibrinolytic protease with potential for thrombus treatment, providing a promising foundation for drug development. Full article

Integrin β4 (ITGB4) mediates lung endothelial cell (EC) inflammation attenuated by simvastatin, an HMG CoA-reductase inhibitor. The cytoplasmic domain of ITGB4 is predicted to bind kindlin-2. Kindlin-2 expression is mediated by SMURF1, an E3 ubiquitin ligase that promotes kindlin-2 ubiquitination and degradation. We hypothesized that increased kindlin-2 expression via the inhibition of SMURF1 mediates EC inflammatory responses relevant to acute lung injury (ALI). To investigate this, human lung ECs were treated with simvastatin (5 µM, 16 h) prior to the immunoprecipitation of kindlin-2 and Western blotting for ITGB4. Next, ECs were treated with a SMURF1 inhibitor, A01, and increased kindlin-2 expression was confirmed. In assays of barrier function, kindlin-2 was silenced (siRNA) in ECs prior to thrombin and measurements of transendothelial resistance (TER) and FITC-dextran transwell flux. Repeat assessments of barrier function were performed in A01-treated ECs. Finally, mice were pretreated with A01 prior to LPS; bronchoalveolar lavage (BAL) fluid was collected, and their lungs were used for histology. Simvastatin increased ITGB4:kindlin-2 association, while A01 increased kindlin-2 expression. Thrombin-induced EC barrier disruption was both increased after kindlin-2 silencing and decreased by A01. Finally, murine ALI was significantly attenuated by A01. Our findings suggest that the augmentation of kindlin-2 may serve as a novel ALI therapeutic strategy. Full article

Bone metastasis and steroids are known to activate the coagulation system and induce osteoporosis, pathological bone fractures, and bone pain. Heparanase is a protein known to enhance the hemostatic system and to promote angiogenesis, metastasis, and inflammation. The objective of the present study was to evaluate the effects of steroids and malignancy on the coagulation factors and osteoblast activity in the bone tissue. The effects of dexacort and malignant medium were evaluated in osteoblasts derived from human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells (HUVECs). The bones of mice treated with dexacort for 1 month were studied. Bone biopsies of ten patients with bone metastasis, ten with steroid-induced avascular necrosis (AVN), and ten with osteoarthritis were compared to ten controls. We found that dexacort and malignant medium significantly increased the heparanase levels in osteoblasts and HUVECs and decreased the levels of alkaline phosphatase (ALKP). Peptide 16AC, derived from heparanase, which interacts with tissue factor (TF), further increased the effect, while peptide 6, which inhibits interactions between heparanase and TF, reversed the effect in these cells. The bone microcirculation of mice treated with dexacort exhibited significantly higher levels of heparanase, TF, TF pathway inhibitor (TFPI), TFPI-2, thrombin, and syndecan-1, but reduced levels of osteocalcin and ALKP. The pathological human bone biopsies’ microcirculation exhibited significantly dilated blood vessels and higher levels of heparanase, TF, TFPI, TFPI-2, and fibrin. In summary, steroids and malignancy increased the activation of the coagulation system in the bone microcirculation and reduced the osteoblast activity. Heparanase inhibitors should be further investigated to attenuate bone fractures and pain. Full article

Background/Objectives: This study aims to investigate the role of congenital single nucleotide thrombophilia in young females with early recurrent pregnancy loss (RPL). Methods: We studied 120 pregnant females with RPL and 80 matched females as a control with no RPL. Females were aged ≤ 35 years, had at least two consecutive first-trimester RPLs, and the acquired cause of RPL was excluded. A matched control group of 80 pregnant women with no RPL was studied. Coagulation tests included prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), a Factor XIII functional assay, and detecting IgM and IgG anti-beta2-Glycoprotein I (β2GPI) antibodies by an ELISA. The DNA samples were tested for Factor V Leiden, Factor II G20210A, Methylenetetrahydrofolate reductase (MTHFR C677T, A1298C), FXIII V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, endothelial protein C receptor (EPCR) A4600G, and endothelial protein C receptor (EPCR) G4678C. Results: Of the single nucleotide gene mutations investigated, the most relevant mutations were MTHFR C677T, MTHFR A1298C, heterozygous FXIII Val34Leu, and heterozygous FXIII 1694 C>T. Each of them conferred a statistically significant effect. There was a statistically significant protective role for the endothelial protein C receptor (EPCR) A2/A2, wild FXIII Val34Leu, and heterozygousFXIII1694 C>T. Conclusions: Our findings suggest the important role of congenital single nucleotide thrombophilia mutations in young Middle Eastern women with early RPL, particularly MTHFR mutations and FXIII Val34Leu. We found a protective effect of EPCR A2/A2, wild FXIIIVal34Leu, and heterozygous FXIII1694 C>T. We recommend additional studies to explore detrimental factors and protective factors. Full article

Background. Alterations induced by direct oral anticoagulants (DOACs) or vitamin K antagonists (VKAs) to thromboelastometry and thrombin generation are not well defined. We performed a simultaneous investigation of thromboelastometry and thrombin generation for patients who were chronically anticoagulated with DOACs or VKAs. Methods. A total of 131 patients on DOACs [apixaban (n = 37), rivaroxaban (n = 34), dabigatran (n = 30), edoxaban (n = 30)] and 33 on VKAs were analyzed. Whole blood was analyzed for thromboelastometry and plasma was analyzed for thrombin generation. Results. While the thromboelastometry clotting time (CT) was responsive to the hypocoagulability induced by DOACs or VKAs, clot formation time and maximal clot formation were not. Cumulatively, the parameters denoting the velocity of thrombin generation (lag time, time-to-peak) were relatively less responsive to the hypocoagulability induced by VKAs than DOACs. Conversely, the parameters denoting the quantity of thrombin generation [peak-thrombin and the endogenous thrombin potential (ETP)] were more responsive to the hypocoagulability induced by VKAs than DOACs. Apixaban showed relatively small differences (peak vs. trough) in the plasma concentration and a relatively small (peak vs. trough) difference of hypocoagulability when assessed by the CT or the ETP. The CT and the ETP were strongly correlated with DOAC concentrations or with the VKA-INR. Conclusions. DOACs and VKAs altered thromboelastometry and thrombin generation to an extent that probably reflects the mode of action of these drugs and may also have practical implications for patients’ management. Apixaban showed a small difference of hypocoagulability (peak vs. trough), suggesting a more stable anticoagulation over the daily course of treatment. Based on the correlations of the CT or the ETP vs. the DOAC concentrations, we estimated that critical values of the CT or the ETP would correspond to DOAC concentrations of 400 or 20 ng/mL. Whenever dedicated tests for DOAC concentrations are not available, the CT or the ETP can be used as surrogates to evaluate the level of anticoagulation induced by DOACs. Full article

Direct oral anticoagulants (DOACs) are increasingly being used due to their improved efficacy/safety ratio and lower clinical and economic burden when compared to vitamin K antagonists. However, bleeding is still the most frequent complication associated with DOACs, and although rare, bleeding episodes can be life-threatening or critical. The impact of DOAC anticoagulation activity during a bleeding event must be evaluated according to patient clinical assessment, dosage and time from last intake, the presence of comorbidities (especially kidney and liver dysfunction), and, whenever possible, coagulation tests. Unfortunately, DOACs’ anticoagulation activity is not easily or usually detectable in routine common coagulation testing. Specific DOAC tests allow for specific drug monitoring, but they are too time consuming, and are usually unavailable in routine emergency practice. If a clinically relevant DOAC plasma concentration is assumed or proven in a severe bleeding scenario, DOAC reversal is needed to restore hemostasis. This experts’ consensus provides a narrative review about DOAC reversal and practical life-threatening bleeding management in several scenarios (trauma, intracranial hemorrhage and gastrointestinal bleeding), focusing on the selection of patients to whom specific reversal agents should be given. Full article

This study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of Exiguobacterium oxidotolerans strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C and pH 3, retaining 85% activity at 50 °C, highlighting its potential stability under broad reaction conditions. SDS-PAGE and LC-MS analysis identified the enzyme as a 32 kDa M4-family metalloprotease. Exiguolysin activity was inhibited by 1,10-phenanthroline, confirming its dependence on metal ions for activity. Zymographic analysis and substrate specificity assays revealed selective hydrolysis of matrix metalloproteinase (MMP) substrates but no activity against elastase substrates. Analysis of the predicted gene sequence and structural predictions using AlphaFold identified the presence and position of HEXXH and Glu-Xaa-Xaa-Xaa-Asp motifs, crucial for zinc binding and catalytic activity, characteristic of ‘Glu-zincins’ and members of the M4 peptidase family. High-throughput screening of a 20 × 20 N-alpha mercaptoamide dipeptide inhibitor library against exiguolysin identified SH-CH₂-CO-Met-Tyr-NH₂ as the most potent inhibitor, with a Ki of 1.95 μM. Notably, exiguolysin selectively inhibited thrombin-induced PAR-1 activation in PC-3 cells, potentially indicating a potential mechanism of virulence in modulating PAR-1 signalling during infection by disarming PARs. This is the first detailed characterization of a peptidase of the M4 (thermolysin) family in the genus Exiguobacterium which may have industrial application potential and relevance as a putative virulence factor. Full article

Background: Andexanet alfa is a specific antidote for factor Xa (FXa) inhibitors. It is licensed to treat patients under FXa inhibitor therapy with life-threatening bleeding. Concomitantly, volume expanders are used to compensate for blood loss and maintain circulation. The competitive binding of andexanet to FXa inhibitors may be disrupted due to hemodilution, as shown by laboratory assays with high sample dilution. This study investigated the efficacy of andexanet for the reversal of FXa inhibitors under hemodilution. Methods: Blood from 10 healthy volunteers was anticoagulated with rivaroxaban and subsequently treated with four different volume expanders (Ringer’s solution, 4% gelatine, 5% and 20% human albumin (HA)) at two dilution levels (20% and 50%). After anticoagulation and hemodilution, andexanet was added according to the high-dose protocol. Blood samples were analyzed using a Russell’s viper venom (RVV) test on a Clot Pro^® device, a thrombin generation assay, a fully automated coagulation analyzer and a chromogenic anti-FXa activity assay. Results: After anticoagulation, the median rivaroxaban concentration was 272 ng/mL (IQR 254–353). Anticoagulation with rivaroxaban caused a significant impairment of all coagulation parameters, which was further aggravated by hemodilution. After the administration of andexanet, coagulation parameters in anticoagulated samples were reversed to near baseline in all groups. Andexanet administration decreased the rivaroxaban plasma concentration in all groups to a median of <10 ng/mL. In the anticoagulated, non-hemodiluted samples, anti-FXa activity was reduced by 98%. The anti-FXa activity in the anticoagulated, hemodiluted samples was reduced by approximately 96% in the 20% diluted samples and by about 93% in the 50% diluted samples. Conclusions: Our data indicate that FXa inhibitor reversal with andexanet is about 5% less effective with 50% hemodilution than in non-hemodiluted samples. Full article

Aeruginosins (AERs) are one of the most common classes of cyanobacterial peptides synthesised through a hybrid non-ribosomal peptide synthase/polyketide synthase pathway. They have been found in Microcystis, Nodularia spumigena, Oscillatoria/Plantothrix, and Nostoc. The presence of AER in Aphanizomenon isolated from the Curonian Lagoon was reported for the first time in our previous work. Here, the structure of aeruginosin 525 (AER525), isolated from Aphanizomenon sp. KUCC C2, was characterised based on high-resolution mass spectrometry. This new AER variant shows potent activity against thrombin. It also inhibits trypsin and carboxypeptidase A but has no effect on elastase and chymotrypsin. In terms of the N-terminal residue and biological activity, AER525 displaces some similarity to dysinosins, which belongs to the most potent inhibitors of thrombin among AERs. The findings underline the potential of AER525 as a new anticoagulant agent. Full article