Search Results (618)

Osteoarthritis (OA) is a major cause of equine lameness, with few effective disease-modifying treatments. This systematic review, conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, evaluated the efficacy of platelet-rich plasma (PRP) for equine OA by analyzing 11 studies (6 clinical, 5 experimental) identified through Web of Science, Scopus, and PubMed (2000–2024). The screening process identified 252 records, of which 136 were duplicates and 105 were excluded based on predefined criteria. The analysis showed that intra-articular PRP injections are generally safe, with transient synovial inflammation occurring mainly when PRP was activated with bovine thrombin. Both leukocyte-rich (L-PRP) and leukocyte-poor (P-PRP) formulations exhibited comparable efficacy, though optimal platelet concentrations (423–658 × 10³/μL) and dosing regimens remain undefined. A PRISMA-based quality assessment highlighted substantial variability in study design, with clinical trials constrained by small sample sizes and high risk of bias. Experimental studies confirmed PRP’s biological activity but showed inconsistencies in preparation methods. The findings indicate that PRP activation is unnecessary and may even be pro-inflammatory, that multiple injections could improve outcomes, and that reporting of cellular composition is inconsistent across studies. The PRISMA framework identified critical evidence gaps, particularly regarding long-term efficacy and protocol standardization. These results emphasize the need for PRISMA-compliant randomized controlled trials featuring standardized PRP protocols, validated outcome measures, and extended follow-up periods to establish evidence-based guidelines for managing equine OA. Full article

Background/Objectives: Vitamin C is frequently used in several dietary supplements due to its proposed health-promoting properties, while phenolic compounds and especially flavonoids have been suggested to provide synergistic antioxidant and cardiovascular benefits. However, the specific interactions between these compounds and their individual contributions to biological activity remain underexplored. This study aimed to evaluate the antioxidant potential and anti-inflammatory and antiplatelet biological effects of a high-dose (1 g) vitamin C–low-dose (50 mg) bioflavonoid (VCF)-based supplement using both in vitro and in vivo approaches in human platelets. Methods: Total phenolic content was quantified and antioxidant capacity was assessed using DPPH, FRAP, and ABTS assays and compared to individual phenolic standard compounds, including (simple phenolics like gallic acid, flavonoids like quercetin and catechin, and polyphenols like curcumin and tannin), and a standard supplement containing only high-dose vitamin C (VC). ATR-FTIR spectroscopy was used to assess molecular interactions between vitamin C and flavonoids. In vitro anti-inflammatory and antiplatelet activities of all supplements and standards were assessed by quantifying their IC₅₀ values against ADP, PAF, and thrombin-induced platelet aggregation. The in vivo evaluation of the efficacy and synergy of VCF supplement versus VC was achieved by a two-arm clinical study in healthy volunteers by quantifying their platelet reactivity, which was measured via EC₅₀ values on the aforementioned platelet agonists (PAF, ADP, and Thrombin) before (t = 0) and after receiving either solely VC or VCF supplementation for four weeks. Results: From all phenolic standards, the flavonoids and especially a mixture of flavonoids (catechin + quercetin) showed higher in vitro antioxidant capacity and anti-inflammatory and antiplatelet efficacy, followed by polyphenols and then simple phenolics. The VCF supplement showed the most potent antioxidant capacity, but also the strongest anti-inflammatory and antiplatelet activities too, in comparison to the VC and the mixture of flavonoids, suggesting higher synergy and thus bio-efficacy as a result of the co-presence of flavonoids and vitamin C in this supplement. Platelet reactivity decreased over time for PAF and thrombin in both arms of the trial, but no significant differences were observed between treatment groups, suggesting that the number of flavonoids used was not sufficient to translate the in vitro findings to the in vivo setting. Conclusions: VC-containing supplements provide antioxidant, anti-inflammatory, and antiplatelet benefits, while the incorporation of flavonoids may provide synergistic health benefits, but more in vivo assessment is needed to fully evaluate the dose efficacy. Full article

Venoms of the Palearctic vipers in the Macrovipera genus cause severe procoagulant clinical effects, yet the precise molecular targets remain incompletely defined. To fill this toxicological knowledge gap, we tested five Macrovipera venoms—M. lebetina cernovi, M. l. obtusa, M. l. turanica (Turkmenistan and Uzbekistan localities), and M. schweizeri—using plasma clotting assays, Factors VII, X, XI, and XII and prothrombin zymogen activation assays, and SDS-PAGE to visualise Factor V (FV) cleavage. All venoms induced extremely rapid clot formation (10.5–12.5 s) compared with the negative control (spontaneous clotting) of 334.6 ± 3.6 s) and the positive control (kaolin trigger) of 55.8 ± 1.9 s. Activation of FVII or FXI was negligible, whereas consistent FX activation and species-variable FXII activation, both moderate, were observed. Prothrombin remained inert in the absence of cofactors, but the presence of FV or FVa elicited potent thrombin generation. SDS-PAGE confirmed proteolytic conversion of the 330 kDa FV zymogen into the ~105 kDa heavy and ~80 kDa light chains of FVa by the venoms of all species. This data demonstrates that Macrovipera venoms rely on a dual enzyme strategy: (i) activation of FV to FVa by serine proteases and (ii) FVa-dependent prothrombin activation by metalloproteases. These results reveal that prothrombin activation is the dominant procoagulant pathway and overshadows the historically emphasised FX activation. This mechanism mirrors, yet is evolutionarily independent from, the FXa:FVa prothrombinase formation seen in Australian elapid venoms, highlighting convergent evolution of cofactor-hijacking strategies among snakes. The discovery of potent FVa-mediated prothrombin activation in Macrovipera challenges existing paradigms of viperid venom action, prompts re-evaluation of related genera (e.g., Daboia), and underpins the design of targeted antivenom and therapeutic interventions. Full article

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

Wound care in military and combat environments poses distinct challenges that set it apart from conventional medical practice in civilian settings. The nature of injuries sustained on the battlefield—often complex, contaminated, and involving extensive tissue damage—combined with limited access to immediate medical intervention, significantly increases the risk of infection, delayed healing, and adverse outcomes. Traditional wound dressings frequently prove inadequate under such extreme conditions, as they have not been designed to address the specific physiological and logistical constraints present during armed conflicts. This review provides a comprehensive overview of recent progress in the development of advanced wound dressings tailored for use in military scenarios. Special attention has been given to multifunctional dressings that go beyond basic wound coverage by incorporating biologically active macromolecules such as collagen, chitosan, thrombin, alginate, therapeutic peptides, and growth factors. These compounds contribute to properties including moisture balance control, exudate absorption, microbial entrapment, and protection against secondary infection. This review highlights the critical role of advanced wound dressings in improving medical outcomes for injured military personnel. The potential of these technologies to reduce complications, enhance healing rates, and ultimately save lives underscores their growing importance in modern battlefield medicine. Full article

Plant latex is a rich source of proteolytic enzymes with potential biomedical applications, particularly in hemostasis. Among them, thrombin-like enzymes (TLEs) have garnered interest in their ability to mimic thrombin by catalyzing the conversion of fibrinogen to fibrin, facilitating clot formation. While TLEs from snake venoms have been well-characterized and applied clinically, their plant-derived counterparts remain underexplored. This review critically examines the structural and functional characteristics of TLEs from plant latex, comparing them to animal-derived TLEs and evaluating their role in both procoagulant and fibrinolytic processes. Emphasis is placed on dual fibrinogenolytic and fibrinolytic activities exhibited by latex proteases, which often vary with concentration, incubation time, and protease type. In vitro coagulation assays and electrophoretic analyses are discussed as critical tools for characterizing their multifunctionality. By addressing the knowledge gaps and proposing future directions, this paper positions plant latex proteases as promising candidates for development in localized hemostatic and thrombolytic therapies. Full article

The development of functional endothelial monolayers on synthetic vascular grafts remains challenging, particularly for small-diameter vessels (<6 mm) prone to thrombosis. Here, we present a pharmacological strategy combining 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate sodium salt (pCPT-cAMP, a tight junction promoter) with nitric oxide/cGMP pathway agonists 3-morpholinosydnonimine (SIN-1), captopril, and sildenafil) to enhance endothelialization. In human umbilical vein endothelial cells (HUVECs), this four-agent cocktail induced a flat, extended phenotype with a 3-fold increased cell area and 57.5% fewer cells required for surface coverage compared to controls. Immunofluorescence analysis revealed enhanced ZO-1 expression and continuous tight junction formation, while sustained nitric oxide (NO) production (3.9-fold increase) and restored prostacyclin (PGI₂) secretion demonstrated preserved endothelial functionality. Anticoagulation assays confirmed a significant reduction in thrombus formation (p < 0.01) via dual inhibition of platelet activation and thrombin binding. These findings establish a synergistic drug combination that promotes rapid endothelialization while maintaining antithrombogenic activity, offering a promising solution for small-diameter vascular grafts. Further studies should validate long-term stability and translational potential in preclinical models. 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

Procoagulant platelets are a specialized subset of activated platelets that externalize phosphatidylserine (PS) on their surface, facilitating the assembly of tenase and prothrombinase complexes and enhancing thrombin generation and clot formation. Although procoagulant platelet formation shares certain features with nucleated cell death pathways, such as mitochondrial dysfunction, calcium (Ca²⁺) overload, membrane blebbing, and microvesiculation, it differs in key molecular mechanisms, notably lacking nuclei and caspase-dependent deoxyribonucleic acid (DNA) fragmentation. Interestingly, molecular components of nucleated cell death pathways in platelets can promote thrombus formation without impacting platelet lifespan. Under pathological conditions, excessive platelet activation may result in platelet lysis, resembling the complete activation of nucleated cell death pathways and contribute to thrombocytopenia. This review compares procoagulant platelet formation with various nucleated cell death pathways, including necrosis, necroptosis, pyroptosis, and ferroptosis, and explores their role in pathological thrombosis and blood clotting. A deeper understanding of mechanisms may help in developing targeted therapies to prevent aberrant blood clotting, platelet death and thrombocytopenia. Full article

In healthy conditions and cardiovascular diseases, the most abundant microparticles (MPs) in the bloodstream are those of platelet origin, but the direct effect of these microparticles on endothelial activation is poorly understood. The objective of this paper is to measure endothelial cell activation, as evaluated by the expression of the adhesion molecules E-selectin, VCAM-1, ICAM-1, and PECAM-1 in endothelial cell line HMEC-1 when stimulated with MPs produced by platelets stimulated in vitro with thrombin (TH), adenosine diphosphate (ADP), calcium ionophore (ICa), N-acetylglucosamine (NAcGlc), and without any stimulus. Platelets from healthy individuals induced the formation of MPs with different agonists. The results from the determination of the phenotype of the MPs showed that the expression of GPIIb/IIIa was significant, with median fold changes of TH = 2.2, ADP = 5.2, Ica = 7.0, and NAcGlc = 10.0. However, in HMEC-1 cells, the expression of adhesion molecules stimulated with MPs had a median change slightly higher for E-Sel expression (ranging from 1.4 to 4.2) and ICAM-1 expression (range 2.2 to 3.0), especially VCAM-1 expression (ranging from 15 to 18.8), all of which were significant. For PECAM-1, only stimulation with ICa (1.5) was significant, demonstrating that MPs elicit stimulus-dependent responses in endothelial cells. Platelet-derived MPs may have a potential role in modulating inflammation and other endothelial functions. Full article

Heparin, a widely used polysaccharidic anticoagulant of animal origin, is associated with risks of contamination and adverse effects, notably bleeding and thrombocytopenia. These limitations have prompted interest in alternative sulfated polysaccharides with anticoagulant properties and improved safety profiles. This study explored the anticoagulant potential of two marine bacterial exopolysaccharides (EPS), infernan and diabolican. It assessed whether chemical modifications (depolymerization, oversulfation) could enhance their anticoagulant properties compared to unfractionated and low molecular weight heparins. Native EPS were depolymerized to generate different molecular weights and then chemically oversulfated to increase negative charge density. Anticoagulant activities were evaluated using clotting and thrombin generation assays (TGA). Molecular docking was performed to model interactions with antithrombin and heparin cofactor II. Only highly sulfated derivatives significantly prolonged activated partial thromboplastin time while showing negligible effect on thrombin time and anti-factor Xa activity. They present different structures, and their binding to antithrombin is not achieved via the classic pentasaccharide motif. In TGA, these derivatives inhibited thrombin formation at higher doses than heparin but induced a marked delay in clot generation. Docking analyses supported their ability to bind serpins, albeit with lower specificity than heparin. Their limited anti-Xa activity and non-animal origin position them as promising anticoagulant candidates. Full article

Beyond haemostasis and thrombosis, platelets are increasingly recognized for playing a crucial role in modulating immunoinflammation. Toll-like receptors (TLRs) constitute the first line of defence against infection and injury, with their engagement stimulating thrombotic and immune responses in platelets. Hence, anti-platelet drugs have been used to treat patients with infections and inflammation. However, due to the increased risk of bleeding with current anti-platelet drugs, alternative therapeutic targets need to be identified to ameliorate the consequences of inflammation-driven platelet hyperactivation. Previously, we demonstrated that resting platelets exhibit a metabolic plasticity that facilitates fuel selection flexibility, while in contrast, thrombin-stimulated platelets become highly glycolytic. Since multiple aspects of platelet activation require energy in terms of ATP, we investigated metabolic alterations in TLR1/TLR2-activated platelets. In this study, we have demonstrated that TLR1/TLR2-induced platelet activation reprogrammed platelets to upregulate glycolysis via CD36-linked mechanisms. In addition, we showed that this glycolytic flux is controlled by hexokinase (HK), which plays a crucial role in TLR1/TLR2-induced platelet aggregation. Targeting platelet metabolism plasticity may offer a novel strategy to inhibit platelet function in TLR-initiated diseases. 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