Clot Structure Research Articles

Advanced Platelet-Rich Fibrin (A-PRF) as Antibiotics Delivery System: In-Vitro Proof-of-Concept Study

Autologous blood centrifugation produces various forms of platelet concentrates widely used in tissue regenerative therapies due to their high concentrations of growth factors and abundance of autologous cells. Advanced Platelet-Rich Fibrin (A-PRF), introduced as a low-speed centrifugation product, contains an even higher concentration of growth factors, a greater number of cells, and a looser fibrin clot structure compared to previous Leukocyte and Platelet-Rich Fibrin (L-PRF). This study aims to assess the potential of A-PRF as a local delivery system for antibiotics. Different concentrations (0.5 mg/mL, 0.25 mg/mL, and 0.125 mg/mL) of injectable amoxicillin (AMX) and metronidazole (MTZ) were preliminarily tested for their impact on A-PRF clot formation, with 0.5 mg/mL selected for subsequent experiments. Blood samples from healthy volunteers were supplemented with antibiotics and centrifuged to form clots. Antibiotic-enriched A-PRF clots were immersed in phosphate-buffered saline (1x PBS) and analyzed at 24 h, 72 h, 7 days, and 14 days. AMX showed a consistent release (mean: 19.9 ± 4.8 ng/mL at 24 h) over 14 days, while MTZ demonstrated greater variability (mean: 12.8 ± 4.5 ng/mL at 24 h). AMX release remained constant over the 14-day period, with no significant variations among patients. In contrast, MTZ displayed a progressively lower release over time. Microbiological analysis revealed bacterial growth inhibition zones for Fusobacterium nucleatum (AMX: 23 mm, MTZ: 28 mm) and Prevotella intermedia (AMX: 34 mm, MTZ: 30 mm) at 24 h. These findings suggest that A-PRF can act as an effective local antibiotic delivery system, maintaining sustained antimicrobial activity and potentially reducing the need for systemic antibiotics.

Dysregulated Clot Mechanics and Kinetics Impacted by Injury Severity, Predict Mortality After Trauma.

Introduction: Coagulopathy following traumatic injury impairs stable blood clot formation and exacerbates mortality from hemorrhage. Understanding how these alterations impact blood clot stability is critical to improving resuscitation. Furthermore, the incorporation of machine learning algorithms to assess clinical markers, coagulation assays and biochemical assays allows us to define the contributions of these factors to mortality. In this study, we aimed to quantify changes in clot formation and mechanics after traumatic injury and their correlation to mortality.Materials and Methods: Plasma was isolated from injured patients upon arrival to the emergency department prior to blood product administration, or procedural intervention. Coagulation kinetics and mechanics of healthy donors and patient plasma were compared with rheological, turbidity and thrombin generation assays. ELISA's were performed to determine tissue plasminogen activator (tPA) and D-dimer concentration. Recursive elimination with random forest models were used to assess the predictive strength of clinical and laboratory factors.Results: Sixty-three patients were included in the study. Median injury severity score (ISS) was 17, median age was 38 years, and mortality was 30%. Trauma patients exhibited reduced clot stiffness, increased fibrinolysis, and reduced thrombin generation compared to healthy donors. Deceased patients exhibited the greatest deviation from healthy levels. Fibrinogen, clot stiffness, D-dimer and tPA all demonstrated significant correlation to ISS. Machine-learning algorithms identified the importance of coagulation kinetics and clot structure on patient outcomes.Conclusions: Rheological markers of coagulopathy and biochemical factors are associated with injury severity and are highly predictive of mortality after trauma, providing evidence for integrated predictive models and therapeutic strategies.

Ultrasound-mediated catheter delivery of tissue plasminogen activator promotes thrombolysis by altering fibrin fibre thickness and clot permeability.

It has been proposed that low power, high frequency ultrasound can augment the ability of thrombolytic agents to dissolve clot in patients with venous thromboembolism. We created a bench model to examine what role and mechanism ultrasound may have in this process. Fibrin polymerisation was analysed through modified light-scattering experiments with the inclusion of catheter-mediated ultrasound application. We studied fibrin fibre diameters through scanning electron microscopy of ultrasound treated fibrin clots. Clot porosity was investigated using permeation tests, while fibrinolysis was analysed through lightscattering experiments, and by changes in porosity of lysing clots under flow. Application of ultrasound did not change initial fibrin polymerisation but did induce a reversible change in maximal turbidity of already formed fibrin clots. This change in turbidity was caused by a reduction in fibrin fibre diameter and was associated with an increase in clot porosity. These reversible structural changes were associated with a linear increase in fibrinolysis rates under static conditions, while an exponential increase in rates was observed under flow. The use of ultrasound augmentation of thrombolysis enhances clot dissolution through greater and more rapid fibrin degradation. This is due to conformational change created by the ultrasound in clot structure, a reversible phenomenon that may increase binding sites for lytic agent and could potentially allow the use of lower doses and shorter infusion times of ultrasound-assisted thrombolytic to treat venous thromboembolism in-vivo.

Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP) and we hypothesise that the Affimer technology, comprising small conformational proteins with two nine amino acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis.Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated of which one, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n=15; from 150.8±100.9 to 109.8±104.8 mins) and those with cardiovascular disease (n=15; 117.6±40.6 to 79.7±33.3 mins); p<0.01 for both groups. The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples.Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), while molecular modelling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15-0.60 mg/ml, had the ability to bind 70-90% of plasma α2AP.In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.

Novel factors affecting fibrin clot formation and their clinical implications.

Fibrin formation is pivotal in hemostasis, serving as a temporary barrier to blood loss following vascular injury, while in thrombosis this process is involved in thrombus progression, stability, and recurrence. Growing evidence shows exceptional complexity of processes that determine fibrin clot structure and function, especially lysability, both in health and disease, which might be relevant in the pathogenesis of arterial and venous thromboembolic diseases. In this review, we summarized available data on novel factors that in recent years have been suggested to contribute to prothrombotic fibrin clot properties, involving formation of compact fibrin networks (reduced clot permeability) displaying impaired susceptibility to lysis (prolonged clot lysis time). The factors discussed in this review encompass elevated levels of factor (F)XI, and its activated form (FXIa), protein carbonylation as the most common type of post‑translational modification, neutrophil extracellular traps formation, increased levels of circulating lipopolysaccharide and zonulin, a marker of gut permeability, along with antithrombin deficiency. These factors have been shown to be not only associated with ischemic stroke, myocardial infarction, pulmonary embolism, and cardiovascular death, but also with unfavorably altered fibrin clot characteristics, which underscores clinical relevance of fibrin clot properties. Given preclinical or ongoing studies aimed at modifying some of these factors, in particular FXI/FXIa inhibitors, recent findings might expand our knowledge on fibrin‑related mechanisms of emerging therapeutic agents tested and stimulate further research into new targets for future therapeutic interventions to prevent thromboembolic events.

Dependence of clot structure and fibrinolysis on apixaban and clotting activator

BackgroundAnticoagulants prevent the formation of potentially fatal blood clots. Apixaban is a direct oral anticoagulant that inhibits factor (F)Xa, thereby impeding the conversion of prothrombin into thrombin and the formation of blood clots. Blood clots are held together by fibrin networks that must be broken down (fibrinolysis) to restore blood flow. Fibrinolysis is initiated when tissue plasminogen activator (tPA) converts plasminogen to plasmin, which binds to and degrades a fibrin fiber. The effects of apixaban on clot structure and lysis have been incompletely studied. ObjectivesWe aimed to study apixaban effects on clot structure, kinetics, and fibrinolysis using thrombin (low or high concentration) or tissue factor (TF) to activate clot formation. MethodsWe used a combination of confocal and scanning electron microscopy and turbidity to analyze the structure, formation kinetics, and susceptibility to lysis when plasma was activated with low concentrations of thrombin, high concentrations of thrombin, or TF in the presence or absence of apixaban. ResultsWe found that the clotting activator and apixaban differentially modulated clot structure and lytic potential. Low thrombin clots with apixaban lysed quickly due to a loose network and FXa cleavage product’s cofactor with tPA; high thrombin clots lysed faster due to FXa cleavage product’s cofactor with tPA; TF generated loose clots with restricted lysis due to their activation of thrombin activatable fibrinolytic inhibitor. ConclusionOur study elucidates the role of apixaban in fibrinolytic pathways with different clotting activators and can be used for the development of therapeutic strategies using apixaban as a cofactor in fibrinolytic pathways.

Hydrodynamic Cavitation‐Induced Thrombolysis on a Clot‐on‐a‐Chip Platform

Complications from thrombosis constitute a massive global burden for human health. Current treatment methods have limitations and can cause serious adverse effects. Hydrodynamic cavitation (HC) is a physical phenomenon where bubbles develop and collapse rapidly within a moving liquid due to sudden pressure changes. These collapsing bubbles provide high targeted energy which can be used in a controlled environment with the help of microfluidic devices. This study introduces a new clot‐on‐a‐chip (CoC) platform based on HC, evaluated for thrombolysis efficacy. The microfluidic device, paired with a polydimethylsiloxane (PDMS) microchip, generates cavitation bubbles at low upstream pressures (≤482 kPa), enabling microscale blood clot erosion. Different HC exposure conditions (varying pressure and duration) are assessed by changes in clot mass, diameter, and scanning electron microscopy (SEM). The largest mass reduction occurs at 482 kPa for 120 s, with a decrease of 6.1 ± 0.12 mg, while the most erosion in diameter of blood clots is obtained 482 kPa for 120 s with complete removal. SEM results show increasing damage to clot structure from less to more intense HC exposures. The CoC platform, at controlled pressures and durations, efficiently disrupts clot structure and offers a promising drug‐free alternative for thrombolysis treatment.

Rupture mechanics of blood clots: Influence of fibrin network structure on the rupture resistance

Embolization is a leading cause of mortality, yet we know little about clot rupture mechanics. Fibrin provides the main structural and mechanical stability to blood clots. Previous studies have shown that altering the concentration of coagulation activators (thrombin or tissue factor (TF)) has a significant impact on fibrin structure and viscoelastic properties, but their effects on rupture properties are mostly unknown. Toughness, which corresponds to the ability to resist rupture, is independent of viscoelastic properties. We used varying TF concentrations to alter the structure and toughness of human plasma clots. We performed single-edge notch rupture tests to examine fibrin toughness under a constant strain rate and we assessed viscoelastic mechanics using rheology. We utilized fluorescent confocal and scanning electron microscopy (SEM) to quantify the fibrin network structure under varying TF concentrations. Our results revealed that increased TF concentration resulted in increased number of fibrin fibers with a reduction in network pore size, thinner and shorter fibrin fibers. Increasing TF concentration yielded a maximum toughness at mid-TF concentration, such that fibrin diameter and number of fibers underlie a complex role in influencing the rupture resistance of blood clots, resulting in a nonmonotonic relationship between TF and toughness. A simple mechanical model, built on our findings from our Fluctuating Spring (FS) computational model, adopted to estimate the fracture toughness (critical energy release rate) as a function of TF predicts trends that are in good agreement with experiments. The differences in mechanical responses point to the importance of studying the structure-function relationships of fibrin networks, which may be predictive of the tendency for embolization. Statement of significanceFibrin, a naturally occurring biomaterial, is the main mechanical and structural scaffold of blood clots that provides the necessary strength and stability to the clot, ensuring effective stemming of bleeding. The rupture of blood clots can result in the blockage of downstream vessels thereby blocking blood flow and oxygen supply. The fibrin network structure has been shown to influence the viscoelastic mechanical properties of clots, but has not been explored for fracture mechanics. Here, we modulate the fibrin network structure by varying the concentration of Tissue Factor (TF). Interestingly, the association between TF concentration and maximum toughness of the clots is non-monotonic. The variations in mechanical responses highlight the importance of studying the structure-function relationships of fibrin networks, as these may predict the tendency for embolization.

Plasmin generation analysis in patients with bleeding disorder of unknown cause

AbstractBleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after evaluation of plasma coagulation and platelet function. Patients with BDUC (n = 375) recorded in the Vienna Bleeding Biobank were analyzed in comparison with healthy controls (HCs; n = 100) in this case-control study. Plasmin generation (PG) parameters were analyzed using calibrated fluorescence detection in citrated plasma. Turbidimetric plasma clot formation/lysis of 293 (78%) patients with BDUC and confocal microscopy of clots from representative patients with BDUC (n = 6) and HCs (n = 9) were assessed. In the PG analysis, patients with BDUC exhibited lower velocity and peak plasmin levels but a higher endogenous plasmin potential than HCs. Peak plasmin levels correlated with maximum clot absorbance but not with clot lysis time. Clot absorbance is an indicator of clot fiber density. Confocal microscopy analysis revealed a tendency towards thicker fibers in clots of patients with BDUC, which negatively correlated with peak plasmin (r = −0.561; P = .030). Peak plasmin correlated weakly with factor XIII, but not with other fibrinolytic factors (alpha2-antiplasmin, thrombin activatable fibrinolysis inhibitor, or plasminogen activator inhibitor 1) or bleeding severity. A model comprising fibrinogen and parameters of PG yielded high predictive power in discriminating between patients with BDUC and HCs across a fivefold stratified cross validation (80% of data; mean area under the curve [AUC], 0.847). The model generalized well to unseen data (20% of data; AUC, 0.856). Overall, patients with BDUC counterintuitively exhibited reduced peak plasmin levels, potentially related to altered clot structure.

Fibrin clot permeability (Ks) in patients on left ventricular assist device

Patients on left ventricular assist devices (LVAD) are prone to excessive hemostasis disturbances due to permanent contact of artificial pump surfaces with blood components. We aimed to investigate if fibrin clot permeability is altered in patients on long-term continuous-flow LVAD therapy and if the clot permeability is associated with clinical characteristics and adverse events. We investigated 85 end-stage heart failure patients (90.6% men, age 48.6–63.8 years) scheduled for continuous flow long-term LVAD support according to current clinical indications. The patients were assessed periodically: prior to LVAD implantation (T1), 3–6 months (T2) after LVAD implantation, 6–12 months after (T3) and then every 6 months. We tested the first three blood samples (T1-T3) and the last available blood sample (T4), but no longer than 5 years after LVAD implantation. We assessed hemostasis parameters (Activated Partial Thromboplastin Time (APTT) Prothrombin Time, Activated Partial Thromboplastin Time, Fibrinogen, d-dimer, Antithrombin, Thrombin Time, Factor VIII, and von Willebrand Factor, aspirin-induced platelet inhibition, adenosine-diphosphate test) changes during the study period. Fibrin Clot Permeability was evaluated using a pressure system and Permeability Coefficient (Ks) was calculated. We observed a decrease in fibrin clot permeability (Ks) between T1, T2, T3 and T4 time periods; P < 0.01 for each comparison. Fibrin clot permeability was negatively correlated with fibrinogen concentration: r = − 0.51, P < 0.001, factor VIII activity r = − 0.42, P < 0.001. There was no association of Ks with age, Left Ventricular Ejection Fraction (LVEF) and medications P > 0.001, however cumulative measurements in patients on aspirin showed shortening of Ks in this group P = 0.0123. Major adverse cardiac and cerebrovascular events (MACCE) occurred in 36.5% patients, bleeding events in 25.9%, Net Adverse Clinical Events (NACE) in 62.4%; 31.7% patients died, and 17.6% underwent transplantation. The transplantation was considered as the endpoint. Discrepancies in Ks were observed between patients with MACCE, bleeding, and NACE, and patients without adverse events. Ks showed a constant trend towards normalization (P < 0.01) only in patients without adverse events. Patients with advanced heart failure have disturbed clot structure. A trend towards normalization of the Ks values is associated with fewer thromboembolic and bleeding complications in this group of patients.

Elevated level of extracellular vimentin is associated with an increased fibrin formation potential in sepsis: ex vivo swine study

BackgroundSepsis can lead to coagulopathy and microvascular thrombosis. Prior studies, including ours, reported an increased level of extracellular vimentin in blood derived from septic patients. Moreover, we identified the contribution of extracellular vimentin to fibrin formation and to the fibrin clot structure ex vivo in plasma from septic patients. Here, we tested the status of plasma vimentin and its impact on fibrin clots using our recently described swine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis-induced coagulopathy.ResultsWe employed ELISA, size-exclusion chromatography, vimentin antibodies, confocal microscopy, and turbidity assays on piglet plasma obtained at pre- and post-MRSA inoculation. Plasma vimentin level at 70 h post-MRSA inoculation was on average twofold higher compared to pre-infection (0 h) level in the same animal. Anti-vimentin antibody effectively reduced fibrin formation ex vivo and increased porosity in the fibrin clot structure generated from septic piglet plasma. In contrast to plasma at 0 h, the size-exclusion chromatography revealed that phosphorylated vimentin was in-complex with fibrinogen in septic piglet plasma.ConclusionsThus, our swine model of sepsis-induced coagulopathy, reproduced increased extracellular circulating vimentin and subsequent potentiation of fibrin formation, often observed in septic patient. These outcomes validate the use of large animal models to investigate the dysregulated host immune response to infection leading to coagulopathy, and to develop new therapies for sepsis-induced disseminated microvascular thrombosis.

Plant-derived compounds normalize platelet bioenergetics and function in hyperglycemia

BackgroundPolyphenols have been shown to decrease oxidative stress and modulate glycemic response. Nevertheless, their effect on platelet bioenergetics and clot structure in diabetes and hyperglycemia is unknown. ObjectivesTo investigate the effect of polyphenols on human platelet bioenergetics and its subsequent effect on clot structure in normoglycemia vs acute hyperglycemia in vitro. MethodsFour polyphenols (resveratrol, hesperetin, epigallocatechin gallate [EGCG], and quercetin) were selected for initial analysis. Healthy volunteers’ isolated platelets/platelet-rich plasma were treated with 5 or 25 mM glucose to represent normoglycemia and acute hyperglycemia, respectively. Platelet-derived reactive oxygen species (ROS), citrate synthase activity (mitochondrial density), mitochondrial calcium flux, and mitochondrial respiration were performed following exposure to polyphenols (20 µM, 1 hour) to determine their effects on platelet bioenergetics. Procoagulant platelets (annexin V) and fibrin fiber density (Alexa Fluor-488 fibrinogen; Invitrogen) were analyzed by laser scanning confocal microscopy, while clot porosity was determined using platelet-rich plasma following exposure to polyphenols (20 µM, 20 minutes). ResultsAcute hyperglycemia increased ROS, mitochondrial calcium flux, maximal respiration, and procoagulant platelet number. Resveratrol, quercetin, and EGCG reduced platelet ROS in normoglycemic and acute hyperglycemic conditions. Mitochondrial density was decreased by quercetin and EGCG in normoglycemia. Resveratrol and EGCG reduced mitochondrial calcium flux in acute hyperglycemia. Resveratrol also decreased procoagulant platelet number and attenuated oxygen consumption rate in normoglycemia and acute hyperglycemia. No effect of hyperglycemia or polyphenols was observed on fibrin fiber density or clot pore size. ConclusionOur results suggest polyphenols attenuate increased platelet activity stemming from hyperglycemia and may benefit thrombosis-preventative strategies in patients with diabetes.

Fibrinogen and plasma clot properties are associated with apolipoprotein B and apolipoprotein B-containing lipoproteins in Africans

Fibrinogen and plasma clot properties are associated with apolipoprotein B and apolipoprotein B-containing lipoproteins in Africans

Influence of haematological parameters on size of the advanced platelet-rich fibrin+ (A-PRF+) in the horse

The advanced-PRF+ (A-PRF+) is a platelet concentrate, showing a higher concentration of growth factors, an increased number of cells and looser structure of the fibrin clot than leukocyte-PRF. A high variability in the size of PRF associated with patients, haematological features and centrifugation protocols was reported. The aims of this study were to evaluate the feasibility of A-PRF+ production in the field and the correlation between haematological parameters, macroscopic and microscopic features in equine A-PRF+. Samples from twenty Standardbred horses (3–7 years) were harvested with glass tubes without anticoagulants, previously heated at 37 °C. Blood samples were centrifugated at 1300 rpm for 8 min with a fixed-angle centrifuge and a horizontal centrifuge in the field, at a temperature of 15–17 °C. Clots were measured and placed on the Wound Box® for a 2-min compression. Membranes were measured and fixed in 10% formalin for histological examination. Clot and membrane surface did not differ between sex and centrifuge. Haematological parameters did not show a significant correlation to clot and membrane size. Membranes obtained from both centrifugation protocols showed a loose fibrin structure and cells evenly distributed throughout the clot. Tubes' warming was effective to obtain A-PRF+ clots from all samples, regardless the environmental temperature. Further studies are needed to evaluate the influence of other blood molecules on the A-PRF+ structure and size.

Blood clot properties, thrombin generation, and platelet activation in patients with dysglycemia and established atherosclerotic cardiovascular disease: The CASCARA study.

There is a strong link between coronary artery disease (CAD), type 2 diabetes (T2D) on one hand, and altered fibrin clot properties, including increased clot density, and unfavorable fibrin clot structure on the other. T2D-related changes in fibrin clots can increase cardiovascular (CV) disease risk, including future CV events. We aimed to assess fibrin clot properties, thrombin generation, and platelet activation in CAD patients with prediabetes (PD) or T2D, compared to CAD patients without glycemic disorders. We allocated patients to three groups: 1) Those with angiographically established CAD but without glycemic abnormalities (CAD group); 2) individuals with PD and established CAD (CAD+PD group); and 3) patients with T2D and CAD (CAD+T2D group). We conducted comparisons across these groups for thrombin generation, fibrin clot permeability, fibrin clot lysis, and platelet activation. The final analysis included 116 eligible patients: 1) CAD group (n = 31); 2) CAD+PD (n = 42); and 3) CAD+T2D (n = 43). The CAD+T2D patients enrolled had well-controlled T2D (median HbA1c level of 5.90%; IQR: 5.7%-6.3%). We found no significant differences in thrombin generation, fibrin clot properties, or platelet activation markers across the three analyzed groups (all P-values >0.20). However, elevated interleukin-6 (IL-6) levels were noted in both the highest and lowest glucose concentration quartiles. Additionally, a substantial increase in endogenous thrombin potential (ETP) was observed in patients in the highest glycated hemoglobin quintile. Individuals with established CAD and concomitant PD or well-controlled T2D exhibited comparable fibrin clot phenotypes, thrombin generation potential, and platelet activation when compared to CAD patients without dysglycemia.

Advancing Thrombosis Research: A Novel Device for Measuring Clot Permeability.

Thromboembolism, a global leading cause of mortality, needs accurate risk assessment for effective prophylaxis and treatment. Current stratification methods fall short in predicting thrombotic events, emphasizing the need for a deeper understanding of clot properties. Fibrin clot permeability, a crucial parameter in hypercoagulable states, impacts clot structure and resistance to lysis. Current clot permeability measurement limitations propel the need for standardized methods. Prior findings underscore the importance of clot permeability in various thrombotic conditions but call for improvements and more precise, repeatable, and standardized methods. Addressing these challenges, our study presents an upgraded, portable, and cost-effective system for measuring blood clot permeability, which utilizes a pressure-based approach that adheres to Darcy's law. By enhancing precision and sensitivity in discerning clot characteristics, this innovation provides a valuable tool for assessing thrombotic risk and associated pathological conditions. In this paper, the authors present a device that is able to automatically perform the permeability measurements on plasma or fibrinogen in vitro-induced clots on specific holders (filters). The proposed device has been tailored to distinguish clot permeability, with high precision and sensitivity, between healthy subjects and high cardiovascular-risk patients. The precise measure of clot permeability represents an excellent indicator of thrombotic risk, thus allowing the clinician, also on the basis of other anamnestic and laboratory data, to attribute a risk score to the subject. The proposed instrument was characterized by performing permeability measurements in plasma and purified fibrinogen clots derived from 17 Behcet patients and 15 sex- and age-matched controls. As expected, our results clearly indicate a significant difference in plasma clot permeability in Behcet patients with respect to controls (0.0533 ± 0.0199 d vs. 0.0976 ± 0.0160 d, p < 0.001). This difference was confirmed in the patient's vs. control fibrin clots (0.0487 ± 0.0170 d vs. 0.1167 ± 0.0487 d, p < 0.001). In conclusion, our study demonstrates the feasibility, efficacy, portability, and cost-effectiveness of a novel device for measuring clot permeability, allowing healthcare providers to better stratify thrombotic risk and tailor interventions, thereby improving patient outcomes and reducing healthcare costs, which could significantly improve the management of thromboembolic diseases.

The impact of von Willebrand factor on fibrin formation and structure unveiled with type 3 von Willebrand disease plasma.

Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (P < 0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (P < 0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (P < 0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (P < 0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.

Reviewing the Rich History of Fibrin Clot Research with a Focus on Clinical Relevance.

Fibrin, described on a single-lens microscopy for the first time by Malpighi in 1666 and named by de Fourcroy, has been extensively studied by biochemists, biophysicists, and more recently by clinicians who recognized that fibrin is the major component of most thrombi. Elucidation of key reactions leading to fibrin clot formation in the 1950s and 1960s grew interest in the clinical relevance of altered fibrin characteristics. Implementation of scanning electron microscopy to image fibrin clots in 1947 and clot permeation studies in the 1970s to evaluate an average pore size enabled plasma clot characterization in cohorts of patients. Unfavorably altered fibrin clot structure was demonstrated by Blombäck's group in coronary artery disease in 1992 and in diabetes in 1996. Fifteen years ago, similar plasma fibrin clot alterations were reported in patients following venous thromboembolism. Multiple myeloma was the first malignant disease to be found to lead to abnormal fibrin clot phenotype in the 1970s. Apart from anticoagulant agents, in 1998, aspirin was first shown to increase fibrin clot permeability in cardiovascular patients. The current review presents key data on the rich history of fibrin research, in particular, those that first documented abnormal fibrin clot properties in a variety of human disease states, as well as factors affecting fibrin phenotype.

VENOUS THROMBUS STIFFNESS IN COMBINATION OF PULMONARY EMBOLISM AND DEEP VEIN THROMBOSIS OF THE LOWER EXTREMITIES (CLINICAL CASE)

Many aspects of pulmonary embolism pathogenesis are still unclear: for example, what is the probability of pulmonary embolization in venous thrombosis and thrombi formation in the pulmonary artery in situ. The origin and timing of thromboemboli formation in the pulmonary artery are of great clinical importance for effective treatment and prognosis of pulmonary embolism outcome. The aim of the study was as part of clinical observation to compare the results of shear wave ultrasound elastography of a venous thrombus and the histological structure of blood clots in the lower extremity and the pulmonary artery obtained during a pathoanatomical examination. Research results. Patient Sh., 67 years old, was hospitalized with a diagnosis of pulmonary embolism. Ultrasound imaging visualized heterogeneous, predominantly hypoechoic non-occlusive thrombotic masses in the lumen of the posterior tibial vein of the left lower limb, extending into the lumen of the popliteal vein for 2 cm with an uneven "ragged" contour of the proximal part of the thrombus, without clear signs of flotation. Shear wave ultrasound elastography determined stiffness of the proximal part of the venous thrombus with an average Young's modulus of 8.5 kPa, which corresponds to the acute stage of thrombosis. Negative dynamics was observed after thrombolytic therapy. Computed tomography angiography of the cerebral vessels revealed signs of intracerebral hemorrhage. On the third day, the patient was pronounced dead. The pathological examination was supplemented with histological data from a fragment of a venous thrombus of the pulmonary artery and lower leg veins. The morphological characteristics of blood clots from the veins of the lower extremities and the pulmonary artery coincided, indicating their common origin and the time of formation (acute stage). Conclusion. A comparative analysis of ultrasound and pathoanatomical data of the clinical case give us the opportunity to recommend shear wave ultrasound elastography of venous thrombi of the lower extremities to assess the state of thromboembolism in the pulmonary artery.

The Interplay between Liver Sinusoidal Endothelial Cells, Platelets, and Neutrophil Extracellular Traps in the Development and Progression of Metabolic Dysfunction-Associated Steatotic Liver Disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a societal burden due to the lack of effective treatment and incomplete pathophysiology understanding. This review explores the intricate connections among liver sinusoidal endothelial cells (LSECs), platelets, neutrophil extracellular traps (NETs), and coagulation disruptions in MASLD pathogenesis. In MASLD's early stages, LSECs undergo capillarization and dysfunction due to excessive dietary macronutrients and gut-derived products. Capillarization leads to ischemic changes in hepatocytes, triggering pro-inflammatory responses in Kupffer cells (KCs) and activating hepatic stellate cells (HSCs). Capillarized LSECs show a pro-inflammatory phenotype through adhesion molecule overexpression, autophagy loss, and increased cytokines production. Platelet interaction favors leucocyte recruitment, NETs formation, and liver inflammatory foci. Liver fibrosis is facilitated by reduced nitric oxide, HSC activation, profibrogenic mediators, and increased angiogenesis. Moreover, platelet attachment, activation, α-granule cargo release, and NETs formation contribute to MASLD progression. Platelets foster fibrosis and microthrombosis, leading to parenchymal extinction and fibrotic healing. Additionally, platelets promote tumor growth, epithelial-mesenchymal transition, and tumor cell metastasis. MASLD's prothrombotic features are exacerbated by insulin resistance, diabetes, and obesity, manifesting as increased von Willebrand factor, platelet hyperaggregability, hypo-fibrinolysis, and a prothrombotic fibrin clot structure. Improving LSEC health and using antiplatelet treatment appear promising for preventing MASLD development and progression.