Fibrinogen Clotting Research Articles

Induction of chronic asthma up regulated the transcription of senile factors in male rats

BackgroundThe main characteristic of asthma is chronic inflammation. We examined cellular senescence by histology and molecular assay in the lungs of a rat model of asthma. This model comprises sensitization by several intraperitoneal injections of ovalbumin with aluminium hydroxide, followed by aerosol challenges every other day.ResultsData showed that asthma induction caused histological changes including, hyperemia, interstitial pneumonia, fibrinogen clots, and accumulation of inflammatory cells in the pleura. There is an elevation of IL-1β and NF-kB proteins in the asthmatic group (P < 0.001) compared to the control group. The expression of ß-galactosidase increased (P < 0.01), while the expression of Klotho and Sox2 genes was decreased in the lung tissue of the asthmatic group (P < 0.01).ConclusionTaken together, these findings suggest that asthmatic conditions accelerated the cellular senescence in the lung tissue.

A Small-Molecule NIR-II Probe for the Diagnosis of Hemorrhagic Diseases.

Numerous hemorrhagic disorders, particularly those presenting deep hemorrhage, pose diagnostic challenges, often leading to delayed treatment and severe outcomes. Near-infrared (NIR)-II fluorescence imaging offers advantages such as deep tissue penetration, real-time visualization, and a high signal-to-background ratio, making it highly suitable for diagnosing hemorrhagic diseases. In this study, an NIR-II fluorescent probe LJ-2P carrying carboxylic and phosphoric acid groups is successfully applied for imaging hemorrhagic diseases. LJ-2P demonstrates a strong affinity for fibrinogen and fibrin clots both computationally and experimentally, thus exhibiting increased brightness upon coagulation. As compared to Indocyanine Green, LJ-2P provides a longer imaging window, higher imaging specificity, and signal-to-background ratio, as well as superior photobleaching resistance in three disease models: gastric, pulmonary, and cerebral hemorrhages. These results reveal that LJ-2P demonstrates enhanced imaging capabilities, enabling precise identification of hemorrhagic sites.

Comparison of the Quantra QPlus and ROTEM Goal-Directed Transfusion Protocols in Cardiothoracic Surgery Patients: A Prospective Observational Study

To compare the designed treatment protocols for the Quantra QPlus and rotational thromboelastometry (ROTEM) with regard to transfusion advice. Prospective observational study. Maastricht University Medical Center, The Netherlands. Adults with elective cardiopulmonary bypass surgery with a ROTEM test. ROTEM tests were performed postoperatively for standard monitoring of coagulation status and clinical decision making. Simultaneously, a concurrent sample was analyzed for the Quantra QPlus. A total of 100 samples were analyzed using both the ROTEM and Quantra QPlus. Agreement between the transfusion advice for the ROTEM and Quantra QPlus protocols were compared using Cohen κ values for i.a. fibrinogen, platelet concentrates, and fresh frozen plasma (FFP). The agreement between ROTEM and Quantra QPlus was poor for overall transfusion (0.174) and fibrinogen transfusion (0.300). The agreement of cutoff values for fibrinogen clot stiffness for the Quantra QPlus and EXTEM A10 for the ROTEM was poor (0.160). The fibrinogen clot stiffness and FIBTEM A10 had a moderate agreement (0.731). A Cohen κ could not be calculated for the agreement of protamine, thrombocytes, FFP or cutoff values for these transfusions since frequencies included zero in these cases. The Quantra QPlus transfusion protocol advises transfusion in many non-bleeders, adjustments appear to be necessary. In a small group of cases in which clinically relevant blood loss was observed, the Quantra QPlus advised administration of transfusion products, whereas the ROTEM tests did not. ROTEM-guided and Quantra-guided transfusion did not correspond in this patient group, and agreement was moderate at best. Specificity and sensitivity for transfusion within protocols were heterogeneous between the methods. More clinical research in high-bleeding risk populations is needed to determine the clinical impact of the different protocols.

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

Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms.

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.

HIV infection and ART use are associated with altered plasma clot characteristics in Black South Africans.

Human immunodeficiency virus (HIV) and antiretroviral treatment (ART) are both associated with hypercoagulability. Altered clot properties could be a potential mechanism thereof. We aimed to investigate the association of HIV and ART, with fibrinogen and plasma clot properties in a group of Black South Africans. At baseline, 151 newly diagnosed people living with HIV (PLWH) and 176 controls were recruited. Some PLWH subsequently commenced with ARTs (n = 70) while others remained ART-naïve (n = 81). Fibrinogen and clot properties (turbidity assay) were investigated from baseline to 5-year follow-up. A sub-group of 21 women (n = 10 ART-treated; n = 11 ART-naïve) with HIV was systematically selected and matched with 12 controls, and additional clot properties (rheometry, permeability and fibre diameter) were investigated. Fibrinogen was lower in the HIV groups compared to the controls, while % γ' fibrinogen was higher. PLWH had shorter lag times and lower maximum absorbance than the controls (p<0.05). Their CLTs on the other hand were longer. Most variables increased over time in all groups, but differences in the degree of change over time was observed for lag time (p = 0.024) and permeability (p = 0.03). Participants who commenced with ART had a tendency of delayed clot formation (p = 0.08) and increased clot permeability (p = 0.005). PLWH had lower total fibrinogen concentration and formed less dense clots. They also formed clots that were more difficult to lyse, which likely not resulted from altered clot properties. ART use (NNRTI's) had a moderately protective effect, delaying clot formation, and increasing clot permeability.

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.

Effect of Seaweed-Derived Fucoidans from Undaria pinnatifida and Fucus vesiculosus on Coagulant, Proteolytic, and Phospholipase A2 Activities of Snake Bothrops jararaca, B. jararacussu, and B. neuwiedi Venom.

Snakebite envenomation (SBE) causes diverse toxic effects in humans, including disability and death. Current antivenom therapies effectively prevent death but fail to block local tissue damage, leading to an increase in the severity of envenomation; thus, seeking alternative treatments is crucial. This study analyzed the potential of two fucoidan sulfated polysaccharides extracted from brown seaweeds Fucus vesiculosus (FVF) and Undaria pinnatifida (UPF) against the fibrinogen or plasma coagulation, proteolytic, and phospholipase A2 (PLA2) activities of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom. The toxicity of FVF and UPF was assessed by the hemocompatibility test. FVF and UPF did not lyse human red blood cells. FVF and UPF inhibited the proteolytic activity of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom by approximately 25%, 50%, and 75%, respectively, while all venoms led to a 20% inhibition of PLA2 activity. UPF and FVF delayed plasma coagulation caused by the venoms of B. jararaca and B. neuwiedi but did not affect the activity of B. jararacussu venom. FVF and UPF blocked the coagulation of fibrinogen induced by all these Bothropic venoms. FVF and UPF may be of importance as adjuvants for SBE caused by species of Bothrops, which are the most medically relevant snakebite incidents in South America, especially Brazil.

Fibrinogen levels and clot properties identify patients who benefit from catheter-directed thrombolysis after DVT

AbstractUltrasound-accelerated catheter–directed thrombolysis (UA-CDT) to improve patency after deep vein thrombosis (DVT) has not conclusively been shown to prevent postthrombotic syndrome (PTS) but might benefit patients who are unlikely to obtain patency with standard treatment. We hypothesized that these patients could be selected based on their fibrin clot properties. To study this, patients with acute iliofemoral DVT from the CAVA (Ultrasound-Accelerated Catheter-Directed Thrombolysis Versus Anticoagulation for the Prevention of Post-thrombotic Syndrome) trial had blood samples taken at inclusion. Fibrin clot properties in plasma were determined by turbidimetric clotting (lag time and maximal turbidity) and lysis assays (time to 50% lysis and lysis rate), permeation assay, and confocal microscopy (fiber density), as well as levels of fibrin clot modifiers fibrinogen and C-reactive protein (CRP). Patency was defined as >90% iliofemoral vein compressibility at 12-month ultrasound. PTS was defined as ≥5 Villalta score at 6 or 12 months. In total, 91 of 152 patients were included, including 43 with additional UA-CDT and 48 with standard treatment. Patients with additional UA-CDT more often obtained patency (55.8 vs 27.1%) Patients who obtained patency had longer lag times and lower maximal turbidity, fibrinogen, and CRP; only maximal turbidity and fibrinogen remained associated when adjusting for treatment, thrombus load, and body mass index. Fibrinogen levels had an optimal cutoff at 4.85 g/L. Low fibrinogen levels best predicted patency. Additional UA-CDT decreased the risk of PTS only in patients with high fibrinogen. Therefore, additional UA-CDT might prevent PTS in selected patients based on routinely measured fibrinogen levels. This study was registered at www.ClinicalTrials.gov as #NCT00970619.

Analyzing and modeling massive transfusion strategies and the role of fibrinogen-How much is the patient actually receiving?

Hemorrhage is a leading cause of preventable death in trauma, cardiac surgery, liver transplant, and childbirth. While emphasis on protocolization and ratio of blood product transfusion improves ability to treat hemorrhage rapidly, tools to facilitate understanding of the overall content of a specific transfusion strategy are lacking. Medical modeling can provide insights into where deficits in treatment could arise and key areas for clinical study. By using a transfusion model to gain insight into the aggregate content of massive transfusion protocols (MTPs), clinicians can optimize protocols and create opportunities for future studies of precision transfusion medicine in hemorrhage treatment. The transfusion model describes the individual round and aggregate content provided by four rounds of MTP, illustrating that the total content of blood elements and coagulation factor changes over time, independent of the patient's condition. The configurable model calculates the aggregate hematocrit, platelet concentration, percent volume plasma, total grams and concentration of citrate, percent volume anticoagulant and additive solution, and concentration of clotting factors: fibrinogen, factor XIII, factor VIII, and von Willebrand factor, provided by the MTP strategy. Transfusion strategies based on a 1:1:1 or whole blood foundation provide between 13.7 and 17.2 L of blood products over four rounds. Content of strategies varies widely across all measurements based on base strategy and addition of concentrated sources of fibrinogen and other key clotting factors. Differences observed between modeled transfusion strategies provide key insights into potential opportunities to provide patients with precision transfusion strategy.

Phototriggered Equilibrated and Transient Orthogonally Operating Constitutional Dynamic Networks Guiding Biocatalytic Cascades.

The photochemical deprotection of structurally engineered o-nitrobenzylphosphate-caged hairpin nucleic acids is introduced as a versatile method to evolve constitutional dynamic networks, CDNs. The photogenerated CDNs, in the presence of fuel strands, interact with auxiliary CDNs, resulting in their dynamically equilibrated reconfiguration. By modification of the constituents associated with the auxiliary CDNs with glucose oxidase (GOx)/horseradish peroxidase (HRP) or the lactate dehydrogenase (LDH)/nicotinamide adenine dinucleotide (NAD+) cofactor, the photogenerated CDN drives the orthogonal operation upregulated/downregulated operation of the GOx/HRP and LDH/NAD+ biocatalytic cascade in the conjugate mixture of auxiliary CDNs. Also, the photogenerated CDN was applied to control the reconfiguration of coupled CDNs, leading to upregulated/downregulated formation of the antithrombin aptamer units, resulting in the dictated inhibition of thrombin activity (fibrinogen coagulation). Moreover, a reaction module consisting of GOx/HRP-modified o-nitrobenzyl phosphate-caged DNA hairpins, photoresponsive caged auxiliary duplexes, and nickase leads upon irradiation to the emergence of a transient, dissipative CDN activating in the presence of two alternate auxiliary triggers, achieving transient operation of up- and downregulated GOx/HRP biocatalytic cascades.

Comprehensive Analysis of the Role of Fibrinogen and Thrombin in Clot Formation and Structure for Plasma and Purified Fibrinogen.

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

Hemostatic bioactivity and mechanism of novel Rubia cordifolia L.-derived carbon dots.

Background: Rubia cordifolia L. (RCL) Carbonisata is a typical calcined natural medicinal plant, which has been used for thousands of years for hemostasis. At present, some studies have shown that some components of processed RCL Carbonisata can enhance hemostasis, but the specific hemostatic material basis is still unclear. Novel carbon dots (CDs) were obtained from Rubia cordifolia L. and named RCL-CDs to explore the hemostatic effect and mechanism of RCL-CDs obtained from Rubia cordifolia L. Methods: RCL-CDs were characterized by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet visible spectroscopy (UV-Vis), fluorescence spectroscopy (FL), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The hemostatic effect of RCL-CDs was evaluated in a mouse tail amputation model and liver scratch model, and the hemostatic mechanism was explored using a capillary coagulation model and coagulation parameters. Results: The particle size distribution of RCL-CDs ranged from 1.74 nm to 9.78 nm, the maximum population was 3-4 nm, and the average particle size was 3.82 nm. The RCL-CDs were approximately spherical with a lattice spacing of 0.206 nm. The quantum yield (QY) of RCL-CDs is 1.09%, and there is a distinct diffraction peak at 2θ = 24.76°. The elemental composition of RCL-CDs was mainly C (65.28%), O (30.10%), and a small amount of N (4.62%). Pharmacological experiments showed that bleeding time and bleeding volume were reduced in mice treated with RCL-CDs. It is worth noting that the low-, medium- and high-dose RCL-CD groups can significantly reduce the blood loss, while the high-dose RCL-CD group can significantly reduce the bleeding time of the mouse tail amputation model and liver scratch model. Additionally, the fibrinogen level (FIB) and platelet counts (PLT) increased and prothrombin time (PT) decreased in rats after treatment with RCL-CDs. Conclusions: RCL-CDs have a significant hemostatic effect, and the mechanism may be exogenous coagulation and activation of fibrinogen. This explains the material basis of the hemostatic effect of RCLC and opens new avenues for more in-depth investigation. In addition, new insights into the potential biomedical applications of CDs in the field of nanohemostasis are provided and a solid foundation for the discovery of novel hemostatic agents is established.

Investigation of acquired dysfibrinogenaemia in adult patients with sepsis using fibrinogen function vs. concentration ratios: a cross-sectional study.

Inherited or acquired molecular abnormalities form a clinically heterogeneous group of fibrinogen disorders called dysfibrinogenaemia. Apart from a pediatric case report and in contrast to other clinical conditions, acquired dysfibrinogenaemia has not been previously reported in septic patients. In an observational cohort study, 79 adult septic patients were investigated for the presence of acquired dysfibrinogenaemia at the time of their admission to the intensive care unit (ICU) of the University Hospital Frankfurt. Following established recommendations, fibrinogen clotting activity vs. antigen ratios were analyzed using Clauss fibrinogen, prothrombin-derived fibrinogen, and radial immunodiffusion (RID) fibrinogen concentration. Prothrombin-derived fibrinogen levels were highest (527 ± 182 mg/dL) followed by Clauss fibrinogen (492 ± 209 mg/dL) and radial immunodiffusion fibrinogen (426 ± 159 mg/dL). Very few cases demonstrated hypofibrinogenaemia making overt disseminated intravascular coagulation (DIC) unlikely in the cohort investigated. Clauss/RID fibrinogen ratios were lower (1.17 ± 0.19) compared to prothrombin time-derived/RID ratios (1.35 ± 0.33). Using the Clauss/RID dataset, 21% of patients (16/76 patients) demonstrated values below a threshold ratio for suspected acquired dysfibrinogenaemia arbitrarily set at 1.0. In contrast, prothrombin-derived ratios were below the threshold in only 7% (4/58 patients). The results point to the presence of acquired dysfibrinogenaemia in part of adult septic patients. If confirmed in further studies, this may form part of a specific laboratory signature of a sepsis-associated coagulation phenotype.

ROS-Driven Structural and Functional Fibrinogen Modifications Are Reverted By JAK Inhibition in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are bone marrow diseases characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Subtypes include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They share clinical features (e.g., splenomegaly, thrombotic complications, risk of leukemic transformation), and a common molecular basis: the dysregulation of the JAK-STAT pathway, which confers a hypersensitivity of hematopoietic stem cells (HSC) to the action of growth factors and cytokines, and, as a result, an overproduction of blood cells. Genetic analysis of patients with MPNs revealed that &amp;gt;95% of patients with PV and about 50% of patients with ET or PMF carry an activating point mutation in the JAK2 gene (JAK2-V617F). Despite clinical evidence indicating a higher susceptibility of MPN patients to develop thrombotic disease, the underlying pathogenetic mechanisms are not fully understood. It appears that thrombosis is the result of a severe inflammatory response associated to remarkable oxidative stress in the presence of endothelial dysfunction and a procoagulant environment. Hypercoagulability is a consequence of disrupted hemostasis and fibrinolysis due to damage of the endothelium and defective fibrinolysis. Evidence suggests that overproduction of ROS and a deprived antioxidant system play a major role in the pathogenesis of MPN, as well as in the resistance to tyrosine-kinase inhibitor therapy. Moreover, systemic inflammation is thought to be the main trigger of thrombosis and seems to be mainly mediated by pro-inflammatory cytokines along with endothelial cell dysfunction. Recent studies have provided evidence that fibrinogen plays a multifaceted role in inflammatory responses and autoimmunity. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with integrins, the leukocyte cell surface adhesion receptors expressed on neutrophils, monocytes, macrophages, and several subsets of lymphocytes. In MPN patients, endothelial cell dysfunction, increased reactive oxygen species (ROS) production, and proinflammatory cytokines have been reported, together with impaired fibrinolysis. We also investigated the potential role of oxidative stress and inflammation in the promotion of thrombosis. In order to study the mechanisms of inflammation-induced thrombosis in MPN patients, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in MPN patients. The eventual counteracting effects of ruxolitinib, a first-in-class JAK inhibitor, were also assessed. 40 MPN patients and 40 age- and sex-matched healthy control subjects were included in the study. We measured specific oxidative stress markers andtheir modifications in plasma, and functional and structural parameters in purified fibrinogen fractions. Marked signs of oxidative stress in plasma (P&amp;lt;0.01 versus controls) and, correspondingly, an increased extent of fibrinogen oxidation (P&amp;lt;0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from MPN patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P&amp;lt;0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture were also identified. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and clot porosity in MPN patients (P&amp;lt;0.01 versus controls). In patient samples, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in MPNs. In patients receiving ruxolitinib, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features, suggesting a potential impactof ruxolitinib on cardiovascular prevention in MPNs.

Dynamic DNA Networks-Guided Directional and Orthogonal Transient Biocatalytic Cascades.

DNA frameworks, consisting of constitutional dynamic networks (CDNs) undergoing fuel-driven reconfiguration, are coupled to a dissipative reaction module that triggers the reconfigured CDNs into a transient intermediate CDNs recovering the parent CDN state. Biocatalytic cascades consisting of the glucose oxidase (GOx)/horseradish peroxidase (HRP) couple or the lactate dehydrogenase (LDH)/nicotinamide adenine dinucleotide (NAD+) couple are tethered to the constituents of two different CDNs, allowing the CDNs-guided operation of the spatially confined GOx/HRP or LDH/NAD+ biocatalytic cascades. By applying two different fuel triggers, the directional transient CDN-guided upregulation/downregulation of the two biocatalytic cascades are demonstrated. By mixing the GOx/HRP-biocatalyst-modified CDN with the LDH/NAD+-biocatalyst-functionalized CDN, a composite CDN is assembled. Triggering the composite CDN with two different fuel strands results in orthogonal transient upregulation of the GOx/HRP cascade and transient downregulation of the LDH/NAD+ cascade or vice versa. The transient CDNs-guided biocatalytic cascades are computationally simulated by kinetic models, and the computational analyses allow the prediction of the performance of transient biocatalytic cascades under different auxiliary conditions. The concept of orthogonally triggered temporal, transient, biocatalytic cascades by means of CDN frameworks is applied to design an orthogonally operating CDN for the temporal upregulated or downregulated transient thrombin-induced coagulation of fibrinogen to fibrin.

DNA and histones impair the mechanical stability and lytic susceptibility of fibrin formed by staphylocoagulase.

Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

Transient Transcription Machineries Modulate Dynamic Functions of G‐Quadruplexes: Temporal Regulation of Biocatalytic Circuits, Gene Replication and Transcription

AbstractNative G‐quadruplex‐regulated temporal biocatalytic circuits, gene polymerization, and transcription processes are emulated by biomimetic, synthetically engineered transcription machineries coupled to reconfigurable G‐quadruplex nanostructures. These are addressed by the following example: (i) A reaction module demonstrates the fuel‐triggered transcription machinery‐guided transient synthesis of G‐quadruplex nanostructures. (ii) A dynamically triggered and modulated transcription machinery that guides the temporal separation and reassembly of the anti‐thrombin G‐quadruplex aptamer/thrombin complex is introduced, and the transient thrombin‐catalyzed coagulation of fibrinogen is demonstrated. (iii) A dynamically fueled transient transcription machinery for the temporal activation of G‐quadruplex‐topologically blocked gene polymerization circuits is introduced. (iv) Transcription circuits revealing G‐quadruplex‐promoted or G‐quadruplex‐inhibited cascaded transcription machineries are presented. Beyond advancing the rapidly developing field of dynamically modulated G‐quadruplex DNA nanostructures, the systems introduce potential therapeutic applications.

Super-high procoagulant activity of gecko thrombin: A gift from sky dragon.

Gecko, the "sky dragon" named by Traditional Chinese Medicine, undergoes rapid coagulation and scarless regeneration following tail amputation in the natural ecology, providing a perfect opportunity to develop the efficient and safe drug for blood clotting. Here, gecko thrombin (gthrombin) was recombinantly prepared and comparatively studied on its procoagulant activity. The 3D structure of gthrombin was constructed using the homology modeling method of I-TASSER. The active gthrombin was prepared by the expression of gecko prethrombin-2 in 293 T cells, followed by purification with Ni2+ -chelating column chromatography prior to activation by snake venom-derived Ecarin. The enzymatic activities of gthrombin were assayed by hydrolysis of synthetic substrate S-2238 and the fibrinogen clotting. The vulnerable nerve cells were used to evaluate the toxicity of gthrombin at molecular and cellular levels. The active recombinant gthrombin showed super-high catalytic and fibrinogenolytic efficiency than those of human under different temperatures and pH conditions. In addition, gthrombin made nontoxic effects on the central nerve cells including neurons, contrary to those of mammalian counterparts, which contribute to neuronal damage, astrogliosis, and demyelination. A super-high activity but safe procoagulant candidate drug was identified from reptiles, which provided a promising perspective for clinical application in rapid blood clotting.

Fabrication of Fibrin/Polyvinyl Alcohol Scaffolds for Skin Tissue Engineering via Emulsion Templating

In the search for a novel and scalable skin scaffold for wound healing and tissue regeneration, we fabricated a class of fibrin/polyvinyl alcohol (PVA) scaffolds using an emulsion templating method. The fibrin/PVA scaffolds were formed by enzymatic coagulation of fibrinogen with thrombin in the presence of PVA as a bulking agent and an emulsion phase as the porogen, with glutaraldehyde as the cross-linking agent. After freeze drying, the scaffolds were characterized and evaluated for biocompatibility and efficacy of dermal reconstruction. SEM analysis showed that the formed scaffolds had interconnected porous structures (average pore size e was around 330 µm) and preserved the nano-scale fibrous architecture of the fibrin. Mechanical testing showed that the scaffolds' ultimate tensile strength was around 0.12 MPa with an elongation of around 50%. The proteolytic degradation of scaffolds could be controlled over a wide range by varying the type or degree of cross-linking and by fibrin/PVA composition. Assessment of cytocompatibility by human mesenchymal stem cell (MSC) proliferation assays shows that MSC can attach, penetrate, and proliferate into the fibrin/PVA scaffolds with an elongated and stretched morphology. The efficacy of scaffolds for tissue reconstruction was evaluated in a murine full-thickness skin excision defect model. The scaffolds were integrated and resorbed without inflammatory infiltration and, compared to control wounds, promoted deeper neodermal formation, greater collagen fiber deposition, facilitated angiogenesis, and significantly accelerated wound healing and epithelial closure. The experimental data showed that the fabricated fibrin/PVA scaffolds are promising for skin repair and skin tissue engineering.