Blood Coagulation Cascade Research Articles

Phytochemical and Biological Investigations of Different Partitioned Extracts of Ficus heteropleura Blume (Family: Moraceae) Leaves

Ficus heteropleura Blume is a plant of the Moraceae family with high medicinal value. Therefore, the plant leaves' fractional extracts have been subjected to preliminary screenings for phytoconstituents and pharmacological studies. The phytochemical analysis was done after fractionating the ethanolic extract with n-hexane, chloroform, and hydro-alcohol. Then, the TLC method, DPPH radical scavenging, disc diffusion, membrane stabilization activity, heat-induced hemolysis, human blood clot lysis, and starch-iodine method were performed. The chemical investigation resulted in the identification of glycosides, amides, reducing sugars, flavonoids, phenols, alkaloids, tannins, saponins, gums, and steroids from the different extracts of the leaves of Ficus heteropleura Blume. In the antioxidant assay, hydro-alcoholic, chloroform, and n-hexane extracts at the dose of 100μg/ml produced significant inhibition of 76.11% (IC50 = 46.47 µg/ml), 72.83% (IC50 = 54.72 µg/ml), and 61.83% (IC50 = 77.95 µg/ml), respectively, as compared to the reference standard drug, ascorbic acid, 85.01% (IC50 = 32.22 µg/ml). However, only moderate anti-inflammatory activity was found in hydro-alcoholic extract (IC50 = 4.24 µg/ml). The extract also showed moderate thrombolytic and membrane-stabilizing effects. Hydro-alcoholic extract had antifungal activity against only Candida albicans (11 mm). However, chloroform plant extract exhibited significant antifungal activity against all included fungi. In the anti-diabetic study, significant inhibition of amylase activity was found by the hydro-alcoholic (73.33%) and chloroform (83.3%) extracts, respectively, whereas the n-hexane was found to have moderate activity (35.44%) compared to acarbose. Therefore, before isolation, characterization, and determination of the mechanism of action of the screened pharmacological activities, further investigation is needed.

A collateral circulation in ischemic stroke accelerates recanalization due to lower clot compaction.

Collaterals improve recanalization in acute ischemic stroke patients treated with intravenous thrombolysis, but the mechanisms are poorly understood. To investigate it, an in vitro flow model of the middle cerebral artery was developed with or without collaterals. An occlusion was achieved using human blood clots. Recanalization time, thrombolysis (clot length decrease and red blood cell (RBC) release), pressure gradient across the clot and clot compaction were measured. Results showed that with or without collateral alteplase-treated RBC dominant clots showed recanalization time 98±23 min vs 130±35 min (difference 32 min, 95% CI -6-58 min), relative clot reduction 31.8±14.9% vs 30.3±13.2% (difference 1.5%, 95% CI 10.4-13.4%) and RBC release 0.30±0.07 vs 0.27±0.09 (difference 0.03, 95% CI 0.04-0.10). Similar results were observed with fibrin-dominant clots. In RBC dominant clots, the presence vs absence of collateral caused different pressure gradients across the clot 0.41±0.09 vs 0.70±0.09 mmHg (difference 0.29 mmHg, 95% CI -0.17-0.41 mmHg), and caused the reduction of initial clot compaction by 5%. These findings align with observations in patients, where collaterals shortened recanalization time. However, collaterals did not increase thrombolysis. Instead, they decreased the pressure gradient across the clot, resulting in less clot compaction and easier distal displacement of the clot.

Fabrication, microstructure and hemostatic activity of Cu-Zn manganite nanoparticles

Copper subbed zinc manganite Zn(1-x)CuxMn2O4 (x = 0.05,0.15,0.25,0.35,0.45,0.55) (BCZMO) NPs were prepared by sol–gel co-precipitation technique at surrounding temperature. BCZMO NPs were analyzed by XRD, SEMfor microstructure characterizations. XRD study confirms the tetragon structure of BCZMO NPs and effect of copper on ZMO NPs. SEM micrographs supports our findings. The BCZMO NPs interfered in blood coagulation and exhibited pro-coagulant property. BCZMO NPs decreased the coagulation time of citrated plasma from control 4.31 min to 1.35 min. In addition, the BCZMO NPs aggregated platelets up to 80 % at a concentration of 600 µg while the agonist epinephrine prompted platelet collection of around 90 % at 10 mM concentration.No kind of hemolytic activity in RBC cells shown by these BCZMO-NPs insisting their non-toxic nature. These BCZMO-NPs exhibited pro-coagulant effect with their interference in blood coagulation cascade and initiation in platelet aggregation. Thus, BCZMO NPs can be a potential candidate in biomedical field as hemostatic agents.

A novel method for the degradation of human blood clot by immobilised bromelain using multi-walled carbon nanotube and polyphenol oxidase

Pathological blood clot in blood vessels, which often leads to cardiovascular diseases, are one of the most common causes of death in humans. Therefore, enzymatic therapy to degrade blood clots is vital. To achieve this goal, bromelain was immobilized and used for the biodegradation of blood clots. Bromelain was extracted from the pineapple fruit pulp (Ananas comosus) and purified by ion exchange chromatography after precipitation with ammonium sulphate (0-80 %), resulting in a yield of 70%, purification fold of 1.42, and a specific activity of 1175 U/mg. Bromelain was covalently immobilized on functionalized multi-walled carbon nanotubes (MWCNT), with an enzyme loading of 71.35%. The results of the characterization of free and immobilized bromelain demonstrated that the optimum pH for free and immobilized bromelain activity was 7.0, while the pH range of stability was from 5.0 to 8.5 and 4.0 to 9.0, respectively. The optimum temperature for free and immobilized bromelain activity was 45ºC, whereas the stability was 15 to 50°C and 15 to 55°C, respectively. The immobilized bromelain activity was decreased after the fifth reuse, and the storage period of the free and immobilized bromelain was decreased after 6 and 123 days, respectively. Casein was the best substrate-free bromelain, and fibrin was the best substrate for immobilized bromelain. The results of the purification of polyphenol oxidases (PPO) from potatoes by ion exchange chromatography gave a yield of about 54 %, a purification fold of 1.27, and a specific activity of 2804 U/mg. The current study showed that the immobilized bromelain can significantly biodegrade human blood clots in vitro, while the PPO enzyme has no significant effect on blood clots.

Coagulo-Net: Enhancing the mathematical modeling of blood coagulation using physics-informed neural networks

Blood coagulation, which involves a group of complex biochemical reactions, is a crucial step in hemostasis to stop bleeding at the injury site of a blood vessel. Coagulation abnormalities, such as hypercoagulation and hypocoagulation, could either cause thrombosis or hemorrhage, resulting in severe clinical consequences. Mathematical models of blood coagulation have been widely used to improve the understanding of the pathophysiology of coagulation disorders, guide the design and testing of new anticoagulants or other therapeutic agents, and promote precision medicine. However, estimating the parameters in these coagulation models has been challenging as not all reaction rate constants and new parameters derived from model assumptions are measurable. Although various conventional methods have been employed for parameter estimation for coagulation models, the existing approaches have several shortcomings. Inspired by the physics-informed neural networks, we propose Coagulo-Net, which synergizes the strengths of deep neural networks with the mechanistic understanding of the blood coagulation processes to enhance the mathematical models of the blood coagulation cascade. We assess the performance of the Coagulo-Net using two existing coagulation models with different extents of complexity. Our simulation results illustrate that Coagulo-Net can efficiently infer the unknown model parameters and dynamics of species based on sparse measurement data and data contaminated with noise. In addition, we show that Coagulo-Net can process a mixture of synthetic and experimental data and refine the predictions of existing mathematical models of coagulation. These results demonstrate the promise of Coagulo-Net in enhancing current coagulation models and aiding the creation of novel models for physiological and pathological research. These results showcase the potential of Coagulo-Net to advance computational modeling in the study of blood coagulation, improving both research methodologies and the development of new therapies for treating patients with coagulation disorders.

1. The attraction of fish blood coagulation system ―Bullhead shark novel blood coagulation cascade―

1. The attraction of fish blood coagulation system ―Bullhead shark novel blood coagulation cascade―

Local delivery of platelet-derived factors mitigates ischemia and preserves ovarian function through angiogenic modulation: A personalized regenerative strategy for fertility preservation

As the most prominent and ideal modality in female fertility preservation, ovarian tissue cryopreservation, and transplantation often confront the challenge of ischemic damage and follicular loss from avascular transplantation. To surmount this impediment, we engineered a novel platelet-derived factors-encapsulated fibrin hydrogel (PFH), a paradigmatic biomaterial. PFH encapsulates autologous platelet-derived factors, utilizing the physiological blood coagulation cascade for precise local delivery of bioactive molecules. In our study, PFH markedly bolstered the success of avascular ovarian tissue transplantation. Notably, the quantity and quality of follicles were preserved with improved neovascularization, accompanied by decreased DNA damage, increased ovulation, and superior embryonic development rates under a Low-concentration Platelet-rich plasma-derived factors encapsulated fibrin hydrogel (L-PFH) regimen. At a stabilized point of tissue engraftment, gene expression analysis mirrored normal ovarian tissue profiles, underscoring the effectiveness of L-PFH in mitigating the initial ischemic insult. This autologous blood-derived biomaterial, inspired by nature, capitalizes on the blood coagulation cascade, and combines biodegradability, biocompatibility, safety, and cost-effectiveness. The adjustable properties of this biomaterial, even in injectable form, extend its potential applications into the broader realm of personalized regenerative medicine. PFH emerges as a promising strategy to counter ischemic damage in tissue transplantation, signifying a broader therapeutic prospect. (197 words)

In vivo and in silico studies of membrane-stabilizing and clot lysis activities of Trachyspermum ammi

Trachyspermum ammi contains many important phytochemicals, including thymol, which is used to treat many diseases. This study aimed to check the membrane-stabilizing and clot-lysis capacity of the ethanol extract of T. ammi (ETA) and its main chemical component, thymol (THY). For this, we used hypotonic solution-induced erythrocyte lysing and egg-albumin protein denaturation for membrane-stabilizing capacity and human blood clot lysis for anti-atherombosis capacity. Further, we observed in silico study of THY and ntric oxide synthase (NOSs) isoform. The stabilizing effects of ETA and THY on the membrane changed depending on the concentration. At the highest concentration (160 μg/mL), they stopped the most bleeding, while the standard drug, acetylsalicylic acid, stopped less bleeding at the same concentration. In the erythrocyte lysing test, ETA showed 102.60 ± 0.01 and 75.83 ± 1.44% membrane protection. In the egg-albumin denaturation test, they showed 27.62 ± 0.02% and 56.71 ± 0.02% membrane protection, respectively. In our clot lysis experiment, ETA and THY also showed significant and concentration-dependent clot lysis capacity. The IC50 and EC50 values of THY were lower than ETA when it came to keeping the membrane stable and breaking up clots, respectively. In our in silico investigation, THY and NOSs showed significant binding interaction. Taken together, ETA and its major component, THY, exhibited potent membrane stabilizing activity and clot lysis activity in in vitro studies. Further studies are required to elucidate its other active principles and their biological effects, along with possible mechanisms.

Investigation of a Serine Protease Inhibitor Active in the Infectious Stage of the Human Liver Fluke Opisthorchis viverrini.

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process.

Recent Advances in Gene Therapy for Hemophilia: Projecting the Perspectives.

One of the well-known X-linked genetic disorders is hemophilia, which could be hemophilia A as a result of a mutation in the F8 (factor VIII) gene or hemophilia B as a result of a mutation in the F9 (factor IX) gene, leading to insufficient levels of the proteins essential for blood coagulation cascade. In patients with severe hemophilia, factor VIII or factor IX activities in the blood plasma are considerably low, estimated to be less than 1%. This is responsible for spontaneous or post-traumatic bleeding episodes, or both, leading to disease complications and death. Current treatment of hemophilia relies on the prevention of bleeding, which consists of expensive lifelong replacement infusion therapy of blood plasma clotting factors, their recombinant versions, or therapy with recombinant monoclonal antibodies. Recently emerged gene therapy approaches may be a potential game changer that could reshape the therapeutic outcomes of hemophilia A or B using a one-off vector in vivo delivery and aim to achieve long-term endogenous expression of factor VIII or IX. This review examines both traditional approaches to the treatment of hemophilia and modern methods, primarily focusing on gene therapy, to update knowledge in this area. Recent technological advances and gene therapeutics in the pipeline are critically reviewed and summarized. We consider gene therapy to be the most promising method as it may overcome the problems associated with more traditional treatments, such as the need for constant and expensive infusions and the presence of an immune response to the antibody drugs used to treat hemophilia.

NanoGraphene Clot: A New Fibrinogen-Mimic Hemostatic Material.

Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 μM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be ∼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Membrane-stabilizing and clot lysis activities of (±) citronellal: In-vitro studies

Numerous plants and their phytochemicals have been identified as having potential medicinal values and are used to treat different ailments worldwide. (±) Citronellal (CTL) is a monoterpene phytochemical with potential therapeutic benefits including anti-inflammatory, antioxidant, antifungal, and antibacterial actions. AimThe present study aimed to evaluate the membrane-stabilizing and clot-lysis activities of CTL through in vitro studies. For this, we performed hypotonic solution-induced erythrocyte lysing and human blood clot lysis methods to check the membrane-stabilizing and clot lysis capacities of CTL using acetylsalicylic acid (ASA) and streptokinase (STK) as standards, respectively. CTL exhibited concentration-dependent membrane-stabilizing activity with an IC50 = 28.83 ± 1.19 µg/mL. In the clot lysis test, CTL also showed a concentration-dependent clot lysing capacity, where it exhibited 50.46 ± 3.81 % clot lysis at a concentration of 160 μg/mL. In the latter case, the IC50 value was I158.22 ± 2.21 µg/mL. CTL exhibit potent membrane-stabilizing and moderate clot-lysis activity. We suppose that CTL may exert these effects, possibly through its capacity to inhibit inflammatory mediators. Further in vivo and in silico studies are required to elucidate CTL’s exact molecular mechanisms behind these biological effects.

Herbal treatments: are there risks for ENT surgery?

When using herbal treatments, we should consider not only their beneficial effects, but also the potential for drug interactions. Serious problems may occur when used in combination with anesthetics and therapeutic agents. Some herbal treatments and biologically active supplements containing herbal components may affect the blood coagulation cascade and increase bleeding during endoscopic procedures in the lumen of ENT organs. They may also interact with later prescribed agents. Therefore, when taking the patient’s history, we should ask not only about the use of pharmaceutical agents, but also about the use of dietary supplements, herbal teas, and some herbal products. In some cases, it is necessary to discontinue herbal treatments in advance to avoid complications during general anesthesia and surgery.

Xa inhibitor edoxaban ameliorates hepatic ischemia-reperfusion injury via PAR-2-ERK 1/2 pathway.

Hepatic ischemia-reperfusion injury causes liver damage during surgery. In hepatic ischemia-reperfusion injury, the blood coagulation cascade is activated, causing microcirculatory incompetence and cellular injury. Coagulation factor Xa (FXa)- protease-activated receptor (PAR)-2 signaling activates inflammatory reactions and the cytoprotective effect of FXa inhibitor in several organs. However, no studies have elucidated the significance of FXa inhibition on hepatic ischemia-reperfusion injury. The present study elucidated the treatment effect of an FXa inhibitor, edoxaban, on hepatic ischemia-reperfusion injury, focusing on FXa-PAR-2 signaling. A 60 min hepatic partial-warm ischemia-reperfusion injury mouse model and a hypoxia-reoxygenation model of hepatic sinusoidal endothelial cells were used. Ischemia-reperfusion injury mice and hepatic sinusoidal endothelial cells were treated and pretreated, respectively with or without edoxaban. They were incubated during hypoxia/reoxygenation in vitro. Cell signaling was evaluated using the PAR-2 knockdown model. In ischemia-reperfusion injury mice, edoxaban treatment significantly attenuated fibrin deposition in the sinusoids and liver histological damage and resulted in both anti-inflammatory and antiapoptotic effects. Hepatic ischemia-reperfusion injury upregulated PAR-2 generation and enhanced extracellular signal-regulated kinase 1/2 (ERK 1/2) activation; however, edoxaban treatment reduced PAR-2 generation and suppressed ERK 1/2 activation in vivo. In the hypoxia/reoxygenation model of sinusoidal endothelial cells, hypoxia/reoxygenation stress increased FXa generation and induced cytotoxic effects. Edoxaban protected sinusoidal endothelial cells from hypoxia/reoxygenation stress and reduced ERK 1/2 activation. PAR-2 knockdown in the sinusoidal endothelial cells ameliorated hypoxia/reoxygenation stress-induced cytotoxicity and suppressed ERK 1/2 phosphorylation. Thus, edoxaban ameliorated hepatic ischemia-reperfusion injury in mice by protecting against micro-thrombosis in sinusoids and suppressing FXa-PAR-2-induced inflammation in the sinusoidal endothelial cells.

Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors

BackgroundDirect oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. ObjectivesThe ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. MethodsHuman FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. ResultsF174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. ConclusionThese human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

FIBROSIS AS AN ADDITIONAL RISK FACTOR FOR STROKE DEVELOPMENT IN HYPERTENSIVE PATIENTS WITH ATRIAL FIBRILLATION (RETROSPECTIVE STUDY)

Objective: Atrial fibrillation (AF) is a common issue that is increasingly prevalent, particularly in hypertensive patients and leads to severe complications, notably stroke. Several prothrombotic factors are elevated in AF, contributing to an increased risk of stroke. Tissue factor (TF) is the main initiator of the blood coagulation cascade, also involved in inflammation and fibrogenesis. Atrial fibrosis plays a central role in creating the substrate for AF, with the Transforming Growth Factor (TGF-B1) identified as a key cytokine involved in the pathogenesis of atrial fibrosis. The aim of this study is to investigate the relationship between atrial fibrosis and the risk of developing transient ischemic attack (TIA) or stroke in hypertensive patients with AF (retrospective study). Design and method: We observed 445 hypertensive patients with nonvalvular AF over three years (2017-2019). Clinical examination included a study of complaints, physical, laboratory, and instrumental examination, along with additional biochemical blood tests, such as TF, hsCRP, IL-6, and TGF-B1. Blood tests were determined using the ELISA method. Treatment regimens for all patients included standard therapy (anticoagulants, antiarrhythmic, and symptomatic medication). Studies were conducted based on open-label protocols, using the universal statistical packages SPSS 13.0. Results: The obtained results have shown that among the 445 hypertensive patients with AF within three years, TIA or stroke was detected in 128 patients (28.8%). We found that in these patients, the level of TF is statistically elevated compared to other patients with AF (1350±65 vs. 850±33 pg/ml). Hypertensive patients undergoing TIA or stroke exhibited a significant increase in levels of inflammatory and fibrosis markers compared with other hypertensive patients with AF. Significant differences between hsCRP levels were observed (8.6±1.4 vs. 4.2±1.6, p=0.002); IL-6 levels (6.4±0.8 vs. 3.6±1.2 pg/ml, p=0.043); and TGF-B1 levels (1240±75 vs. 900±43 pg/ml, p=0.02). Moreover, plasma levels of hs-CRP were higher among AF patients at high risk of stroke (3 or more) according to CHA2DS2-VASc (p=0.002). Conclusions: We have demonstrated that fibrosis and inflammation markers, along with coagulation cascade markers, are additional prognostic criteria contributing to the development of TIA or stroke in hypertensive patients with AF.

Contribution of Red Blood Cells and Platelets to Blood Clot Computed Tomography Imaging and Compressive Mechanical Characteristics

Thrombus computed tomography (CT) imaging characteristics may correspond with thrombus mechanical properties and thus predict thrombectomy success. The impact of red blood cell (RBC) content on these properties (imaging and mechanics) has been widely studied. However, the additional effect of platelets has not been considered. The objective of the current study was to examine the individual and combined effects of blood clot RBC and platelet content on resultant CT imaging and mechanical characteristics. Human blood clot analogues were prepared from a combination of preselected RBC volumes and platelet concentrations to decouple their contributions. The resulting clot RBC content (%) and platelet content (%) were determined using Martius Scarlet Blue and CD42b staining, respectively. Non-contrast and contrast-enhanced CT (NCCT and CECT) scans were performed to measure the clot densities. CECT density increase was taken as a proxy for clinical perviousness. Unconfined compressive mechanics were analysed by performing 10 cycles of 80% strain. RBC content is the major determinant of clot NCCT density. However, additional consideration of the platelet content improves the association. CECT density increase is influenced by clot platelet and not RBC content. Platelet content is the dominant component driving clot stiffness, especially at high strains. Both RBC and platelet content contribute to the clot’s viscoelastic and plastic compressive properties. The current in vitro results suggest that CT density is reflective of RBC content and subsequent clot viscoelasticity and plasticity, and that perviousness reflects the clot’s platelet content and subsequent stiffness. However, these indications should be confirmed in a clinical stroke cohort.

Dragon's Blood-Loaded Mesoporous Silica Nanoparticles for Rapid Hemostasis and Antibacterial Activity.

Dragon's blood (DB) is a traditional Chinese medicine (TCM) with hemostatic effects and antibacterial properties. However, it is still challenging to use for rapid hemostasis because of its insolubility. In this study, different amounts of DB were loaded on mesoporous silica nanoparticles (MSNs) to prepare a series of DB-MSN composites (5DB-MSN, 10DB-MSN, and 20DB-MSN). DB-MSN could quickly release DB and activate the intrinsic blood coagulation cascade simultaneously by DB and MSN. Hemostasis tests demonstrated that DB-MSN showed superior hemostatic effects than either DB or MSNs alone, and 10DB-MSN exhibited the best hemostatic effect. In addition, the antibacterial activities of DB-MSN against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) improved with the increase in DB. Furthermore, the hemolysis assay and cytocompatibility assay demonstrated that all DB-MSNs exhibited excellent biocompatibility. Based on these results, 10DB-MSN is expected to have potential applications for emergency hemostatic and antibacterial treatment in pre-hospital trauma.

Immunogenic and toxic effects of graphene oxide nanoparticles in mouse skeletal muscles and human red blood cells

To investigate immunogenic and toxic effects of graphene oxide (GO) nanoparticles in mouse skeletal muscles and in human blood in vitro. GO nanoparticles prepared using a probe sonicator were supended in deionized H2O or PBS, and particle size and surface charge of the nanoparticles were measured with dynamic light scattering (DLS). Different concentrations (0.5, 1.0 and 2.0 mg/mL) of GO suspension or PBS were injected at multiple sites in the gastrocnemius muscle (GN) of C57BL/6 mice, and inflammatory response and immune cell infiltrations were detected with HE and immunofluorescence staining. We also examined the effects of GO nanoparticles on human red blood cell (RBC) morphology, hemolysis and blood coagulation using scanning electron microscope (SEM), spectrophotometry, and thromboelastography (TEG). GO nanoparticles suspended in PBS exhibited better colloidal dispersity, stability and surface charge effects than those in deionized H2O. In mouse GNs, injection of GO suspensions dose- and time-dependently resulted in sustained muscular inflammation and myofiber degeneration at the injection sites, which lasted till 8 weeks after the injection; immunofluorescence staining revealed obvious infiltration of monocytes, macrophages, dendritic cells and CD4+ T cells around the injection sites in mouse GNs. In human RBCs, incubation with GO suspensions at 0.2, 2.0 and 20 mg/mL, but not at 0.002 or 0.02 mg/mL, caused significant alterations of cell morphology and hemolysis. TEG analysis showed significant abnormalities of blood coagulation parameters following treatment with high concentrations of GO. GO nanoparticles can induce sustained inflammatory and immunological responses in mouse GNs and cause RBC hemolysis and blood coagulation impairment, suggesting its muscular toxicity and hematotoxicity at high concentrations.