A method to quantitatively characterize the formation and dissociation of tumor cell clusters using light transmission aggregometry.

In this paper, we have modified the workflow of the traditional light transmission aggregometry (LTA) protocol to characterize tumor cell clusters in vitro in a quantifiable and multifaceted manner. Circulating tumor cell (CTC) clusters have high metastatic potential compared to single tumor cells traveling in the bloodstream. Thus, engineering new therapeutic strategies that specifically target this CTC population is essential. To accomplish this, quantifiable methods to characterize their therapeutic effect on tumor cell clusters is a prerequisite. The method presented here enables the user to precisely quantify the dissociation of cancer cell clusters in the presence of clinically relevant fibrinolytic agents, such as alteplase and tenecteplase. The efficacy of the fibrinolytic agents can be quantified using this in vitro assay, prior to conducting preclinical studies. Here, we have obtained the fibrinolytic activity data in terms of lag time to the initiation of tumor cell dissociation, time to 25% dissociation, and trend of dissociation over time. To validate the assay, cell counts and phase-contrast microscopy images were recorded over time. Further, we explored an LTA-assisted preparation of platelet-tumor-cell clusters of calibrated size for potential downstream testing/applications. To assess whether the assay is applicable to characterize the dissociation of cancer cell clusters in the presence of platelets, we added low (50 000 platelets·µL-1), normal (200 000 platelets·µL-1) and high (450 000 platelets·µL-1) concentrations of platelets to the tumor cell clusters. In addition to dissociation parameters, microcopy images were recorded over time to validate the assay and enabled the enumeration of clusters and single cells. The correlative light electron microscopy (CLEM) technique was utilized to visualize the morphology and composition of platelet-tumor cell clusters.

Empirical and modified hemostatic resuscitation for liver blast injury combined with seawater immersion: A preliminary study.

PURPOSE: To compare the effects of empirical and modified hemostatic resuscitation for liver blast injury combined with seawater immersion. METHODS: Thirty rabbits were subjected to liver blast injury combined with seawater immersion, and were then divided into 3 groups randomly (n = 10 each): group A (no treatment after immersion), group B (empirical resuscitation with 20 mL hydroxyethyl starch, 50 mg tranexamic acid, 25 IU prothrombin complex concentrate and 50 mg/kg body weight fibrinogen concentrate), and group C (modified resuscitation with additional 10 IU prothrombin complex concentrate and 20 mg/kg body weight fibrinogen concentrate based on group B). Blood samples were gathered at specified moments for assessment of thromboelastography, routine coagulation test, and biochemistry. Mean arterial pressure, heart rate, and survival rate were also documented at each time point. The Kolmogorov-Smirnov test was used to examine the normality of data distribution. Multigroup comparisons were conducted with one-way ANOVA. RESULTS: Liver blast injury combined with seawater immersion resulted in severe coagulo-fibrinolytic derangement as indicated by prolonged prothrombin time (s) (11.53 ± 0.98 vs. 7.61 ± 0.28, p＜0.001), activated partial thromboplastin time (APTT) (s) (33.48 ± 6.66 vs. 18.23 ± 0.89, p＜0.001), reaction time (R) (min) (5.85 ± 0.96 vs. 2.47 ± 0.53, p＜0.001), decreased maximum amplitude (MA) (mm) (53.20 ± 5.99 vs. 74.92 ± 5.76, p＜0.001) and fibrinogen concentration (g/L) (1.188 ± 0.29 vs. 1.890 ± 0.32, p = 0.003), and increased D-dimer concentration (mg/L) (0.379 ± 0.32 vs. 0.051 ± 0.03, p = 0.005). Both empirical and modified hemostatic resuscitation could improve the coagulo-fibrinolytic states and organ function, as indicated by shortened APTT and R values, decreased D-dimer concentration, increased fibrinogen concentration and MA values, lower concentration of blood urea nitrogen and creatine kinase-MB in group B and group C rabbits in comparison to that observed in group A. Further analysis found that the R values (min) (4.67 ± 0.84 vs. 3.66 ± 0.98, p = 0.038), APTT (s) (23.16 ± 2.75 vs. 18.94 ± 1.05, p = 0.001), MA (mm) (60.10 ± 4.74 vs. 70.21 ± 3.01, p < 0.001), and fibrinogen concentration (g/L) (1.675 ± 0.21 vs. 1.937 ± 0.16, p = 0.013) were remarkably improved in group C than in group B at 2 h and 4 h after injury. In addition, the concentration of blood urea nitrogen (mmol/L) (24.11 ± 1.96 vs. 21.00 ± 3.78, p = 0.047) and creatine kinase-MB (U/L) (85.50 ± 13.60 vs. 69.74 ± 8.56, p = 0.013) were lower in group C than in group B at 6 h after injury. The survival rates in group B and group C were significantly higher than those in group A at 4 h and 6 h after injury (p < 0.001), however, there were no statistical differences in survival rates between group B and group C at each time point. CONCLUSIONS: Modified hemostatic resuscitation could improve the coagulation parameters and organ function better than empirical hemostatic resuscitation.

Fibrinolysis is impaired in patients with primary immune thrombocytopenia.

BACKGROUND: Patients with primary immune thrombocytopenia (ITP) have an increased risk of thrombosis, which may be due to altered fibrinolysis. OBJECTIVES: To elucidate the clinical impact of delayed fibrinolysis in ITP patients. METHODS: A turbidimetric clot formation and lysis assay and a fluorometric plasmin generation (PG) assay were performed and levels of plasminogen-activator-inhibitor-1 (PAI-1), tissue-plasminogen-activator (tPA), tPA-PAI-1 complexes, α2-antiplasmin, TAFI and D-Dimer were assessed in 86 adult primary ITP patients and 78 healthy controls (HC). RESULTS: ITP patients showed significantly delayed clot formation, increased clot density, and prolonged clot lysis time (CLT) compared to HC with a median (IQR) CLT of 28.0 (13.7-34.7) minutes in patients and 17.3 (12.0-28.0) minutes in HC, while in PG only the lag time was prolonged. In ITP patients compared to controls, PAI-1 was higher (1.2 (0.8-2.6) vs 1.1 (0.6-2.1) U/mL), and tPA antigen and activity were lower (tPA antigen: 2.6 (1.1-4.4) vs 3.7 (3.2-4.7) ng/mL; tPA activity ≤0 U/mL: 26% vs 7%). TPA-PAI-1 complex levels were positively associated with CLT in multiple linear regression analysis (ß=0.241, p=0.019), whereas PG parameters were not associated with CLT. Six patients, who developed thrombosis during follow-up, had higher levels of tPA-PAI-1 complexes. CONCLUSION: Prolonged CLT and delayed onset of PG may indicate a hypofibrinolytic tendency in ITP patients, as also indicated by high PAI-1 and low tPA levels. No association was found between fibrinolytic potential and the bleeding phenotype, whereas higher tPA-PAI-1 complex levels were associated with prolonged CLT and increased in patients with future thrombosis.

The Use of Fibrinolytic Agents in the Salvage of Free Flaps: A Systematic Review.

BACKGROUND: Microvascular thrombosis following free tissue transfer presents a complex challenge for surgeons and carries the potential risk of flap failure. The application of fibrinolytic agents represents a robust therapeutic option. The aim of this systematic review is to provide a comprehensive overview of the clinical use of fibrinolytic drugs in the rescue of compromised free flaps. METHODS: A systematic literature search for clinical studies detailing the utilization of fibrinolytic agents for salvaging free flaps was conducted using the PubMed and Web of Science databases. The inclusion criteria encompassed English-language publications that specifically addressed the clinical application of fibrinolytic agents for free-flap salvage. RESULTS: A total of 331 articles were screened after excluding duplicates, with 56 meeting the inclusion criteria. Among these, 21 were clinical trials (evidence level III), and 35 were case studies (evidence level IV/V). In total, 459 flaps underwent treatment with fibrinolytic agents. CONCLUSION: The application of fibrinolytic agents appears to be a valuable intervention for rescuing compromised free flaps attributable to microvascular compromise. Notably, no prospective randomized trials have been published on this subject, and the evidence within the existing literature is characterized by its limited and heterogeneous nature. Further research is imperative to gather data on the efficacy, dosage, and safety profile of fibrinolytic agents.

Protection of Tight Junctional Complexes between hCMEC/D3 Cells by Deep-Sea Fibrinolytic Compound FGFC1.

Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood-brain barrier (BBB), whose regulation closely correlates to the BBB's integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of the BBB via the construction of an in vitro model. This study aims to investigate the protective effect of the deep-sea-derived fibrinolytic compound FGFC1 against H2O2-induced dysfunction of TJCs and to elucidate the underlying mechanism. The barrier function was shown to decline following exposure to 1 mM H2O2 in an in vitro model of hCMEC/D3 cells, with a decreasing temperature-corrected transendothelial electrical resistance (tcTEER) value. The decrease in the tcTEER value was significantly inhibited by 80 or 100 µM FGFC1, which suggested it efficiently protected the barrier integrity, allowing it to maintain its function against the H2O2-induced dysfunction. According to immunofluorescence microscopy (IFM) and quantitative real-time polymerase chain reaction (qRT-PCR), compared to the H2O2-treated group, 80~100 µM FGFC1 enhanced the expression of claudin-5 (CLDN-5) and VE-cadherin (VE-cad). And this enhancement was indicated to be mainly achieved by both up-regulation of CLDN-5 and inhibition of the down-regulation by H2O2 of VE-cad at the transcriptional level. Supported by FGFC1's molecular docking to these proteins with reasonable binding energy, FGFC1 was proved to exert a positive effect on TJCs' barrier function in hCMEC/D3 cells via targeting CLDN-5 and VE-cad. This is the first report on the protection against H2O2-induced barrier dysfunction by FGFC1 in addition to its thrombolytic effect. With CLDN-5 and VE-cad as the potential target proteins of FGFC1, this study provides evidence at the cellular and molecular levels for FGFC1's reducing the risk of bleeding transformation following its application in thrombolytic therapy for cerebral thrombosis.

Reduction in the need for surgery and mortality after early administration of fibrinolytics following empyema drainage.

OBJECTIVES: Although intrapleural administration of fibrinolytics is an important treatment option for the management of empyema, the addition of fibrinolytics failed to reduce the need for surgery and mortality in previous randomized controlled trials. This study aimed to investigate the effects of administrating fibrinolytics in the early phase (within 3 days of chest tube insertion) of empyema compared with late administration or no administration. METHODS: We used the Japanese Diagnosis Procedure Combination Inpatient Database to identify patients aged ≥16 years who were hospitalized and underwent chest tube drainage for empyema. A 1:2 propensity score matching and stabilized inverse probability of treatment weighting were conducted. RESULTS: Among the 16 265 eligible patients, 3082 and 13 183 patients were categorized into the early and control group, respectively. The proportion of patients who underwent surgery was significantly lower in the early fibrinolytics group than in the control group; the odds ratio (95% confidence interval) was 0.69 (0.54-0.88) in the propensity score matching (P = 0.003) and 0.64 (0.50-0.80) in the stabilized inverse probability of treatment weighting analysis (P < 0.001). All-cause 30-day in-hospital mortality, length of hospital stay, duration of chest tube drainage, and total hospitalization costs were also more favourable in the early fibrinolytics group. CONCLUSIONS: The early administration of fibrinolytics may reduce the need for surgery and death in adult patients with empyema.

Computational analysis to comprehend the structure-function properties of fibrinolytic enzymes from Bacillus spp for their efficient integration into industrial applications.

Background: The fibrinolytic enzymes from Bacillus sp. are proposed as therapeutics in preventing thrombosis. Computational-based analyses of these enzymes' amino acid composition, basic physiological properties, presence of functional domain and motifs, and secondary and tertiary structure analyses can lead to developing a specific enzyme with improved catalytic activity and other properties that may increase their therapeutic potential. Methods: The nucleotide sequences of fibrinolytic enzymes produced by the genus Bacillus and its corresponding protein sequences were retrieved from the NCBI database and aligned using the PRALINE programme. The varied physiochemical parameters and structural and functional analysis of the enzyme sequences were carried out with the ExPASy-ProtParam tool, MEME server, SOPMA, PDBsum tool, CYS-REC tool, SWISS-MODEL, SAVES servers, TMHMM program, GlobPlot, and peptide cutter software. The assessed in-silico data were compared with the published experimental results for validation. Results: The alignment of sixty fibrinolytic serine protease enzymes (molecular mass 12-86 kDa) sequences showed 49 enzymes possess a conserved domain with a catalytic triad of Asp196, His242, and Ser569. The predicted instability and aliphatic indexes were 1.94-37.77, and 68.9-93.41, respectively, indicating high thermostability. The random coil means value suggested the predominance of this secondary structure in these proteases. A set of 50 amino acid residues representing motif 3 signifies the Peptidase S8/S53 domain that was invariably observed in 56 sequences. Additionally, 28 sequences have transmembrane helices, with two having the most disordered areas, and they pose 25 enzyme cleavage sites. A comparative analysis of the experimental work with the results of in-silico study put forward the characteristics of the enzyme sequences JF739176.1 and MF677779.1 to be considered when creating a potential mutant enzyme as these sequences are stable at high pH with thermostability and to exhibit αß-fibrinogenase activity in both experimental and in-silico studies.

Fibrinolytic Platelet Decoys Reduce Cancer Metastasis by Dissociating Circulating Tumor Cell Clusters.

During metastasis, circulating tumor cells (CTCs) can travel in the bloodstream as individual cells or clusters, associated with fibrin and platelets. Clusters have a higher metastatic potential due to their increased ability to withstand shear stress and arrest in small vessels. Moreover, CTC-platelet interaction protects CTCs from shear stress and immune detection. The objective of this project is to develop a fibrinolytic platelet system to leverage platelet-CTC interactions and dissociate CTC clusters. For this approach, tissue plasminogen activator (tPA) is loaded onto two modified platelet systems: platelet Decoys and lyophilized platelets. The activities of the systems are characterized using a Förster Resonance Energy Transfer-based assay and an angiogenic assay. Furthermore, the ability of the system to dissociate cancer cell clusters in vitro is assessed using light transmission aggregometry. The data demonstrates that the fibrinolytic platelets can maintain tPA activity, interact with CTCs, and dissociate cancer cell clusters. Finally, fibrinolytic platelets are assessed in vivo, demonstrating a decreased tumor load and increased survival with tPA-Decoy treatment, which is selected as the optimal treatment based on favorable in vitro results and in vivo trials. Therefore, this fibrinolytic platelet approach is a promising method for leveraging platelet-CTC interactions to disperse CTC clusters and reduce metastasis.

Management of Previously Failed Coiling and Clipping of a Middle Cerebral Artery Aneurysm.

Endovascular coiling techniques have emerged as an alternative and effective approach for treating intracranial aneurysms. However, in some cases, previously coiled aneurysms may require secondary treatment with surgical clipping, presenting a more complex challenge compared with the initial intervention.1,2 We present the case of a 39-year-old man with a residual class III Raymond-Roy occlusion partially coiled aneurysm at the left middle cerebral artery bifurcation (Video 1). Faced with the risks of rerupture, the patient underwent microsurgical treatment after providing consent. Despite successful initial microsurgical clipping, postoperative complications arose due to coil protrusion into the middle cerebral artery bifurcation, resulting in thrombotic occlusion of the frontal M2 branch. Emergency repeat microsurgical intervention and administration of a thrombolytic agent were performed to address complications, ultimately preserving blood flow. Subsequent endovascular placement of a flow-diverting stent 7 weeks after discharge confirmed complete occlusion of the aneurysm. The patient had no neurological deficit on follow-up. When planning microsurgical clipping of an aneurysm previously treated with coils, it is critical to consider coil placement, as there is a risk of prolapse if the coil is in the neck of the aneurysm. Thrombosis of the cerebral arteries is a potential complication of microsurgical clipping of partially coiled intracranial aneurysms, and injection of a fibrinolytic agent into thrombosed arterial branches may be an effective intraoperative method for treating intra-arterial thrombosis.3 This case illustrates the challenges associated with treating partially coiled aneurysms, highlighting the significance of careful planning when considering microsurgical treatment.

A Protease from Moringa oleifera Lam. Exhibits In-vitro Blood Clot Solubilization and Fibrin Hydrolysis.

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.

CHI3L1 on fibrinolytic system imbalance in chronic rhinosinusitis with nasal polyp.

Background: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting more than 10% of the global adult population. It is classified into Th1, Th2, and Th17 endotypes and eosinophilic and non-eosinophilic types. Th2-based inflammation and eosinophilic CRS (ECRS) are associated with tissue remodeling and fibrinolytic system impairment. Objective: To elucidate the role of eosinophils in inducing fibrin deposition in CRS nasal polyp tissues and explore potential regulatory mechanisms. Methods: We analyzed the expression of genes related to the serpin family and fibrinolytic system using Gene Expression Omnibus and Next-generation sequencing data. Differentially expression genes (DEGs) analysis was used to compare control and nasal polyp tissues, followed by KEGG and Gene ontology (GO) analysis. We measured the expression and correlation of plasminogen activator-1 (PAI-1), tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and urokinase plasminogen activator surface receptor (u-PAR) in CRS tissues, and evaluated the effect of eosinophils on the fibrinolytic system using a cytokine array and co-culture. Results: Nasal polyp tissues showed upregulated PAI-1, u-PA, and u-PAR expression and downregulated t-PA expression. Fibrinolytic system-related genes positively correlated with Th2 cytokines, except for t-PA. Eosinophil-derived Chitinase-3-like protein 1 (CHI3L1) increased PAI-1 expression and decreased t-PA levels in fibroblasts and epithelial cells. The inhibition of CHI3L1 suppresses these alterations. Conclusion: CHI3L1 contributes to fibrin deposition by impairing the fibrinolytic system during nasal polyp formation. The regulation of CHI3L1 expression may inhibit fibrin deposition and edema in ECRS, presenting a potential treatment for this condition.

Oral fibrinolytic agent in hyper-coagulopathy in severe COVID - 19 ARDS Patients: two case reports.

Abstract: The use of oral fibrinolytic agent (DLBS1033) has been proven for adjuvant treatment in venous thromboembolism, however until now there is no published report about its uses and effectiveness as an addition to the standard therapy of severe COVID-19 cases and hypercoagulopathy. We present two cases of severe confirmed COVID-19 from PCR tests, seen at Ngimbang Hospital, Lamongan, East Java in October and November, 2020. The first patient was a 51-year-old male who presented to ER with fever, dyspnoea, cough, and oxygen desaturation (SpO2 room air 87%), with comorbids of pulmonary hypertension (PH), atrial fibrillation, heart failure secondary to corpulmonale, and hypercoagulopathy. The second patient was a 56-yearold female who presented with fever, dyspnoea, and oxygen desaturation (Sp02 room air 88%), with comorbid ARDS, hypertension, hyperglycaemia, hypercoagulopathy, heart failure, and CAD. Both of the patients were treated with standard treatment therapy for severe COVID-19 and comorbid therapy, and DLBS1033 in addition to fondaparinux due to limited hospital resources. Both patients showed good clinical outcomes after the course of treatment and had no adverse effects. CONCLUSIONS: Our two case reports were the first that showed good clinical outcome and safety of DLBS1033 treatment in addition to fondaparinux for hypercoagulopathy therapy.

Integrating Metabolic Oligosaccharide Engineering and SPAAC Click Chemistry for Constructing Fibrinolytic Cell Surfaces.

To effectively solve the problem of significant loss of transplanted cells caused by thrombosis during cell transplantation, this study simulates the human fibrinolytic system and combines metabolic oligosaccharide engineering with strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry to construct a cell surface with fibrinolytic activity. First, a copolymer (POL) of oligoethylene glycol methacrylate (OEGMA) and 6-amino-2-(2-methylamido)hexanoic acid (Lys) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization, and the dibenzocyclooctyne (DBCO) functional group was introduced into the side chain of the copolymer through an active ester reaction, resulting in a functionalized copolymer DBCO-PEG4-POL with ε-lysine ligands. Then, azide functional groups were introduced onto the surface of HeLa model cells through metabolic oligosaccharide engineering, and DBCO-PEG4-POL was further specifically modified onto the surface of HeLa cells via the SPAAC "click" reaction. In vitro investigations revealed that compared with unmodified HeLa cells, modified cells not only resist the adsorption of nonspecific proteins such as fibrinogen and human serum albumin but also selectively bind to plasminogen in plasma while maintaining good cell viability and proliferative activity. More importantly, upon the activation of adsorbed plasminogen into plasmin, the modified cells exhibited remarkable fibrinolytic activity and were capable of promptly dissolving the primary thrombus formed on their surfaces. This research not only provides a novel approach for constructing transplantable cells with fibrinolytic activity but also offers a new perspective for effectively addressing the significant loss of transplanted cells caused by thrombosis.

Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea.

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 µM and 49.0 µmol min-1 mg-1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema.

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Potential antiplatelet agents with grape seed - backbone polyphenols: computational studies.

The study focused on grape seed-derived polyphenols for their antiplatelet, anti-inflammatory, and fibrinolytic properties through molecular docking and dynamics simulations. Compounds were evaluated for their effects on P2Y12, PTP1B, thromboxane A2, and other targets. Compounds 1 and 6 showed strong inhibitory potential on P2Y12. Compounds 2 and 7, plus epigallocatechin gallate, demonstrated effective inhibition on NF-KB and COX1. The compounds exhibited drug-like properties and potential for new thrombotic disease therapies. The research sheds light on the interactions between polyphenols and target proteins, paving the way for novel antiplatelet strategies.

Surgery compared to fibrinolytic therapy for symptomatic left-sided prosthetic heart valve thrombosis (SAFE-PVT): Rationale and design of a randomized controlled trial.

BACKGROUND: Left-sided mechanical prosthetic heart valve thrombosis (PVT) occurs because of suboptimal anticoagulation and is common in low-resource settings. Urgent surgery and fibrinolytic therapy (FT) are the two treatment options available for this condition. Urgent surgery is a high-risk procedure but results in successful restoration of valve function more often and is the treatment of choice in developed countries. In low-resource countries, FT is used as the default treatment strategy, though it is associated with lower success rates and a higher rate of bleeding and embolic complications. There are no randomized trials comparing the two modalities. METHODS: We performed a single center randomized controlled trial comparing urgent surgery (valve replacement or thrombectomy) with FT (low-dose, slow infusion tissue plasminogen activator, tPA) in patients with symptomatic left-sided PVT. The primary outcome was the occurrence of a complete clinical response, defined as discharge from hospital with completely restored valve function, in the absence of stroke, major bleeding or non-CNS systemic embolism. Outcome assessment was done by investigators blinded to treatment allocation. The principal safety outcome was the occurrence of a composite of in-hospital death, non-fatal stroke, non-fatal major bleed or non-CNS systemic embolism. Outcomes will be assessed both in the intention-to-treat, and in the as-treated population. We will also report outcomes at one year of follow-up. The trial has completed recruitment. CONCLUSION: This is the first randomized trial to compare urgent surgery with FT for the treatment of left-sided PVT. The results will provide evidence to help clinicians make treatment choices for these patients. (Clinical trial registration: CTRI/2017/10/010159).

Clot-Targeted Nanogels for Dual-Delivery of AntithrombinIII and Tissue Plasminogen Activator to Mitigate Disseminated Intravascular Coagulation Complications.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Spontaneous intracerebral hemorrhage - patients retrospectively consent to fibrinolytic surgery despite poor neurological outcome and reduced health-related quality of life.

Spontaneous intracerebral hemorrhage (ICH) might lead to devastating consequences. Nonetheless, subjective interpretation of life circumstances might vary. Recent data from ischemic stroke patients show that there might be a paradox between clinically rated neurological outcome and self-reported satisfaction with quality of life. Our hypothesis was that minimally invasive surgically treated ICH patients would still give their consent to stereotactic fibrinolysis despite experiencing relatively poor neurological outcome. In order to better understand the patients' perspective and to enhance insight beyond functional outcome, this is the first study assessing disease-specific health-related quality of life (hrQoL) in ICH after fibrinolytic therapy. We conducted a retrospective analysis of patients with spontaneous ICH treated minimally invasive by stereotactic fibrinolysis. Subsequently, using standardized telephone interviews, we evaluated functional outcome with the modified Rankin Scale (mRS), health-related Quality of Life with the Quality of life after Brain Injury Overall scale (QOLIBRI-OS), and assessed retrospectively if the patients would have given their consent to the treatment. To verify the primary hypothesis that fibrinolytic treated ICH patients would still retrospectively consent to fibrinolytic therapy despite a relatively poor neurological outcome, we conducted a chi-square test to compare good versus poor outcome (mRS) between consenters and non-consenters. To investigate the association between hrQoL (QOLIBRI-OS) and consent, we conducted a Mann-Whitney U-test. Moreover, we did a Spearman correlation to investigate the correlation between functional outcome (mRS) and hrQoL (QOLIBRI-OS). The analysis comprised 63 data sets (35 men, mean age: 66.9 ± 11.8 years, median Hemphill score: 3 [2-3]). Good neurological outcome (mRS 0-3) was achieved in 52% (33/63) of the patients. Patients would have given their consent to surgery retrospectively in 89.7% (52/58). These 52 consenting patients comprised all 33 patients (100%) who achieved good functional outcome and 19 of the 25 patients (76%) who achieved poor neurological outcome (mRS 4-6). The mean QOLIBRI-OS value was 49.55 ± 27.75. A significant association between hrQoL and retrospective consent was found (p = 0.004). This study supports fibrinolytic treatment of ICH even in cases when poor neurological outcome would have to be assumed since subjective perception of deficits could be in contrast with the objectively measured neurological outcome. HrQoL serves as a criterion for success of rtPa lysis therapy in ICH.