Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Sarcocinerenolides A, an open-loop decarbonizing cembranolide, and sarcocinerenolides B-I, eight polyoxygenated cembranolides with anti-thrombotic activity from the South China Sea soft coral Sarcophyton cinereum.

A previously undescribed open-loop decarbonizing cembranolide, sarcocinerenolide A, and eight undescribed cembranolides, sarcocinerenolides B-I, characterized by poly-membered oxygen ring fragments were isolated from the soft coral Sarcophyton cinereum collected from the South China Sea. The structures and absolute configurations of these previously undescribed compounds were precisely determined by analysis of NMR data, DP4+ and ECD spectra. The bioactivities of the compounds were evaluated using zebrafish models and sarcocinerenolides C and H exhibited anti-thrombotic activity.

Advances in Nano-Functional Materials in Targeted Thrombolytic Drug Delivery.

Thrombotic disease has been listed as the third most fatal vascular disease in the world. After decades of development, clinical thrombolytic drugs still cannot avoid the occurrence of adverse reactions such as bleeding. A number of studies have shown that the application of various nano-functional materials in thrombus-targeted drug delivery, combined with external stimuli, such as magnetic, near-infrared light, ultrasound, etc., enrich the drugs in the thrombus site and use the properties of nano-functional materials for collaborative thrombolysis, which can effectively reduce adverse reactions such as bleeding and improve thrombolysis efficiency. In this paper, the research progress of organic nanomaterials, inorganic nanomaterials, and biomimetic nanomaterials for drug delivery is briefly reviewed.

Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 µg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Platelet Membrane-Coated r-SAK Improves Thrombolytic Efficacy by Targeting Thrombus.

Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.

A Nonbactericidal Anionic Antimicrobial Peptide Provides Prophylactic and Therapeutic Efficacies against Bacterial Infections in Mice by Immunomodulatory-Antithrombotic Duality.

Although bactericidal cationic antimicrobial peptides (AMPs) have been well characterized, less information is available about the antibacterial properties and mechanisms of action of nonbactericidal AMPs, especially nonbactericidal anionic AMPs. Herein, a novel anionic antimicrobial peptide (Gy-CATH) with a net charge of -4 was identified from the skin of the frog Glyphoglossus yunnanensis. Gy-CATH lacks direct antibacterial effects but exhibits significantly preventive and therapeutic capacities in mice that are infected with Staphylococcus aureus, Enterobacteriaceae coli, methicillin-resistant Staphylococcus aureus (MRSA), or carbapenem-resistant E. coli (CREC). In vitro and in vivo investigations proved the regulation of Gy-CATH on neutrophils and macrophages involved in the host immune defense against infection. Moreover, Gy-CATH significantly reduced the extent of pulmonary fibrin deposition and prevented thrombosis in mice, which was attributed to the regulatory role of Gy-CATH in physiological anticoagulants and platelet aggregation. These findings show that Gy-CATH is a potential candidate for the treatment of bacterial infection.

Network pharmacology analysis and experimental verification of the antithrombotic active compounds of trichosanthis pericarpium (Gualoupi) in treating coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichosanthis pericarpium (TP; Gualoupi, pericarps of Trichosanthes kirilowii Maxim) has been used in traditional Chinese medicine (TCM) to reduce heat, resolve phlegm, promote Qi, and clear chest congestion. It is also an essential herbal ingredient in the "Gualou Xiebai" formula first recorded by Zhang Zhongjing (from the Eastern Han Dynasty) in the famous TCM classic "Jin-Guì-Yào-Lüe" for treating chest impediments. According to its traditional description, Gualou Xiebai is indicated for symptoms of chest impediments, which correspond to coronary heart diseases (CHD). AIM OF THE STUDY: This study aimed to identify the antithrombotic compounds in Gualoupi for the treatment of CHD. MATERIALS AND METHODS: A CHD rat model was established with a combination of high-fat diet and isoproterenol hydrochloride (ISO) administration via subcutaneous multi-point injection in the back of the neck. This model was used to evaluate the antithrombotic effect of two mainstream cultivars of TP ("HaiShi GuaLou" and "WanLou") by analyzing the main components and their effects. Network pharmacology, molecular docking-based studies, and a zebrafish (Danio rerio) thrombosis model induced by phenylhydrazine was used to validate the antithrombosis components of TP. RESULTS: TP significantly reduced the body weight of the CHD rats, improved myocardial ischemia, and reduced collagen deposition and fibrosis around the infarcted tissue. It reduced thrombosis in a dose-dependent manner and significantly reduced inflammation and oxidative stress damage. Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as candidate active TP compounds with antithrombotic effects. The key potential targets of TP in thrombosis treatment were initially identified by molecular docking-based analysis, which showed that the candidate active compounds have a strong binding affinity to the potential targets (protein kinase C alpha type [PKCα], protein kinase C beta type [PKCß], von Willebrand factor [vWF], and prostaglandin-endoperoxide synthase 1 [PTGS1], fibrinogen alpha [Fga], fibrinogen beta [Fgb], fibrinogen gamma [Fgg], coagulation factor II [F2], and coagulation factor VII [F7]). In addition, the candidate active compounds reduced thrombosis, improved oxidative stress damage, and down-regulated the expression of thrombosis-related genes (PKCα, PKCß, vWF, PTGS1, Fga, Fgb, Fgg, F2, and F7) in the zebrafish model. CONCLUSION: Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as the active antithrombotic compounds of TP used to treat CHD. Mechanistically, the active compounds were found to be involved in oxidative stress injury, platelet activation pathway, and complement and coagulation cascade pathways.

Selective binding of cationic fibrinogen-mimicking chitosan nanoparticles to activated platelets and efficient drug release for antithrombotic therapy.

Thrombosis is the main cause of catastrophic events including ischemic stroke, myocardial infarction and pulmonary embolism. Acetylsalicylic acid (ASA) therapy offers a desirable approach to antithrombosis through a reduction of platelet reactivity. However, major bleeding complications, severe off-target side effects, and resistance or nonresponse to ASA greatly attenuate its clinical outcomes. Herein, we report a cationic fibrinogen-mimicking nanoparticle, denoted as ASA-RGD-CS@TPP, to achieve activated-platelet-targeted delivery and efficient release of ASA for safer and more effective antithrombotic therapy. This biomimetic antithrombotic system was prepared by one-pot ionic gelation between cationic arginine-glycine-aspartic acid (RGD)-grafted chitosan (RGD-CS) and anionic tripolyphosphate (TPP). The platform exhibited selective binding to activated platelets, leading to efficient release of ASA and subsequent attenuation of platelet functions, including the remarkable inhibition of platelet aggregation through a potent blockage of cyclooxygenase-1 (COX-1). After intravenous administration, ASA-RGD-CS@TPP displayed significantly prolonged circulation time and successful prevention of thrombosis in a mouse model. ASA-RGD-CS@TPP was demonstrated to significantly enhance antithrombotic therapy while showing minimal coagulation and hemorrhagic risks and excellent biocompatibility in vivo as compared to free ASA. This platform provides a simple, safe, effective and targeted strategy for the development of antithrombotic nanomedicines.

Layer-by-layer self-assembly embedding of nattokinase in chitosan/γ-polyglutamic acid: Preparation, fibrinolytic activity, stability, and in vitro digestion study.

Nattokinase (NK) is a thrombolytic enzyme extracted from natto, which can be used to prevent and treat blood clots. However, it is sensitive to the environment, especially the acidic environment of human stomach acid, and its effect of oral ingestion is minimal. This study aims to increase NK's oral and storage stability by embedding NK in microcapsules prepared with chitosan (CS) and γ-polyglutamic acid (γ-PGA). The paper prepared a double-layer NK oral delivery system by layer self-assembly and characterized its stability and in vitro simulated digestion. According to the research results, the bilayer putamen structure has a protective effect on NK, which not only maintains high activity in various environments (such as acid-base, high temperature) and long-term storage (60 days), but also effectively protects the loaded NK from being destroyed in gastric fluid and achieves its slow release. This work has proved the feasibility of the design of bilayer putamen structure in oral administration and has good fibrolytic activity. Therefore, the novel CS/γ-PGA microcapsules are expected to be used in nutraceutical delivery systems.

Intelligent Design of Antithrombotic Peptide Targeting Collagen.

Binding of blood components to collagen was proved to be a key step in thrombus formation. Intelligent Design of Protein Matcher (IDProMat), a neural network model, was then developed based on the principle of seq2seq to design an antithrombotic peptide targeting collagen. The encoding and decoding of peptide sequence data and the interaction patterns of peptide chains at the interface were studied, and then, IDProMat was applied to the design of peptides to cover collagen. The 99.3% decrease in seq2seq loss and 58.3% decrease in MLP loss demonstrated that IDProMat learned the interaction patterns between residues at the binding interface. An efficient peptide, LRWNSYY, was then designed using this model. Validations on its binding on collagen and its inhibition of platelet adhesion were obtained using docking, MD simulations, and experimental approaches.

Anti-hemostatic, antithrombotic, and chemical profiles of a curly-leaf variety of Petroselinum crispum (Apiaceae), a food and medicinal aromatic herb.

Thrombosis is currently among the major causes of morbidity and mortality in the World. New prevention and therapy alternatives have been increasingly sought in medicinal plants. In this context, we have been investigating parsley, Petroselinum crispum (Mill.) Nym, an aromatic herb with two leaf varieties. We report here the in vitro, in vivo, and ex vivo anti-hemostatic and antithrombotic activities of a parsley curly-leaf variety. Aqueous extracts of aerial parts (PCC-AP), stems (PCC-S), and leaves (PCC-L) showed significant in vitro antiplatelet activity. PCC-AP extract exhibited the highest activity (IC50 2.92 mg/mL) when using ADP and collagen as agonists. All extracts also presented in vitro anticoagulant activity (APTT and PT) and anti-thrombogenic activity. PCC-S was the most active, with more significant interference in the factors of the intrinsic coagulation pathway. The oral administration of PCC-AP extract in rats caused a greater inhibitory activity in the deep vein thrombi (50%; 65 mg/kg) than in arterial thrombi formation (50%; 200 mg/kg), without cumulative effect after consecutive five-day administration. PCC-AP extract was safe in the induced bleeding time test. Its anti-aggregating profile was similar in ex vivo and in vitro conditions but was more effective in the extrinsic pathway when compared to in vitro results. Apiin and coumaric acid derivatives are the main compounds in PCC-AP according to the HPLC-DAD-ESI-MS/MS profile. We demonstrated for the first time that extracts from different parts of curly parsley have significant antiplatelet, anticoagulant, and antithrombotic activity without inducing hemorrhage, proving its potential as a source of antithrombotic compounds.

Purification and Properties of a Plasmin-like Marine Protease from Clamworm (Perinereis aibuhitensis).

Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC-MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7-11 (50%) or 30-60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bß- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified from Perinereis aibuhitensis, a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.

Dual-targeting fucoidan-based microvesicle for arterial thrombolysis and re-occlusion inhibition.

Arterial thrombosis is a critical thrombotic disease that poses a significant threat to human health. However, the existing clinical treatment of arterial thrombosis lacks effective targeting and precise drug release capability. In this study, we developed a system for targeted delivery and on-demand release in arterial thrombosis treatment. The carrier was constructed using chitosan (CS) and fucoidan (Fu) through layer-by-layer assembly, with subsequent surface modification using cRGD peptide. Upon encapsulation of urokinase-type plasminogen activator (uPA), the resulting therapeutic drug delivery system, uPA-CS/Fu@cRGD, demonstrated dual-targeting abilities towards P-selectin and αIIbß3, as well as pH and platelet-responsive release properties. Importantly, we have demonstrated that the dual targeting effect exhibits higher targeting efficiency at shear rates simulating thrombosed arterial conditions (1800 s-1) compared to single targeting for the first time. In the mouse common iliac artery model, uPA-CS/Fu@cRGD exhibited great thrombolytic capability while promoting the down-regulation of coagulation factors (FXa and PAI-1) and inflammatory factors (TNF-α and IL-6), thus improving the thrombus microenvironment and exerting potential in preventing re-occlusion. Our dual-target and dual-responsive, fucoidan-based macrovesicle represent a promising platform for advanced drug target delivery applications, with potential to prevent coagulation tendencies as well as improving thrombolytic and reducing the risk of re-occlusion.

Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model.

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4-11 and 37-60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bß, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague-Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.

A erythrocyte-platelet hybrid membrane coated biomimetic nanosystem based on ginsenosides and PFH combined with ultrasound for targeted delivery in thrombus therapy.

Thrombus is one of the culprits for global health problems. However, most current antithrombotic drugs are limited by restricted targeting ability and a high risk of systemic bleeding. A hybrid cell membrane-coated biomimetic nanosystem (PM/RM@PLGA@P/R) was constructed in this paper to fulfil the targeted delivery of ginsenoside (Rg1) and perfluorohexane (PFH). Poly lactic-co-glycolic acid (PLGA) is used as carriers to coat Rg1 and PFH. Thanks to the camouflage of erythrocyte membrane (RM) and platelet membrane (PM), the nanosystem in question possesses remarkable features including immune escape and self-targeting. Therefore, a compact nano-core with PLGA@P/R was formed, with a hybrid membrane covering the surface of the core, forming a "core-shell" structure. With its "core-shell" structure, this nanoparticle fancifully combines the advantages of both PFH (the low-intensity focused ultrasound (LIFU)-responsive phase-change thrombolysis) and Rg1(the antioxidant, anti-inflammatory and anticoagulant abilities). Meanwhile, PM/RM@PLGA@P/R nanoparticles exhibits superior in-vitro performance in terms of ROS scavenging, anticoagulant activity and immune escape compared with those without cell membranes (PLGA@P/R). Furthermore, in the animal experiment in which the tail vein thrombosis model was established by injecting k-carrageenan, the combined treatment of LIFU and PM/RM@PLGA@P/R showed a satisfactory antithrombotic efficiency (88.20 %) and a relatively higher biological safety level. This strategy provides new insights into the development of more effective and safer targeted biomimetic nanomedicines for antithrombotic treatments, possessing potential application in synergistic therapy field.

Plasma-Derived Nanoclusters for Site-Specific Multimodality Photo/Magnetic Thrombus Theranostics.

Traditional thrombolytic therapeutics for vascular blockage are affected by their limited penetration into thrombi, associated off-target side effects, and low bioavailability, leading to insufficient thrombolytic efficacy. It is hypothesized that these limitations can be overcome by the precisely controlled and targeted delivery of thrombolytic therapeutics. A theranostic platform is developed that is biocompatible, fluorescent, magnetic, and well-characterized, with multiple targeting modes. This multimodal theranostic system can be remotely visualized and magnetically guided toward thrombi, noninvasively irradiated by near-infrared (NIR) phototherapies, and remotely activated by actuated magnets for additional mechanical therapy. Magnetic guidance can also improve the penetration of nanomedicines into thrombi. In a mouse model of thrombosis, the thrombosis residues are reduced by ≈80% and with no risk of side effects or of secondary embolization. This strategy not only enables the progression of thrombolysis but also accelerates the lysis rate, thereby facilitating its prospective use in time-critical thrombolytic treatment.

Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid-state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20-fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders.

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and 19,200 U/mL, respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Biomimetic Antithrombotic Tissue-Engineered Vascular Grafts for Converting Cholesterol and Free Radicals into Nitric Oxide.

Small-diameter tissue-engineered vascular grafts (sdTEVGs) are essential materials used in bypass or replacement surgery for cardiovascular diseases; however, their application efficacy is limited because of patency rates, especially under hyperlipidemia, which is also clinically observed in patients with cardiovascular diseases. In such cases, improving sdTEVG patency is challenging because cholesterol crystals easily cause thrombosis and impede endothelialization. Herein, the development of a biomimetic antithrombotic sdTEVG incorporating cholesterol oxidase and arginine into biomineralized collagen-gold hydrogels on a sdTEVG surface is described. Biomimetic antithrombotic sdTEVGs represent a multifunctional substrate for the green utilization of hazardous substances and can convert cholesterol into hydrogen peroxide, which can react with arginine to generate nitric oxide (NO). NO is a vasodilator that can simulate the antithrombotic action of endothelial cells under hyperlipidemic conditions. In vivo studies show that sdTEVGs can rapidly produce large amounts of NO via a cholesterol catalytic cascade to inhibit platelet aggregation, thereby improving the blood flow velocity and patency rates 60 days after sdTEVG transplantation. A practical and reliable strategy for transforming "harmful" substances into "beneficial" factors at early transplantation stages is presented, which can also promote vascular transplantation in patients with hyperlipidemia.

Isolation and Characterization of One New Natural Compound with Other Potential Bioactive Secondary Metabolites from Glycosmis cyanocarpa (Blume) Spreng. (Family: Rutaceae).

Glycosmis cyanocarpa (Blume) Spreng is a plant in the Rutaceae family and a species in the Glycosmis genus that has received little attention. Therefore, this research aimed to report the chemical and biological analysis of Glycosmis cyanocarpa (Blume) Spreng. The chemical analysis involved the isolation and characterization of secondary metabolites through an extensive chromatographic study, and the structures of these metabolites were elucidated on the basis of a detailed analysis of NMR and HRESIMS spectroscopic data and by comparison with those of related compounds reported in the literature. Different partitions of the crude ethyl acetate (EtOAc) extract were evaluated for antioxidant, cytotoxic, and thrombolytic potentials. In chemical analysis, one new phenyl acetate derivative, namely 3,7,11,15-tetramethylhexadec-2-en-1-yl 2-phenylacetate (1), along with four known compounds N-methyl-3-(methylthio)-N-(2-phenylacetyl) acrylamide (2), penangin (3), ß-Caryophyllene oxide (4), and acyclic diterpene-phytol (5) were isolated for the first time from the stem and leaf of the plant. The ethyl acetate fraction showed significant free radical scavenging activity with an IC50 value of 11.536 µg/mL compared to standard ascorbic acid (4.816 µg/mL). In the thrombolytic assay, the dichloromethane fraction showed the maximum thrombolytic activity of 16.42% but was still insignificant compared to the standard streptokinase (65.98%). Finally, in a brine shrimp lethality bioassay, the LC50 values of dichloromethane, ethyl acetate, and aqueous fractions were found to be 0.687 µg/mL, 0.805 µg/mL, and 0.982 µg/mL which are significant compared to the standard vincristine sulfate of 0.272 µg/mL.