Potent Anticoagulant Activity Research Articles

Atrase A, a P-III class metalloproteinase purified from cobra venom, exhibits potent anticoagulant activity by inhibiting coagulation pathway and activating the fibrinolytic system

Snake venoms, comprising a complex array of protein-rich components, an important part of which are snake venom metalloproteinases (SVMPs). These SVMPs, which are predominantly isolated from viperid venoms, are integral to the pathology of snakebites. However, SVMPs derived from elapid venoms have not been extensively explored, and only a handful of SVMPs have been characterized to date. Atrase A, a nonhemorrhagic P-III class metalloproteinase from Naja atra venom, exhibits weak proteolytic activity against fibrinogen in vitro but has pronounced anticoagulant effects in vivo. This contrast spurred investigations into its anticoagulant mechanisms. Research findings indicate that atrase A notably extends the activated partial thromboplastin time, diminishes fibrinogen levels, and impedes platelet aggregation. The anticoagulant action of atrase A primarily involves inhibiting coagulation factor VIII and activating the endogenous fibrinolytic system, which in turn lowers fibrinogen levels. Additionally, its effect on platelet aggregation further contributes to its anticoagulant profile. This study unveils a novel anticoagulant mechanism of atrase A, significantly enriching the understanding of the roles of cobra venom metalloproteinases in snake venom. Furthermore, these findings underscore the potential of atrase A as a novel anticoagulant drug, offering insights into the functional evolutions of cobra venom metalloproteinases.

Ascorbic acid-mediated selenium nanoparticles as potential antihyperuricemic, antioxidant, anticoagulant, and thrombolytic agents

Abstract Selenium (Se) is an important trace element that is involved in controlling oxidative stress and inflammatory disorders. Gouty arthritis is the inflammation and pain within the joints and tissues caused due to the accumulation of monosodium urate (MSU) crystals. This study aimed to investigate the antigout, antioxidant, anticoagulant, and thrombolytic potential of ascorbic acid-mediated Se nanoparticles (A-SeNPs). Different analytical techniques were used to investigate the formation of A-SeNPs. The antigout potential of the nanoparticles was carried out using MSU crystal dissolution, uric acid (UA) degradation assay, and xanthine oxidase inhibition (XOI). A-SeNPs exhibited excellent antihyperurecemic activity in a concentration-dependent manner. It was observed that at the tested concentration of 20 mg·mL−1, the A-SeNPs demonstrated significant breakage and dissolution of MSU crystals and resulted in UA degradation of 67.76%. Similarly, A-SeNPs resulted in 76% XOI with an excellent IC50 of 140 µg·mL−1. Furthermore, considerable antioxidant activity was noted for the A-SeNPs as evaluated with multiple antioxidant assays. Finally, the NPs were found to have significant anticoagulant and thrombolytic potential. Thus, it was concluded that A-SeNPs have potent antihyperuricemic, antioxidant, anticoagulant, and thrombolytic activities, making them an ideal choice for future biomedical applications.

Proteomic Comparisons of Caprine Milk Whole Cream Buttermilk Whey and Cheese Whey Cream Buttermilk.

Buttermilk, a potential material used to produce milk fat globule membrane (MFGM), is obtained as a byproduct of butter making from milk whole cream and cheese whey cream. This study investigated the effects of rennet and acid coagulation on the protein profiles of buttermilk rennet-coagulated whey (BRW) and buttermilk acid-coagulated whey (BAW). They were compared to those of whey cream buttermilk (WCB). Rennet coagulation was more efficient in removing casein, while retaining more IgG and lactoferrin than acid coagulation. BRW had more MFGM than BAW. Butyrophilin, xanthine dehydrogenase, and mucin1 were significantly higher (P < 0.05) in BRW, while fatty acid-binding protein 3 was enriched in BAW. KEGG analysis showed that complement and coagulation cascades had the greatest differences, and the abundance of proteins involved in this signaling pathway in BRW and BAW was higher, suggesting their potential anticoagulant and anti-inflammatory activity. BAW had higher apolipoprotein A4 and transcobalamin 2, which are essential carriers for transporting long-chain fatty acids and vitamin B12 from the intestine to the blood. Therefore, BAW intake might improve lipids and vitamin B12 absorption. This study can help deepen the understanding of protein composition of MFGM-enriched whey and facilitate the production of MFGM proteins for infants and old-aged populations.

Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Swim Bladder: Isolation, Structural Analysis, and Anticoagulant Activity.

Glycosaminoglycans (GAGs) with unique structures from marine animals show intriguing pharmacological activities and negligible biological risks, providing more options for us to explore safer agents. The swim bladder is a tonic food and folk medicine, and its GAGs show good anticoagulant activity. In this study, two GAGs, CMG-1.0 and GMG-1.0, were extracted and isolated from the swim bladder of Cynoscion microlepidotus and Gadus morhua. The physicochemical properties, precise structural characteristics, and anticoagulant activities of these GAGs were determined for the first time. The analysis results of the CMG-1.0 and GMG-1.0 showed that they were chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains with molecular weights of 109.3 kDa and 123.1 kDa, respectively. They were mainly composed of the repeating disaccharide unit of -{IdoA-α1,3-GalNAc4S-β1,4-}- (DS-A). The DS-B disaccharide unit of -{IdoA2S-α1,3-GalNAc4S-β1,4-}- also existed in both CMG-1.0 and GMG-1.0. CMG-1.0 had a higher proportion of CS-O disaccharide unit -{-GlcA-β1,3-GalNAc-β1,4-}- but a lower proportion of CS-E disaccharide unit -{-GlcA-β1,3-GalNAc4S6S-β1,4-}- than GMG-1.0. The disaccharide compositions of the GAGs varied in a species-specific manner. Anticoagulant activity assay revealed that both CMG-1.0 and GMG-1.0 had potent anticoagulant activity, which can significantly prolong activated partial thromboplastin time. GMG-1.0 also can prolong the thrombin time. CMG-1.0 showed no intrinsic tenase inhibition activity, while GMG-1.0 can obviously inhibit intrinsic tenase with EC50 of 58 nM. Their significantly different anticoagulant activities may be due to their different disaccharide structural units and proportions. These findings suggested that swim bladder by-products of fish processing of these two marine organisms may be used as a source of anticoagulants.

Structural characterization of a water-soluble acidic polysaccharide CSP-IV with potential anticoagulant activity from fruit pulp of Clausena lansium (Lour.) Skeels Guifei

Four main water-soluble wampee fruit pulp polysaccharides, named CSP-I, CSP-II, CSP-III and CSP-IV, were isolated from Clausena lansium (Lour.) Skeels Guifei, therein CSP-IV content was higher than the others. All components possess certain anticoagulant activity demonstrated by prolonged activated partial thromboplastin time, especially CSP-IV, which suggests that CSP-IV plays anticoagulant effect through disturbing intrinsic coagulation pathway. The wampee polysaccharide CSP-IV with Mw of 510.1 kDa was mainly composed of Gal, Ara and GalA. Backbone of CSP-IV contains Gal, Ara and GalA, two kinds of side chains contain one monosaccharide Gal or Ara, both branch on Gal residue of backbone. CSP-IV has no the conformation of triple helix demonstrated by Congo red test. These results showed that CSP-IV is an acidic polysaccharide with potential anticoagulant activity via targeting intrinsic coagulation pathway.

Development of heparinized and hepatocyte growth factor-coated acellular scaffolds using porcine carotid arteries.

Tissue-engineered blood vessel substitutes have been developed due to the lack of suitable small-diameter vascular grafts. Xenogeneic extracellular matrix (ECM) scaffolds have the potential to provide an ideal source for off-the-shelf vascular grafts. In this study, porcine carotid arteries were used to develop ECM scaffolds by decellularization and coating with heparin and hepatocyte growth factor (HGF). After decellularization, cellular and nucleic materials were successfully removed with preservation of the main compositions (collagen, elastin, and basement membrane) of the native ECM. The ultimate tensile strength, suture strength, and burst pressure were significantly increased after cross-linking. Pore size distribution analysis revealed a porous structure within ECM scaffolds with a high distribution of pores larger than 10 μm. Heparinized scaffolds exhibited sustained release of heparin in vitro and showed potent anticoagulant activity by prolonging activated partial thromboplastin time. The scaffolds showed an enhanced HGF binding capacity as well as a constant release of HGF as a result of heparin modification. When implanted subcutaneously in rats, the modified scaffolds revealed good biocompatibility with enzyme degradation resistance, mitigated immune response, and anti-calcification. In conclusion, heparinized and HGF-coated acellular porcine carotid arteries may be a promising biological scaffold for tissue-engineered vascular grafts.

Branch distribution pattern and anticoagulant activity of a fucosylated chondroitin sulfate from Phyllophorella kohkutiensis

Fucosylated chondroitin sulfate (FCS) extracted from Phyllophorella kohkutiensis (PkFCS) is composed of d-GalNAc, d-GlcA, l-Fuc and -SO42−. According to the defined structures revealed by NMR spectra of the branches released by mild acid hydrolysis and oligosaccharides generated by β-eliminative depolymerization, the backbone of PkFCS is CS-E, and the branch types attached to C-3 of d-GlcA include l-Fuc2S4S, l-Fuc3S4S, l-Fuc4S, and the disaccharide α-d-GalNAc-1,2-α-l-Fuc3S4S with the ratio of 43:13:22:22. Notably, novel heptasaccharide and hendecasaccharide were identified that are branched with continuous distribution of the disaccharide. The structural sequences of the oligosaccharides indicate that three unique structural motifs are present in the entire PkFCS polymer, including a motif branched with randomly distributed different sulfated l-Fuc units, a motif containing regular l-Fuc2S4S branches and a motif enriched in α-d-GalNAc-1,2-α-l-Fuc3S4S. This is the first report about the distribution pattern of diverse branches in natural FCS. Natural PkFCS exhibited potent anticoagulant activity on APTT prolonging and anti-iXase activity. Regarding the structurally defined oligosaccharides with sulfated fucosyl side chains, octasaccharide (Pk4b) is the minimum fragment responsible for its anticoagulant activity correlated with anti-iXase. However, further glycosyl modification with a non-sulfated d-GalNAc at the C-2 position of l-Fuc3S4S could significantly decrease the anticoagulant and anti-iXase activity.

Integrated Analysis of Metabolomics Combined with Network Pharmacology and Molecular Docking Reveals the Effects of Processing on Metabolites of Dendrobium officinale.

Dendrobium officinale (D. officinale) is a precious medicinal species of Dendrobium Orchidaceae, and the product obtained by hot processing is called "Fengdou". At present, the research on the processing quality of D. officinale mainly focuses on the chemical composition indicators such as polysaccharides and flavonoids content. However, the changes in metabolites during D. officinale processing are still unclear. In this study, the process was divided into two stages and three important conditions including fresh stems, semiproducts and "Fengdou" products. To investigate the effect of processing on metabolites of D. officinale in different processing stages, an approach of combining metabolomics with network pharmacology and molecular docking was employed. Through UPLC-MS/MS analysis, a total of 628 metabolites were detected, and 109 of them were identified as differential metabolites (VIP ≥ 1, |log2 (FC)| ≥ 1). Next, the differential metabolites were analyzed using the network pharmacology method, resulting in the selection of 29 differential metabolites as they have a potential pharmacological activity. Combining seven diseases, 14 key metabolites and nine important targets were screened by constructing a metabolite-target-disease network. The results showed that seven metabolites with potential anticoagulant, hypoglycemic and tumor-inhibiting activities increased in relative abundance in the "Fengdou" product. Molecular docking results indicated that seven metabolites may act on five important targets. In general, processing can increase the content of some active metabolites of D. officinale and improve its medicinal quality to a certain extent.

BIO26: Engineering Anti-platelet polymers for regenerative cardiovascular applications

Thrombogenesis is the main failure cause of implantable cardiovascular devices. Thrombus formation not only occludes devices but exacerbates immune responses responsible for foreign body reaction. Biodegradable polymers modified with anticoagulating moieties represent a strategy to overcome these limitations. Herein, we propose the use of a novel polyester urethane urea (PEUU) with pendant carboxyl groups for functionalization with different anti-platelet deposition molecules. PEUU was synthesized from PCL (Polycaprolactone), 1,4-diisocyanatobutane and putrescine, and then chemically oxidized to introduce carboxyl groups in the polymer backbone (PEUU-COOH). PEUU-COOH was functionalized in-situ with Polyethyleneimine mW 400 (PEI), 4 arm Polyethylene Glycol (PEG-4-Arm-NH2), seleno-L-cystine, heparin sodium and fondaporinux as anti-platelet deposition molecules. After solvent casting, functionalization was confirmed via Fourier-transformed infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Metabolic activity (MTT) and hemolysis assays were performed to validate biocompatibility. Anti-platelet activity of PRP exposed films was examined via a platelet aggregation assay, through an activation assay via lactate dehydrogenase (LDH), and by surface imaging with scanning electron microscopy (SEM). Whole-blood clotting time quantification assay was carried out to evaluate anticogulating properties. Upon oxidation, PEUU exhibited COOH pendant groups that were successfully employed to conjugate the anti-platelet molecules, obtaining homogeneous films. Overall cytotoxicity was below 20% and the hemolysis was below 5%, suggesting high biocompatibility and hemocompatibility. The most potent anticoagulant activity was obtained for heparin-functionalized materials followed by PEG-4-Arm-NH2 and PEI. Surprisingly, Seleno-L-cystine and Fondaporinux showed no anti-platelet activity. Results indicate that the polymers introduced here are a promising strategy to avoid thrombogenesis in vascular grafts.

New Hybrid Tetrahydropyrrolo[3,2,1-ij]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation.

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa.

Prediction of a Stable Complex of Compounds in the Ethanol Extract of Celery Leaves (Apium graveolens L.) Function as a VKORC1 Antagonist

The vitamin K cycle, specifically the protein VKORC1 (Vitamin K epoxide reductase complex subunit 1), is closely linked to coagulation mechanisms in the body. Disruptions in this cycle will be associated with vascular diseases such as myocardial infarction and stroke. Warfarin is commonly used as an anticoagulant, but it still has side effects in interactions with numerous drug compounds; thus, it is necessary to search for safer candidate compounds. The ethanol extract of celery leaves (Apium graveolens L.) has potential anticoagulant activity, but the compound responsible for this activity has not been identified yet. The CADD method has been developed to predict in silico a compound's potential to interact with binding sites. This study aimed to predict interactions and obtain a stable complex of the compounds in the ethanol extract of celery leaves, which function as a VKORC1 antagonist. A total of 23 compounds were simulated using Autodock 4.2, and AMBER 18 was then used to simulate the stability of the five compounds with the best interactions. The docking simulation results of 17 test compounds (ligands) yielded five selected compounds, namely 6-isopentenyloxy-isobergapten (S1), Heratomin (S2), Apigenin (S3), Lanatin (S4), and Isoimperatorin (S5), with G values of −9.27, −9.26, −9.22, −9.13, and −8.94 kcal/mol, respectively. The MD simulation continued to produce 6-isopentenyloxy-isobergapten as the most effective ligand for stabilizing the complex for 100 ns from these five ligands. In conclusion, the 6-isopentenyloxy-isobergapten from celery leaf has the potential as a candidate anticoagulant, VKORC1.

Design of an Ultralow Molecular Weight Heparin That Resists Heparanase Biodegradation.

Heparanase, an endo-β-d-glucuronidase produced by a variety of cells and tissues, cleaves the glycosidic linkage between glucuronic acid (GlcA) and a 3-O- or 6-O-sulfated glucosamine, typified by the disaccharide -[GlcA-GlcNS3S6S]-, which is found within the antithrombin-binding domain of heparan sulfate or heparin. As such, all current forms of heparin are susceptible to degradation by heparanase with neutralization of anticoagulant properties. Here, we have designed a heparanase-resistant, ultralow molecular weight heparin as the structural analogue of fondaparinux that does not contain an internal GlcA residue but otherwise displays potent anticoagulant activity. This heparin oligosaccharide was synthesized following a chemoenzymatic scheme and displays nanomolar anti-FXa activity yet is resistant to heparanase digestion. Inhibition of thrombus formation was further demonstrated after subcutaneous administration of this compound in a murine model of venous thrombosis. Thrombus inhibition was comparable to that observed for enoxaparin with a similar effect on bleeding time.

A Nonsynonymous-Single-Nucleotide Polymorphism (ns-SNP) in TFPI, the Gene Encoding Tissue Factor Pathway Inhibitor (TFPI), is Pleiotropically Associated with Increased Plasma TFPI Levels and a Decreased Risk of Type 2 Diabetes (T2D) in Mexican Americans of South Texas

The ns-SNP, rs7586970 (T>C), which results in an asparagine (Asn [N]) to serine (Ser [S]) substitution at amino acid 221 (221 N>S) in TFPI, has been shown to be associated with coronary heart disease and T2D. We performed an exome scan of plasma TFPI levels in 706 participants in our study of genetic determinants of cardiovascular disease in Mexican Americans of South Texas. Using a linear mixed model approach, while accounting for age, sex, and their interactions, we found a heritability of 34% for plasma TFPI levels (p=4.4E-08). The only variant showing exome-wide significance was the rs7586970 (T>C) variant on chromosome 2 at the TFPI gene (p=5.1E-07; Figure 1A). The quantile-quantile distribution of the observed p-values from all the exome-wide tests against the p-values expected under the null hypothesis of no association shows an overall agreement, indicating that the p-value for the association test for the 221 N>S variant is likely not an artifact (data not shown). The regression coefficient for the polymorphism as a predictor of TFPI levels showed them to be increasing from the individuals homozygous for the major T-allele (T/T: N/N), to the heterozygous individuals (T/C: N/S), to the individuals homozygous for the minor C-allele (C/C: S/S) (Figure 1B). Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the rs7586970 (T>C) polymorphism is significantly associated with a reduction in the risk of T2D (p=0.009) (data not shown). While not shown, the effect of the TFPI variant is to upwardly displace the liability threshold to the right, which results in a reduced prevalence of T2D from 6% to just below 3% in the rare allele homozygotes. While the reduction is a small change, it is statistically significant, nonetheless. Moreover, our observed effect size in terms of T2D risk is consistent with the widely held view that T2D has a multifactorial etiology involving a complex combination of environmental risk factors and genes with small effects sizes. A plausible mechanism for the reduced risk of T2D involves tissue factor (TF), which is the physiologic initiator of coagulation that is normally found outside the vasculature. As such, coagulation is initiated when the blood vessel is disturbed or when vascular and blood cells are induced to secrete and express surface membrane TF, as occurs in inflammation. TF then binds its primary cofactor, FVIIa, which circulates in the blood, TF-FVIIa activates FIX and FX, and the small amount FXa generated converts prothrombin to thrombin. TFPI is the primary inhibitor of TF-induced coagulation by binding and inhibiting TF-FVIIa in a FXa-dependent manner. Coagulation only proceeds when enough FXa is generated to overwhelm the inhibitory potential of TFPI. The rs7586970 ns-SNP conferred substitution of Ser for Asn at amino acid 221, the putative glycosyl-phosphatidylinositol anchor site of TFPI-β, its phospholipid bound form, potentially disrupts the binding of TFPI-β to the surface of endothelial cells resulting in higher levels of free form which has more potent anticoagulant activity. In conclusion, we found that TFPI 221 N>S ns-SNP is pleiotropically associated with plasma TFPI levels and risk of T2D. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Vivo Anticoagulant and Antithrombic Activity of Depolymerized Glycosaminoglycan from Apostichopus japonicus and Dynamic Effect-Exposure Relationship in Rat Plasma.

Glycosaminoglycan from Apostichopus japonicus (AHG) and its depolymerized fragments (DAHGs) are anticoagulant fucosylated chondroitin sulfate. The aim of this study was to further evaluate the anticoagulant and antithrombic activity of AHG and DAHGs, as well as reveal the dynamic relationship between exposure and effect in vivo. The results demonstrated that AHG100 (Mw~100 kDa), DAHG50 (Mw~50 kDa), and DAHG10 (Mw~10 kDa) exhibited potent anticoagulant activity by inhibiting intrinsic factor Xase complex (FXase) as well as antithrombin-dependent factor IIa (FIIa) and factor Xa (FXa). These glycosaminoglycans markedly prevented thrombosis formation and thrombin-induced platelet aggregation in a dose- and molecular weight-dependent manner in vitro and in vivo. The further bleeding time measurement indicated that DAHG10 exhibited obviously lower hemorrhage risks than native AHG100. Following oral administration, DAHG10 could be absorbed into blood, further dose-dependently prolonging activated partial thromboplastin time (APTT) and thrombin time (TT) as well as inhibiting FXa and FIIa partially through FXase. Anticoagulant activity was positively associated with plasma concentration following oral administration of DAHG10. Our study proposed a new point of view to understand the correlation between effects and exposure of fucosylated chondroitin sulfate as an effective and safe oral antithrombotic agent.

Eco-friendly preparation of Bi2O3, Ag-Bi2O3 and Ag-Bi2O3-rGO nanomaterials and their photocatalytic H2 evolution, dye degradation, nitrite sensing and biological applications

The current studies demonstrate the preparation of Bi2O3, Ag-Bi2O3, Ag-Bi2O3-rGO nanocomposite using a clear and concise, environmentally sustainable, low-cost green combustion method employing citrus limon as fuel. The synthesised materials were characterised through XRD, UV-DRS, FT-IR, TGA, TEM, SEM and Raman studies. The synthesised Ag-Bi2O3-rGO composite exhibited much improved photocatalytic H2 evolution activity (432.7 μmolg-1h−1) which is 5.5 times greater than bare-Bi2O3 (78.6 μmolg-1h−1) and 4.2 times greater than Ag-Bi2O3 (101.2 μmolg-1h−1) NPs. Further, Bi2O3, Ag-Bi2O3 and Ag-Bi2O3-rGO composite degraded the MB dye by 63.96, 74.9 and 100 % respectively in 90 min. Ag-Bi2O3-rGO exhibited superior electrochemical nitrite sensing property (with the LOD of 19.792 μM). Ag-Bi2O3-rGO nanocomposite exhibited potential antiradical property with IC50 of 900 μg/mL Ag-Bi2O3-rGO was found to have potent direct haemolytic, anticoagulant, antiplatelet and antibacterial activities. This study illustrates a simple, environmental friendly green synthetic technique for large-scale synthesis of multifunctional Ag-Bi2O3-rGO nanocomposite.

Molecular Characterization of Kunitz-Type Protease Inhibitors from Blister Beetles (Coleoptera, Meloidae).

Protease inhibitors are widely studied since the unrestricted activity of proteases can cause extensive organ lesions. In particular, elastase activity is involved in the pathophysiology of acute lung injury, for example during SARS-CoV-2 infection, while serine proteases and thrombin-like proteases are involved in the development and/or pathology of the nervous system. Natural protease inhibitors have the advantage to be reversible and with few side effects and thus are increasingly considered as new drugs. Kunitz-type protease inhibitors (KTPIs), reported in the venom of various organisms, such as wasps, spiders, scorpions, and snakes, have been studied for their potent anticoagulant activity and widespread protease inhibitor activity. Putative KTPI anticoagulants have been identified in transcriptomic resources obtained for two blister beetle species, Lydus trimaculatus and Mylabris variabilis. The KTPIs of L. trimaculatus and M. variabilis were characterized by combined transcriptomic and bioinformatics methodologies. The full-length mRNA sequences were divided on the base of the sequence of the active sites of the putative proteins. In silico protein structure analyses of each group of translational products show the biochemical features of the active sites and the potential protease targets. Validation of these genes is the first step for considering these molecules as new drugs for use in medicine.

Bioactive Peptides and Proteins from Wasp Venoms

Wasps, members of the order Hymenoptera, use their venom for predation and defense. Accordingly, their venoms contain various constituents acting on the circulatory, immune and nervous systems. Wasp venom possesses many allergens, enzymes, bioactive peptides, amino acids, biogenic amines, and volatile matters. In particular, some peptides show potent antimicrobial, anti-inflammatory, antitumor, and anticoagulant activity. Additionally, proteinous components from wasp venoms can cause tissue damage or allergic reactions in organisms. These bioactive peptides and proteins involved in wasp predation and defense may be potential sources of lead pharmaceutically active molecules. In this review, we focus on the advances in bioactive peptides and protein from the venom of wasps and their biological effects, as well as the allergic reactions and immunotherapy induced by the wasp venom.

Potent antiplatelet aggregation, anticoagulant and antioxidant activity of aerial Canna x generalis L.H Bailey & E.Z Bailey and its phytoconstituents

This study evaluated for the first time the inhibitory effect on human platelet aggregation, blood coagulation, and the antioxidant activity of Canna x generalis fractions and its phytoconstituents. The antiplatelet effect was evaluated using the turbidimetric method to determine the percentage inhibition (%I), the area under aggregation curve (AUC) and the aggregation velocity (slope). Prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were measured to assess the inhibitory effect on blood coagulation. ABTS and DPPH assays were performed for evaluation of the antioxidant effect. Compounds structures were determined by NMR and MS spectroscopic methods. The ethyl acetate fraction exhibited the most potent antiplatelet (%I, 98.2 ± 1.7% at 4 mg/mL), anticoagulation (mean APTT, 42.97 s) and antioxidant activity (IC50, 58 ± 0.1 and 12.5 ± 2.0 μg/mL for DPPH and ABTS, respectively). Thirteen compounds were found for the first time from aerial CG: 16α-hydro‑ent-kauran-3β-ol-7,19-dioic acid, named cannagelic acid (a new ent‑kaurane diterpenoid) (1), 16α-hydro‑ent-kauran-17,19-dioic acid (2), ent‑kauran-15-en-17,19-dioic acid (3), 3-methoxybenzaldehyde (4), 2-methoxynaphtalene (5), 4-ketopinoresinol (6), p-coumaric acid (7), indole-3-carboxylic acid (8), procatechuic aldehyde (9), astragalin (10), isoquercitrin (11), corchoionol C (12) and rutin (13). Compounds 7, 9–11, 13 possess good antiplatelet, anticoagulant and antioxidant activity. This study demonstrated that compound 1 did not show any tested effects. Compounds 4 and 5 had a very weak antiplatelet effect. Compound 8 expressed the highest capacity to inhibit platelet aggregation for both ADP and collagen (%I = 70.2 ± 1.8 and 79.2 ± 1.4 at 0.4 mg/mL, respectively), followed by compound 6. Moreover, these two compounds at 0.4 mg/mL could prolong APTT compared to the negative control (p < 0.05). Compound 12 had weak antiplatelet activity, but its antioxidant effect was better than other tested compounds. In conclusion, CG would be a good natural resource of bioactive molecules or dietary supplements to prevent and/or treat cardiovascular and free radicals-associated diseases.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman

Ethnopharmacological relevanceThe prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. Aim of the studyWe aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. Materials and methodsW. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. ResultsThe anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2–8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. ConclusionOur results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

An integration strategy combined progressive multivariate statistics with anticoagulant activity evaluation for screening anticoagulant quality markers in Chinese patent medicine

Ethnopharmacological relevanceThe cardiovascular and cerebrovascular diseases affect human health globally. Naoxintong capsules (NXTs), a famous Chinese Patent Medicine, has been especially applied to treat cerebral infarction and coronary heart disease in clinical practice. The anticoagulant activity of this prescription plays an important role in this course of treatment. Aim of the studyThrombin and factor Xa (FXa) are two key targets considering the anticoagulant activity. The purpose of this investigation is to screen the quanlity markers as key thrombin and FXa inhibitors for the anticoagulant activity oriented quality control of Chinese patent medicine. Materials and methodsSimple multi-polar solvent extraction processes using various proportions of solvents were conducted and their thrombin/FXa inhibitory activities were evaluated in vitro. Bivariate correlation analysis (BCA), grey correlation analysis (GCA), and orthogonal partial least squares discriminate analysis (OPLS-DA) were adopted for screening the potential active markers related to the anticoagulant activity. The chemical structures of these active compounds were identified by UHPLC–Q-TOF-MS/MS and their thrombin/FXa inhibitory activity was determined. The molecular docking technology was applied to explore the interaction between the compounds and targets. The contribution of these anticoagulant active ingredients in NXT was also investigated. Last but not the least, the contents of these markers in NXT were determined by liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method. ResultsThe results showed that the NXT extract exhibited great activity against thrombin and FXa, especially extracted by 75% methanol (v/v). Six marker compounds with potential anticoagulant activity were screened out. Therein, four of the active compounds owing thrombin inhibitory activity (paeoniflorin, lithospermic acid, salvianolic acid B, Z-ligustilide) and five of the active compounds owing FXa inhibitory activity (3,5-dicaffeoylquinic acid, rosmarinic acid, lithospermic acid, salvianolic acid B and Z-ligustilide). In addition, these active compounds accounted for a large proportion of thrombin/FXa inhibitory activity of NXTs. The binding energy also showed the strong interaction formed by close connection of the compounds to the residues of targets. ConclusionsThe proposed integrated stategy could be an efficient strategy to screen potential thrombin/FXa inhibitors for the bioactivity related quanlity control of Chinese patent medicine.