Theoretical exploring of potential mechanisms of antithrombotic ingredients in danshen-chishao herb-pair by network pharmacological study, molecular docking and zebrafish models.

BACKGROUND: Salvia miltiorrhiza (Danshen, DS) and Radix Paeoniae Rubra (Chishao, CS) herbal pair (DS-CS) is a famous traditional Chinese combination which has been used as antithrombotic formular for centuries. However, there is still lack of sufficient scientific evidence to illustrate its underlying mechanisms. The purpose of this study is to investigate the antithrombotic effects of DS-CS extract in zebrafish and explore its possible mechanism of action. METHODS: The quality of traditional Chinese medicines DS and CS granules was evaluated using High Performance Liquid Chromatography (HPLC). Subsequently, the therapeutic effect of the DS-CS combination and its components, Salvianolic Acid A (SAA) and Paeoniflorin (PF), in various concentrations on thrombosis was experimentally validated. Moreover, the interaction between DS-CS and the thrombosis disease targets was analyzed through network pharmacology, predicting the potential antithrombotic mechanism of DS-CS. Molecular docking and in vivo zebrafish experiments were conducted to validate the predicted targets, with qRT-PCR utilized for target validation. RESULTS: DS-CS exhibited anti-thrombotic effect in zebrafish with concentrations ranging from 25 to 300 µg/mL. The co-administration of PF and SAA at 25 µg/mL each revealed a synergistic antithrombotic effect exceeding that of individual components when contrasted with PHZ treatment. Protein-protein interaction (PPI) analysis identified key genes, including Albumin (ALB), Proto-oncogene tyro-sine-protein kinase Src (SRC), Matrix metalloproteinase-9 (MMP9), Caspase-3 (CASP3), Epidermal growth factor receptor (EGFR), Fibroblast growth factor 2 (FGF2), Vascular endothelial growth factor receptor 2 (KDR), Matrix metalloprotein-ase-2(MMP2), Thrombin (F2), and Coagulation factor Xa (F10), associated with the antithrombotic action of PF and SAA. Furthermore, KEGG pathway analysis indicated involvement of lipid metabolism and atherosclerosis pathways. Molecular docking revealed strong binding of PF and SAA to pivotal hub genes, such as SRC, EGFR, and F10. The experimental findings demonstrated that DS-CS could upregulate the mRNA expression levels of EGFR while inhibiting F10 and SRC mRNA levels, thereby ameliorating thrombotic conditions. CONCLUSION: This research provided valuable insights into the potential mechanisms underlying the antithrombotic activity of DS-CS. Our findings suggested that PF and SAA could be the key active ingredients responsible for this activity. The antithrombotic effects of DS-CS appeared to be mediated through the regulation of mRNA expression of SRC, EGFR, and F10. These results enhanced our understanding of DS-CS's therapeutic potential and lay the groundwork for future studies to further elucidate its mechanisms of action.

Antiplatelet and Antithrombotic Properties of Compound L-36, a 6H-1,3,4-Thiadiazine Derivative.

Compound L-36, a new derivative of 6H-1,3,4-thiadiazine, was studied in in vitro and in vivo experiments. This compound exhibits high antiplatelet and antithrombogenic activity. In in vitro experiments, compound L-36 by its antiplatelet activity (by IC50) was superior to acetylsalicylic acid by 9.4 times. In in vivo experiments, compound L-36 by its ED50 value was close to the comparison drug. On the model of pulmonary artery thrombosis, compound L-36 ensured better survival of experimental animals than acetylsalicylic acid. Morphological studies showed that compound L-36 effectively attenuated the thrombosis processes in the pulmonary tissue induced by intravenous injection of a thrombogenic mixture (epinephrine and collagen).

Comparison of the prophylactic antithrombotic effect of indobufen and warfarin in patients with nephrotic syndrome: a randomized controlled trial.

PURPOSE: The risk of thromboembolic events is elevated in patients with nephrotic syndrome, and warfarin use has been associated with an increased risk of bleeding. Indobufen, a selective cyclooxygenase-1 inhibitor, is currently being evaluated for the prevention of thromboembolic events in nephrotic syndrome. This study aimed to compare the efficacy and safety of indobufen with that of warfarin in patients with nephrotic syndrome. MATERIALS AND METHODS: This multicenter, randomized, three-arm, open-label, parallel controlled trial involved a total of 180 adult patients with nephrotic syndrome from four centers in China. Patients were randomly assigned to receive 100 mg indobufen (bid), 200 mg indobufen (bid), and 3 mg warfarin (qd) daily for 12 weeks. The primary endpoints included thromboembolic and bleeding events, while laboratory results and adverse events constituted secondary endpoints. RESULTS: No thromboembolic events occurred in the high-/low-dose indobufen and warfarin groups. Moreover, the use of a low dose of indobufen significantly reduced the risk of minor bleeding events compared with warfarin use (2% versus 18%, p < .05). Finally, adverse events were more frequent in warfarin-treated patients. CONCLUSIONS: This study found that indobufen therapy provided equivalent effects in preventing thromboembolic events compared with warfarin therapy, while low dose of indobufen was associated with a reduced risk of bleeding events, thus it should be recommended for the prevention of thromboembolic events in clinical practice in patients with nephrotic syndrome. TRIAL REGISTRATION NUMBER: ChiCTR-IPR-17013428.

The Antithrombotic Effect of Recombinant Neorudin on Thrombi.

Introduction: Recombinant neorudin (EPR-hirudin, EH) was developed through the addition of an EPR (Glu-Pro-Arg) peptide to the amino terminus of hirudin, which can be recognized and cut by coagulation factors XIa (FXIa) and/or Xa (FXa). In this study, the low-bleeding antithrombotic effects of EH were evaluated utilizing experimental models of thrombosis in rabbits and rats to provide a test basis for clinical trials. Methods: The bleeding risks of EH and hirudin were first compared in mice by the tail-clipping method, and then the antithrombotic activity of EH was investigated in a rabbit model of arteriovenous bypass thrombosis and a rat model of thrombotic cerebral infarction. Results: In mice, intravenous administration of EH at 1.5 mg/kg and 3 mg/kg did not affect the bleeding time compared with normal saline, while the administration of hirudin at 1.5 mg/kg prolonged the bleeding time by over 3 times the administration of normal saline. Furthermore, intravenous administration of EH had a significant dose-dependent inhibitory effect on the formation and development of arteriovenous bypass thrombosis and thrombotic cerebral infarction. Compared with an equimolar dose of hirudin, the antithrombotic effect of EH was similar, while the bleeding side effects were significantly attenuated. Moreover, when the antithrombotic effects were similar, EH had a shorter bleeding time and was associated with less bleeding than low molecular weight heparin (LMWH). EH had a therapeutic effect on thrombotic cerebral infarction without increasing the occurrence of cerebral hemorrhage. Conclusion: The findings from the preclinical animal models used in this study showed that EH could not only effectively inhibit thrombus formation but also reduce the risk of bleeding.

Development of drug loaded cardiovascular prosthesis for thrombosis prevention using 3D printing.

Cardiovascular disease (CVD) is a general term for conditions which are the leading cause of death in the world. Quick restoration of tissue perfusion is a key factor to combat these diseases and improve the quality and duration of patients' life. Revascularization techniques include angioplasty, placement of a stent, or surgical bypass grafting. For the latter technique, autologous vessels remain the best clinical option; however, many patients lack suitable autogenous due to previous operations and they are often unsuitable. Therefore, synthetic vascular grafts providing antithrombosis, neointimal hyperplasia inhibition and fast endothelialization are still needed. To address these limitations, 3D printed dipyridamole (DIP) loaded biodegradable vascular grafts were developed. Polycaprolactone (PCL) and DIP were successfully mixed without solvents and then vascular grafts were 3D printed. A mixture of high and low molecular weight PCL was used to better ensure the integration of DIP, which would offer the biological functions required above. Moreover, 3D printing technology provides the ability to fabricate structures of precise geometries from a 3D model, enabling to customize the vascular grafts' shape or size. The produced vascular grafts were fully characterized through multiple techniques and the last step was to evaluate their drug release, antiplatelet effect and cytocompatibility. The results suggested that DIP was properly mixed and integrated within the PCL matrix. Moreover, these materials can provide a sustained and linear drug release without any obvious burst release, or any faster initial release rates for 30 days. Compared to PCL alone, a clear reduced platelet deposition in all the DIP-loaded vascular grafts was evidenced. The hemolysis percentage of both materials PCL alone and PCL containing 20% DIP were lower than 4%. Moreover, PCL and 20% DIP loaded grafts were able to provide a supportive environment for cellular attachment, viability, and growth.

Saponins as Modulators of the Blood Coagulation System and Perspectives Regarding Their Use in the Prevention of Venous Thromboembolic Incidents.

Saponins comprise a heterogenous group of chemical compounds containing a triterpene or steroid aglycone group and at least one sugar chain. They exist as secondary metabolites, occurring frequently in dicotyledonous plants and lower marine animals. Plant saponin extracts or single saponins have indicated antiplatelet and anticoagulant activity. Venous thromboembolism (VTE), including deep venous thrombosis and pulmonary embolism, is a multifactorial disease influenced by various patient characteristics such as age, immobility, previous thromboembolism and inherited thrombophilia. This mini-review (1) evaluates the current literature on saponins as modulators of the coagulation system, (2) discusses the impact of chemical structure on the modulation of the coagulation system, which may further provide a basis for drug or supplement design, (3) examines perspectives of their use in the prevention of VTE. It also describes the molecular mechanisms of action of the saponins involved in the prevention of VTE.

Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish.

Rubiae Radix et Rhizoma (called "Qiancao", QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called "Qiancaotan", QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC-MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile-water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.

Biological Activities of Rhamnan Sulfate Extract from the Green Algae Monostroma nitidum (Hitoegusa).

Monostroma nitidum is a green single-cell layered algae that grows on the southwest coast of Japan. It is often used for salad ingredients, boiled tsukudani, soups, etc., due to its health benefits. M. nitidum is composed of many cell aggregates, and the various substances that fill the intercellular space are dietary fibers, vitamins, and minerals. Rhamnan sulfate (RS), a sulfated polysaccharide, is main the component of the fiber extracted from M. nitidum. Recently, some biological properties of RS have been demonstrated by in vitro and in vivo studies that probably protect human subjects from viruses and ameliorate vascular dysfunction caused by metabolic disorders, especially lifestyle-related diseases. In this review, we focus on the antithrombotic effects of RS and introduce its antiviral and other biological activities.

Effect of water-extractive components from funazushi, a fermented crucian carp, on the activity of fibrinolytic factors.

BACKGROUND: Japanese fermented foods, including funazushi, have have been studied insufficiently. Related research into fermented products has led to the hope that they might have positive effects on blood circulation, including anti-thrombosis effects. The possible antithrombotic effects of funazushi on the fibrinolytic system were examined. RESULTS: The administration of extracts from funazushi increased the activity of plasmin and tissue plasminogen activators in the fibrinolytic system but decreased the activity of plasminogen activator inhibitor type-1 (PAI-1). This decrease was positively correlated with the decreased plasma triglyceride levels. Funazushi extract directly inhibited PAI-1 activity in vitro despite alimentary enzyme digestion, although direct PAI-1 inhibition was not observed in an extract from salted crucian carp. CONCLUSION: These results suggest that funazushi extracts are closely involved in the antithrombotic effects of the fibrinolytic system, and that they exert their effect through a reduction in PAI-1 activity. The findings also indicate that fermentation processing is necessary to achieve the antithrombotic effects of funazushi. © 2020 Society of Chemical Industry.

Influence of ceftriaxone on pharmacokinetics, anticoagulation andantithrombotic effects of phenolic acids from Danhong injection in rats / 药学学报

Danhong injection (DHI) and ceftriaxone sodium were used in combination based on their experimental uses in clinic. This study was designed to investigate the impact of ceftriaxone on pharmacokinetics and pharmacodynamics of the phenolic acids from DHI. After administration of DHI for 7 d, ceftriaxone (CFTX) was combined with DHI for the next 7 d in adult male Sprague-Dawley (SD) rats. All the drugs were administered through caudal vein. UHPLC-TQ-MS was applied in determining the plasma concentration of p-coumaric acid (p-CA), salvianolic acid D (SaD), rosmarinic acid (RA) and salvianolic acid B (SaB). The pharmacokinetic parameters of the combination group or the Danhong injection alone group were calculated by statistical moment method, Cmax and the average of the area under the curve AUC0-t using 90% confidence interval of the bioequivalence and bioavailability degree module in DAS 3.2.8 statistic software. The results showed that Cmax of p-CA, SaD, RA and SaB were unqualified within 90% confidence intervals for bioequivalence statistics. And the results showed that AUC0-t of SaD, RA and SaB within 90% confidence intervals for bioequivalence statistics were unqualified. There were no significant difference in the tmax (P>0.05). The results of anticoagulation in vivo showed that the international normalized ratio (INR), prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were significantly increased when combined with CFTX (P<0.05 or P<0.01). The results in antithrombotic effects revealed that the thromboxane B2 (TXB2) level in serum was significantly decreased (P<0.01) in the combination group compared with Danhong injection alone. However, there was no significant difference in antiplatelet effects. These results suggest that CFTX may enhance the anticoagulation and antithrombotic effects of DHI through altering pharmacokinetics and pharmacodynamics in SD rats.

Screening and validation of antithrombotic effective components group from Trichosanthes extract / 药学学报

To screen the antithrombotic effective components group of Trichosanthes extract, and to verify its pharmacodynamics and analyze its mechanism, the HPLC fingerprint of Trichosanthes extract (0.09, 0.45, 0.9 g·kg-1) was established, and the pharmacodynamic indexes of antithrombosis in rats with aspirin (0.01 g·kg-1) as positive control group were determined (the animals used in this experiment were approved by the Medical Ethics Committee of Wannan Medical College). The antithrombotic spectrum-activity relationship of Trichosanthes extract was studied and the effective antithrombotic ingredients group was screened by grey relational analysis. The monomer compound mixed solution (0.006, 0.03, 0.06 g·kg-1) was prepared according to the content of each component in the active component group, and the pharmacodynamics and action mechanism were studied to verify the correctness of the spectrum-effect relationship. The correlation between the 22 components of Trichosanthes extract and antithrombotic efficacy was different and showed dose-effect relationship. Cytosine, uracil, guanine, hypoxanthine, xanthine, adenine, guanosine, and adenosine are the main antithrombotic components of Trichosanthes extract. The ratio of cytosine, uracil, guanine, hypoxanthine, xanthine, adenine, guanosine and adenosine was 3∶12∶10∶5∶2∶8∶13∶14. Compared with the model group, the thrombus dry weight of each effective components group could be effectively reduced (P<0.01 or P<0.05), but there was no significant difference between each effective components group and the Trichosanthes extract group. Compared with the model group, the TXB2 content in group (0.06 g·kg-1, 0.03 g·kg-1) could be effectively reduced (P<0.01 or P<0.05), and the content of 6-keto-PGF1α could be increased in each group (P<0.01), and the TXB2/6-keto-PGF1α tended to be normal and showed a dose-effect relationship. The effect was better than that in the Trichosanthes extract group (0.45 g·kg-1) (P<0.01). The effective ingredients group has a good antithrombotic effect, its mechanism is to inhibit platelet aggregation and improve vascular endothelial function.

Aqueous Extract of Taegeuk Ginseng Inhibits Platelet Aggregation and Thrombus Formation.

The inhibitory effects of taegeuk ginseng extract (TGE) on platelet aggregation and thrombus formation were investigated. The TGE significantly inhibited collagen- and adenosine 5'-diphosphate (ADP)-induced platelet aggregation in vitro in a dose-dependent manner. Also oral administration of TGE to rats significantly prevented ADP- and collagen-induced platelet aggregation ex vivo, but it did not affect the plasma coagulation system. The oral administration of TGE significantly delayed the occlusion of the carotid artery in ferrous chloride-treated rats in vivo. These results suggest that in vivo antithrombotic effect of TGE may be due to its inhibitory activity on platelet aggregation rather than on plasma coagulation.

Preparation and characterization of acetylsalicylic acid/chitosan nanoparticles and its antithrombotic effects.

Chitosan (CS)-acetylsalicylic acid (ASA) nanoparticles, which are well dispersed and stable in aqueous solution, have been prepared by interpolymer complexation of ASA in CS solution. The physicochemical properties of nanoparticles were investigated by using FT-IR, 1H NMR, scanning electron microscope（SEM）, dynamic light scattering, and UV spectrophotometer. It was found that the carboxyl group of the ASA had firmly integrated on the amino group of CS and the ASA-CS nanoparticles were almost spherical in shape with an average diameter of less than (79.3 ± 24.6) nm in high reproducibility and showed high chemical stability against environmental changes. It was also found that the prepared nanoparticles carried a positive charge and showed the size in the range from 700 to 150 nm. The surface structure and zeta potential of nanoparticles can be controlled by different preparation processes. The factor experiment results indicated that the ASA-CS nanoparticles had satisfactory loading capacity (LC) and encapsulation efficiency (EE), 27.27% and 46.88% (data not shown), respectively. The experiments of in vitro ASA release showed that these nanoparticles provided a sustained and pH-dependent drug release manner, and the release behavior was influenced by the pH value of the medium. Preliminary pharmacology experiment exhibited prolonged circulation and higher bioavailability than that of ASA. All the results indicated that ASA/CS nanoparticles may have promising pharmaceutical application, and further pharmacological research is needed to confirm these beneficial effects.

Chemical analysis, pharmacological activity and process optimization of the proportion of bilobalide and ginkgolides in Ginkgo biloba extract.

Variations on the efficacy of commercial Ginkgo biloba preparations have been reported, although all the products follow the same standards. Terpene trilactones (TTLs), including bilobalide (BB) and ginkgolides, are one of the main active components in G. biloba extract and have been received the most attention due to their chemical uniqueness and their importance for quality control. A plenty of studies demonstrated that BB and ginkgolides display differential activities on various biological processes. However, the influence of different ratios of BB and ginkgolides on the efficacy of TTLs has not been detected yet. The aims of this study were: (1) to test whether different ratios of BB and ginkgolides existed in commercial G. biloba preparations; (2) to detect the influence of different ratios of BB and ginkgolides on the in vivo efficacy of TTLs; and (3) to optimize the extraction process of G. biloba to approach the better BB and ginkgolides ratio with the maximum in vivo effects. First, the content and proportion of BB and ginkgolides in various G. biloba preparations were quantified by HPLC-MS analysis. As the results, an obvious fluctuation in the proportion of BB and ginkgolides was observed in the preparations from different commercial suppliers. The ratio was ranged from 0.3 to 0.8. Second, a zebrafish thrombosis model was used to evaluate the antithrombotic effects of different ratios of BB and ginkgolides. The result showed that the proportion of BB and ginkgolides at 1:2 produced the maximum antithrombotic effects. Third, the extraction process of G. biloba was optimized using a design space technique aiming to approach the best BB and ginkgolides ratio obtained from zebrafish experiment. The extraction process was modeled based on the results of Box-Behnken designed experiments. Design space was then calculated using a probability-based method. Within this design space, G. biloba extraction process can be guaranteed to achieve the better BB and ginkgolides ratio with high assurance. Normal operation space for G. biloba extraction process was recommended as ethanol concentration of 50% to 70%, liquid-to-solid ratio of 5.6 mL/g to 7.3 mL/g, and extraction time of 2.2 h to 3.0 h. This work not only suggest that the proportion of BB and ginkgolides should be used as a quality control index in ginkgo preparations besides the content of TTLs, but also provide a way to approach it with the extraction process parameters controlled in the normal operation ranges.

Antithrombotic effects of cilostazol combined with clopidogrel and aspirin in senile patients with cerebrovas-cular disease after PCI surgery / 中国基层医药

Objective To analyze the antithrombotic effects of cilostazol combined with aspirin and clopi-dogrel in elderly patients with cerebrovascular disease after PCI .Methods 100 elderly patients with cerebrovascular diseases who treated with coronary artery interventional therapy ( PCI) were randomly divided into the control group and the observation group according to the digital table ,50casess in each group.The two groups were given control of blood pressure ,blood lipids ,blood sugar ,improve circulation and other conventional treatment .The control group was treated with aspirin combined with clopidogrel ,the observation group was treated with cilostazol based on the treatment of control group.Before and after treatment for 1,4 and 8 weeks,the platelet aggregation degree was detected by PL-11 automatic platelet analyzer .During 2 months of follow-up,the degree of platelet aggregation ,the volume of platelets,the efficacy of treatment and the incidence of adverse reactions were compared .Results The platelet aggre-gation rate between the two groups had no statistically significant difference before treatment (t0 =2.782,P>0.05). After treatment,the platelet aggregation rate of the two groups decreased significantly ,but after treatment for 1,4 and 8 weeks,the platelet aggregation rates of the observation group were significantly lower than those of the control group [(51.87 ±9.65)％,(40.85 ±10.24)％,(38.52 ±9.64)％;(69.25 ±8.41)％,(62.43 ±9.22)％,(58.46 ± 10.18)％],the differences were statistically significant (t1 =5.693,t4 =4.846,t8 =6.719,all P<0.05).Before treatment,the mean platelet volume between the two groups had statistically significant difference ( t0 =2.146,P>0.05).After treatment,the platelet volume of the two groups decreased significantly ( t1 =1.656,t4 =1.438,t8 =2.189,all P<0.05).There were no statistically significant differences between the observation group and the control group (t1 =3.716,t4 =1.271,t8 =2.523,all P>0.05).The effective rate of the observation group was 94.00％(47/50),which was significantly higher than that of the control group [82.00％(41/50)],the difference was statis-tically significant (χ2 =4.683,P<0.05).The incidence rates of adverse reactions in the observation group and the control group were 10.00％(5/50) and 8.00％(4/50),respectively,there was no statistically significant difference between the two groups (χ2 =1.947,P=0.136).Conclusion Cilostazol combined with clopidogrel and aspirin in the treatment of elderly patients with cerebrovascular disease after PCI can significantly reduce platelet aggregation rate,improve clinical curative effect ,and has certain clinical value .

Reduced metabolic activity of gut microbiota by antibiotics can potentiate the antithrombotic effect of aspirin.

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

Anticoagulant properties of enoxaparin 400 IU/mL-40 % ethanol catheter lock solution.

Unfractionated heparin (UFH) is the most widely used interdialytic lock solution but has no anti-infectious properties. Ethanol at a content ≥40 %v/v eradicates experimental biofilm but has no anticoagulant properties. In contrast to UFH, enoxaparin (Enox) can be combined with 40 % ethanol without precipitation. Enoxaparin 400 UI/mL-40 % ethanol (Enox/Eth) has antibiofilm properties and therefore has promise as an alternative lock solution. This study assessed the anticoagulant properties of Enox/Eth. Enox and Enox/Eth were diluted in whole blood at a final Enox concentration of 0.5, 1 (N = 6 samples), 1.5 (N = 4) and 2 (N = 6) IU/mL. Anti-Xa activity was determined by chromogenic assay and the inhibition of endogenous thrombin potential (ETP) by thrombinography. Quantitative data were compared by the Mann-Withney U test. For Enox concentrations of 0.5, 1, 1.5 and 2 UI/mL in whole blood samples, the mean ± SD values of the anti-Xa activity were 0.68 ± 0.09, 1.26 ± 0.14, 1.73 ± 0.30, 2.35 ± 0.32 UI/mL for Enox/Eth and 0.94 ± 0.15, 1.80 ± 0.22, 2.74 ± 0.23, 3.54 ± 0.44 UI/mL for Enox (P = 0.03, P = 0.03, P = 0.13, P = 0.03); and of the percentage of ETP inhibition was 17.36 ± 9.65, 30.27 ± 17.06, 36.5 ± 17.06, 57.82 ± 15.42 for Enox/Eth, and 42.96 ± 15.68, 68.93 ± 10.01, 83.5 ± 8.81, 91.19 ± 4.67 for Enox (P = 0.03, P = 0.03, P = 0.13, P = 0.03), respectively. The median and IQR values of Enox concentration inhibiting 50 % of ETP (IC50 ETP) were 1.8 [1.1-2.4] IU/mL for Enox/Eth and 0.7 [0.3-0.9] IU/mL for Enox, P = 0.03. Enox/Eth has strong anticoagulant activity, albeit lower than that of Enox, but with an extremely low IC50 ETP compared to the Enox concentration of non-diluted Enox/Eth.

Lignans and aromatic glycosides from Piper wallichii and their antithrombotic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper wallichii (Miq.) Hand.-Mazz. is a medicinal plant used widely for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina in China. Previous study showed that lignans and neolignans from Piper spp. had potential inhibitory activities on platelet aggregation. In the present study, we investigated the chemical constituents of Piper wallichii and their antithrombotic activities, to support its traditional uses. MATERIALS AND METHODS: The methanolic extract of the air-dried stems of Piper wallichii was separated and purified using various chromatographic methods, including semi-preparative HPLC. The chemical structures of the isolates were determined by detailed spectroscopic analysis, and acidic hydrolysis in case of the new glycoside 2. Determination of absolute configurations of the new compound 1 was facilitated by calculated electronic circular dichroism using time-dependent density-functional theory. All compounds were tested for their inhibitory effects on platelet aggregation induced by platelet activating factor (PAF) in rabbits׳ blood model, from which the active ones were further evaluated the in vivo antithrombotic activity in zebrafish model. RESULTS: A new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A-D (2-5) were isolated from the stems of Piper wallichii, along with 25 known compounds, including 13 lignans, six aromatic glycosides, two phenylpropyl aldehydes, and four biphenyls. Five known compounds (6-10) showed in vitro antiplatelet aggregation activities. Among them, (-)-syringaresinol (6) was the most active compound with an IC50 value of 0.52 mM. It is noted that in zebrafish model, the known lignan 6 showed good in vivo antithrombotic effect with a value of 37% at a concentration of 30 µM, compared with the positive control aspirin with the inhibitory value of 74% at a concentration of 125µM. CONCLUSION: This study demonstrated that lignans, phenylpropanoid and biphenyl found in Piper wallichii may be responsible for antithrombotic effect of the titled plant.

Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats.

BACKGROUND: Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants. OBJECTIVES: To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage. METHODS: Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail. RESULTS: In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose-response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin. CONCLUSIONS: These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.

The antithrombotic and fibrinolytic effect of natto in hypercholesterolemia rats.

Antithrombotic and fibrinolytic activity of natto was evaluated on platelet aggregation in vitro and in vivo. Natto showed inhibitory effects on platelet aggregation induced by adenosine 5'diphosphate (ADP) and collagen. Orally administered natto also showed fibrinolytic activity in hypercholesterolemia rats. Normal levels of natto, when administered for four weeks, shortened euglobulin clot lysis time (ECLT) and prolonged partial thromboplastin time (PATT) significantly compared to non-treated group. In addition, the natto treatment decreased total cholesterol in serum. These results showed that intake of normal levels of natto can elicit antithrombotic and fibrinolytic effects, suggesting its consumption may improve blood circulation.