Antiplatelet and Anticoagulant Effects of Two New Phenylpropanoid Sucrose Esters and Other Secondary Metabolites from the Aerial Part of Canna edulis.

This study isolated pure compounds from Canna edulis aerial parts and assessed their antiplatelet and anticoagulant potential. Structural elucidation resulted in the identification of two new compounds: caneduloside A (1) and caneduloside B (2), and eleven known compounds: 6'-acetyl-3,6,2'-tri-p-coumaroyl sucrose (3), 6'-acetyl-3,6,2'-triferuloyl sucrose (4), tiliroside (5), afzelin (6), quercitrin (7), 2-hydroxycinnamaldehyde (8), cinnamic acid (9), 3,4-dimethoxycinnamic acid (10), dehydrovomifoliol (11), 4-hydroxy-3,5-dimethoxybenzaldehyde (12), and (S)-(-)-rosmarinic acid (13). Compounds 3, 4, 6-9, 13 were previously reported for antithrombotic properties. Hence, antithrombotic tests were conducted for 1, 2, 5, 10-12. All tested compounds demonstrated a dose-dependent antiaggregatory effect, and 10 and 12 were the most potent for both ADP and collagen activators. Additionally, 10 and 12 showed anticoagulant effects, with prolonged prothrombin time and activated partial thromboplastin time. The new compound 1 displayed antiplatelet and anticoagulant activity, while 2 mildly inhibited platelet aggregation. C. edulis is a potential source for developing antithrombotic agents.

Overview of piperlongumine analogues and their therapeutic potential.

Natural products have long been an important source for discovery of new drugs to treat human diseases. Piperlongumine (PL) is an amide alkaloid isolated from Piper longum L. (long piper) and other piper plants and has received widespread attention because of its diverse biological activities. A large number of PL derivatives have been designed, synthesized and assessed in many pharmacological functions, including antiplatelet aggregation, neuroprotective activities, anti-diabetic activities, anti-inflammatory activities, anti-senolytic activities, immune activities, and antitumor activities. Among them, the anti-tumor effects and application of PL and its derivatives are most extensively studied. We herein summarize the development of PL derivatives, the structure and activity relationships (SARs), and their therapeutic potential on the treatments of various diseases, especially against cancer. We also discussed the challenges and future directions associated with PL and its derivatives in these indications.

Antiplatelet activity and chemical analysis of leaf and fruit extracts from Aristotelia chilensis.

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.

In vitro antiplatelet activity of extract and its fractions of Paulownia Clone in Vitro 112 leaves.

BACKGROUND: Paulownia Clone in Vitro 112, also known as Oxytree is a hybrid of Paulownia elongata and Paulownia fortunei, developed under laboratory conditions. Its seeds are sterile, making it a noninvasive variety that can only be propagated in the laboratory. In China, species from the Paulownia genus (Paulowniaceae) are widely used in traditional medicine for the treatment of infectious diseases, such as gonorrhea and erysipelas. It has a broad spectrum of bioactivity, including neuroprotective, antioxidant, antibacterial, antiphlogistic, antiviral, and cytotoxic actions. However, the antiplatelet potential of Paulownia Clone in Vitro 112 has not yet been described. STUDY DESIGN: The aim of our study was thus to examine the effect of an extract and four fractions from leaves of Paulownia Clone in Vitro 112 on various parameters of platelet activation in an in vitro model. METHODS: Composition of the investigated extract and fractions was determined by UHPLC-UV-MS. The following parameters of platelet activation were investigated: nonenzymatic lipid peroxidation in resting platelets; enzymatic lipid peroxidation (AA metabolism) in platelets activated by thrombin; superoxide anion (O2-.) generation in the resting and activated platelets; platelet adhesion to collagen type I and fibrinogen; platelet aggregation stimulated by various physiological agonists, such as ADP, collagen, and thrombin. The effect of the extract and fractions on extracellular LDH activity, a marker of cell damage, was also determined. RESULTS: Verbascoside a phenylethnanoid glycoside, was the main secondary metabolite of the extract from leaves of oxytree (constituting approximately 45 % of all compounds). There were also iridoids, such as catalpol, aucubin, and 7-hydroxytomentoside, as well as flavonoids, such as luteolin and apigenin glycosides. Moreover, the extract had stronger antiplatelet properties than the fractions. For example, the extract at 10 µg/mL inhibited five parameters of platelet activation. CONCLUSIONS: Our results show that Paulownia Clone in Vitro 112 leaves are a new valuable source of compounds with antiplatelet potential.

Callicarpa nudiflora Hook. & Arn.: A comprehensive review of its phytochemistry and pharmacology.

ETHNOPHARMALOGICAL RELEVANCE: Callicarpa nudiflora Hook. & Arn. is a perennial evergreen shrub or low arbor in the Genus Callicarpa. Its dried aerial parts are used as traditional Chinese herbal medicine, Luo-hua-zi-zhu (Callicarpa nudiflora), which has been widely used in anti-bacteria and anti-ulcer in China (Commission, 2015; Development, 1994; Ming-Sheng, 2008). AIM OF THE STUDY: The present paper reviewed findings in phytochemistry and pharmacology of Callicarpa nudiflora. METHODS: Chinese and English studies on Callicarpa nudiflora were collected from databases including Web of Science, SciFinder, PubMed, Elsevier, and CNKI (Chinese), and the phytochemical and pharmacological studies of Callicarpa nudiflora were reviewed. RESULTS: A total of 300 small molecules, 173 of which are volatile oils, have been isolated from Callicarpa nudiflora. These small molecules could be divided into seven structural types - phenylpropanoids, flavonoids, triterpenes, diterpenes, iridoid glycosides, volatile oils, and other small molecules. Different types of compounds in Callicarpa nudiflora were summarized as follow: a) diterpenoid compounds can inhibit the generation of nitric oxide (NO) for exerting the function of anti-inflammation; b) triterpene compounds can play a role of anti-thrombus via inhibiting platelet aggregation and oleanane type and arbutane type pentacyclic triterpenes have the hepatoprotective activities; c) iridoid glycosides have cytotoxicity to tumor cells, and phenylpropanoids compounds have an antioxidant effect and could improve the function of memory. Our group further studied the antiviral activities of Callicarpa nudiflora finding that it has significant effects on RSV, EV71, COXB5, and HSV-1.

Extracts from Uncaria tomentosa as antiplatelet agents and thrombin inhibitors - The in vitro and in silico study.

ETHNOPHARMACOLOGICAL RELEVANCE: A wide range of traditional medicine applications of Uncaria tomentosa (Willd. ex Schult.) DC., commonly known as 'vilcacora' or 'cat's claw', includes blood purification, its anticoagulant properties and its use in haemorrhage therapy. AIM OF THE STUDY: Our work is devoted to the effects of ethanol and aqueous extracts (1-50 µg/ml) from U. tomentosa leaves and bark on the haemostatic system. The study is based on two main questions: Can these extracts influence the coagulation cascade of blood plasma or the activation of blood platelets? Do they feature any anticoagulant properties? MATERIALS AND METHODS: Blood platelet aggregation was measured in human platelet-rich plasma; the anticoagulant tests were based on the thrombin, prothrombin and the activated partial thromboplastin time. For the thrombin (TH)-inhibitory activity evaluation, the chromogenic substrate S-2238 and fibrinogen, i.e. physiological substrate for this enzyme, were used. In silico studies included the interactions of TH and the main components of the extracts. RESULTS: The examined extracts demonstrated slight antiplatelet activity. The thrombin time was slightly prolonged. The most efficient TH inhibitor was the ethanolic fraction from leaves (IC50 = 5.86 and 12.48 µg/ml, for the amidolytic and proteolytic assay, respectively). The plant ingredients interacted with TH within and outside the active site, dependently on the compound. The higher binding affinity was found for procyanidins B2 and C1. CONCLUSIONS: The examined extracts demonstrated slight antiplatelet effects; however, they may be promising candidates for the natural inhibitors of TH, which is critical for the formation of fibrin clot.

Lagopsis supina extract and its fractions exert prophylactic effects against blood stasis in rats via anti-coagulation, anti-platelet activation and anti-fibrinolysis and chemical characterization by UHPLC-qTOF-MS/MS.

Lagopsis supina (Steph.) IK. -Gal. ex Knorr. has been used for centuries as an empiric treatment for blood stasis syndrome in China without scientific validation. The aim of this study was to evaluate for the first time the chemical profiling, efficacy and mechanism of L. supina ethanol extract (LS) and its four fractions (LSA∼D) in Dextran 500-induced acute blood stasis model rats. Oral administration of LS (229.0∼916.0 mg/kg) and LSC (17.6∼70.4 mg/kg) once daily for seven consecutive days significantly improved microcirculation hemodynamics function (blood flow, blood concentration and blood flow velocity), hemorheological parameters (whole blood viscosity, whole blood reduced viscosity, plasma viscosity, platelet aggregation rate, hematokrit, erythrocyte assembling index and erythrocyte deformation index), and coagulation parameters (thrombin time, prothrombin time, activated partial thromboplastin time, fibrinogen and antithrombin III). Furthermore, their markedly down-regulated thromboxane B2 and 6-keto-prostaglandin F1α levels. In addition, it significantly decreased tissue-type plasminogen activator (t-PA), urokinasetype plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) levels, as well as PAI-1/t-PA and PAI-1/u-PA rations. In parallel, 51 chemical constituents were identified from LS based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS), and quantitative analysis showed that the two major constituents of stachysoside A and acteoside were present in 0.90 ± 0.01 and 1.36 ± 0.01 mg/g of the L. supina whole plant, respectively. These findings suggest that LS and LSC possess prominent anti-blood stasis effect on rats by modulating the anti-coagulation, anti-platelet activation and anti-fibrinolysis, and supports the traditional folk use of this plant.

Isolation and Functional Identification of an Antiplatelet RGD-Containing Disintegrin from Cerastes cerastes Venom.

The current report focuses on purification, structural and functional characterization of Cerastategrin from Cerastes cerastes venom, a novel basic disintegrin (pI 8.36) with 128 amino acid residues and a molecular weight of 13 835.25 Da measured by MALDI-MSMS. The 3D structure of Cerastategrin is organized as α-helix (13%), ß-strand (15%) and disordered structure (30%) and presents homologies with several snake venom disintegrins. Structural modeling shows that Cerastategrin presents an RGD motif that connects specifically to integrin receptors. Cerastategrin exhibits the inhibition of ADP induced platelets with an IC50 of 0.88 µg/mL and shows in vivo long stable anticoagulation effect 24 h post-injection of increasing doses ranging from 0.2 to 1 mg/kg, therefore, Cerastategrin maintained irreversibly the blood incoagulable. Moreover, Cerastategrin decreases the amount of bounded αIIbß3 and reduced significantly the quantity of externalized P-Selectin. Cerastategrin acts as a molecule targeting specifically the receptor αIIbß3; therefore, it behaves as a potent platelet activation inhibitor. As a new peptide with promising pharmacological properties, Cerastategrin could have a potential therapeutical effect in the vascular pathologies and may be a new effective treatment approach.

Inhibitory effect on human platelet aggregation and coagulation and antioxidant activity of C. edulis Ker Gawl rhizome and its secondary metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: Although Canna edulis Ker Gawl rhizome has been used in Traditional Vietnamese Medicine to prevent and treat heart diseases without thorough scientific evidence, limited intensive search for the bioactivities and useful substances has been done. AIM OF THE STUDY: This study aims to investigate the antiplatelet aggregation, anticoagulant and antioxidant activity of extracts from C. edulis rhizome, separate and purify its compounds from the most active fraction and evaluate the antiplatelet aggregation, anticoagulant and antioxidant activity of isolated compounds. MATERIALS AND METHODS: C. edulis rhizome was extracted with ethanol, then fractionated with n-hexane, ethyl acetate and water. The inhibitory effect on adenosine diphosphate- and collagen-induced human platelet aggregation was evaluated. Prothrombin time, activated partial thromboplastine time and thrombine time were measured to examine the anticoagulant activity. The free radical scavenging ability was assessed with DPPH and ABTS assays. The fraction that showed the most active was used to separate and purify compounds. The structures of compounds were elucidated by NMR and MS spectroscopic methods. Isolated compounds were also tested for antiplatelet, anticoagulant and antioxidant activity. RESULTS: The ethyl acetate fraction showed the most potent antiplatelet aggregation, anticoagulant and antioxidant activity. Subsequent fractionation of this active fraction resulted in the isolation of seven known compounds: 5-hydroxy-6-methyl-2H-pyran-2-one (1), epimedokoreanone A (2), nepetoidin B (3), ferulic acid (4), caffeic acid (5), hydroxytyrosol (6), and 1H-indole-3-carboxaldehyde (7). Previous studies reported the antiplatelet, anticoagulant and antioxidant activity of ferulic acid (4), caffeic acid (5) and hydroxytyrosol (6) and the antioxidant activity of nepetoidin B (3). This study demonstrated that both epimedokoreanone A (2) and nepetoidine B (3) also exhibited good antiplatelet effect and epimedokoreanone A (2) also had effective anticoagulant and antioxidant activity, while 5-hydroxy-6-methyl-2H-pyran-2-one (1) showed weaker antiplatelet and antioxidant activity but no anticoagulant effect. CONCLUSIONS: This is the first experimental study to demonstrate the potent dose-dependent antiplatelet aggregation, anticoagulant and antioxidant activity of C. edulis rhizome and its pure compounds, supporting the traditional use of this plant for the treatment of heart diseases. The C. edulis rhizome is a potential source of bioactive compounds or functional food for treatment and/or prevention of heart- and oxidative stress-related diseases and its bioactive compounds are good candidates for drug development of anti-thrombosis and antioxidant agents.

Purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract that exhibits anticoagulant and antiplatelet activity.

The current study deals with the purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract. Sephadex G-100 and DEAE-A25 column chromatography techniques were employed to isolate NEGp. NEGp showed single sharp band at 29 kDa region on 10% SDS-PAGE, and under reduced and non-reduced conditions revealed its monomeric nature. Besides, NEGp taken up the PAS stain at 29 kDa region reveals the presence of carbohydrate moiety. Purity of NEGp was adjudged by RP-HPLC, as it revealed a single sharp peak at the retention time of 3.4 min. The exact molecular mass of NEGp was found to be 26 kDa which was confirmed by MALDI-TOF. Circular di-chromism spectra of NEGp showed 12.0% α-helix, 24.3% α-helix turn and 63.7% random coils without beta pleated sheets. NEGp was found to exhibit anticoagulant activity by extending clotting time of both platelet rich plasma and platelet poor plasma from control 240 s to 1800 s and 280 s to 2100 s respectively at the concentration of 8 µg. NEGp inhibited the agonists such as ADP, epinephrine and arachidonic acid induced platelet aggregation in washed platelets. The percentage of inhibition was found to be 70%, 80% and 60% respectively. While, it did not interfere in thrombin, PAF and collagen induced platelet aggregation. NEGp did not hydrolyse RBC membrane, devoid of haemorrhagic and edema inducing properties in experimental mice.

Cytotoxic property of Streptococcus mitis strain producing two different types of cholesterol-dependent cytolysins.

Streptococcus mitis strain Nm-65 secretes an atypical 5-domain-type cholesterol-dependent cytolysin (CDC) called S. mitis-derived human platelet aggregation factor (Sm-hPAF) originally described as a platelet aggregation factor. Sm-hPAF belongs to Group III CDC that recognize both membrane cholesterol and human CD59 as the receptors, and shows preferential activity towards human cells. Draft genome analyses have shown that the Nm-65 strain also harbors a gene encoding another CDC called mitilysin (MLY). This CDC belongs to Group I CDC that recognizes only membrane cholesterol as a receptor, and it is a homolog of the pneumococcal CDC, pneumolysin. The genes encoding each CDC are located about 20 kb apart on the Nm-65 genome. Analysis of the genomic locus of these CDC-encoding genes in silico showed that the gene encoding Sm-hPAF and the region including the gene encoding MLY were both inserted into a specific locus of the S. mitis genome. The results obtained using deletion mutants of the gene(s) encoding CDC in Nm-65 indicated that each CDC contributes to both hemolysis and cytotoxicity, and that MLY is the major hemolysin/cytolysin in Nm-65. The present study aimed to determine the potential pathogenicity of an S. mitis strain that produces two CDC with different receptor recognition properties and secretion modes.

Antiplatelet and antibacterial activities of Essential Oils obtained from rhizomes and leaves of Hedychium coronarium J. Koening.

Hedychium coronarium J. Koening, belonging to Zingiberaceae family, is a perennial herb with fleshly aromatic rhizomes. There are no information about the antiplatelet properties of essential oils (EOs) from rhizomes (HCR) and leaves (HCL) of this herb, additionally, there are reports about the antibacterial activity of the Zingiberaceae species, however, no studies have been carried out in the Colombian Amazon Region. The EOs were characterized by GC-MS, the antiaggregant activity was assessed by ADP and Collagen as platelet agonist and the antibacterial activity against E. faecalis and S. aureus were evidenced by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A high content of oxygenated monoterpenes were found in HCL essential oil (EO) and 20 compounds were identified in HCR EO. The HCL EO showed antiaggregant activity when collagen was used and HCR EO showed a concentration-dependent activity against ADP and collagen, meanwhile only the HCR EO showed antibacterial activity against E. faecalis and S. aureus.

Exploration in the mechanism of rhubarb for the treatment of hyperviscosity syndrome based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperviscosity syndrome (HVS) is a major risk factor for thrombotic diseases. Rhubarb, well-known as a traditional Chinese medicine, exhibits multiple pharmacological activities, especially for promoting blood circulation to remove blood stasis (PBRB), which has been become a functional health food for decreasing the risk of cardiovascular diseases. However, due to the complexity of rhubarb components, it is still difficult to clarify the specific targets of effective substances in PBRB, and the pharmacodynamic mechanism needs to be further probed. MATERIALS AND METHODS: The "compound-target-cell-disease" network analysis was initially used to predict potential targets and bioactive compounds. The effect of rhubarb for the treatment of HVS was examined by histopathology and biochemical assays based on the HVS rat model. RESULTS: Through the "compound-target-cell-disease" network analysis, eight potential therapeutic targets were eventually screened out, and platelets were predicted as the main effector cells of rhubarb in PBRB. Among targets coagulation factor II (prothrombin, F2) and fibrinogen gamma chain (FGG) were closely related to platelets, and five compounds associated with F2 and FGG were predicted including emodin-8-O-beta-D-glucopyranoside (Emo), physcion-8-O-beta-D-glucopyranoside (Phy), procyanidin B-5,3'-O-gallate, torachrysone-8-O-beta-D-(6'-oxayl)-glucoside and epicatechin. Furthermore, thoracic aorta histopathology and biochemical examinations showed middle dose of rhubarb (0.42 g/kg/day) significantly ameliorated pathological changes, hemorheology parameters, as well as levels of representative biomarkers such as plasma P-selectin (P-sel) and thromboxane (TXB2) in platelet activation compared to HVS rat model, whose effects were comparable to the positive drug aspirin or even better. Finally, it was further validated F2 and FGG as the major effective targets of rhubarb as well as its two active ingredients Emo and Phy in PBRB. CONCLUSIONS: This study may provide an innovative way and scientific information to further understand the main effective components of rhubarb and its mechanisms about targets of F2 and FGG in PBRB, especially the new therapeutic target FGG, which also provide a basis for establishing a quality control for rhubarb by bioassays that could correlate the clinical efficacy and its mechanism.

Effect of Ancylostoma ceylanicum hookworm platelet inhibitor on platelet adhesion and peripheral blood mononuclear cell proliferation.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Passiflora edulis Leaf Extract: Evidence of Antidiabetic and Antiplatelet Effects in Rats.

Different Passiflora species have been appointed as a promising herbal medicine due to antioxidant properties; however, their effect on oxidative process induced by diabetes is still controversial. We aimed to evaluate effects of hydroethanolic extract 70% from P. edulis leaf on biochemical blood markers, collagen glycation, production of oxidant species and platelet aggregation in diabetic rats. The phytochemical analysis of the extract was performed by dereplication using LC coupled to the Photodiode Array Detector and Mass Spectrometer detector. Male Wistar rats were assigned to the control group and groups treated with alloxan (150 mg/kg) intraperitoneally, extract (200 mg/kg/d, for 90 d) and combination of alloxan and extract. The phytochemical analysis suggested the presence of flavonoids C-glycosides in the extract. The diabetic animals treated with the extract presented improvement in glycaemic control, reduced glycation collagen, levels of non-high density lipoprotein (non-HDL) cholesterol, total cholesterol and creatinine, production of oxidant species and aggregation in platelet in relation to diabetic animals non-treated. Our results showed that P. edulis leaf extract presents a health benefit to the diabetic state, preventing the appearance of its complications. Its effect can be associated with flavonoids, among which is the flavonoid C-glycoside isoorientin.

Anti-Platelet Properties of Phenolic Extracts from the Leaves and Twigs of Elaeagnus rhamnoides (L.) A. Nelson.

Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a small tree or bush. It belongs to the Elaeagnaceae family, and has been used for many years in traditional medicine in both Europe and Asia. However, there is no data on the effect of sea buckthorn leaves and twigs on the properties of blood platelets. The aim of the study was to analyze the biological activity of phenolic extracts from leaves and twigs of sea buckthorn in blood platelets in vitro. Two sets of extracts were used: (1) phenolic compounds from twigs and (2) phenolic compounds from leaves. Their biological effects on human blood platelets were studied by blood platelet adhesion, platelet aggregation, arachidonic acid metabolism and the generation of superoxide anion. Cytotoxicity was also evaluated against platelets. The action of extracts from sea buckthorn twigs and leaves was compared to activities of the phenolic extract (a commercial product from the berries of Aronia melanocarpa (Aronox®) with antioxidative and antiplatelet properties. This study is the first to demonstrate that extracts from sea buckthorn leaves and twigs are a source of bioactive compounds which may be used for the prophylaxis and treatment of cardiovascular pathologies associated with blood platelet hyperactivity. Both leaf and twig extracts were found to display anti-platelet activity in vitro. Moreover, the twig extract (rich in proanthocyanidins) displayed better anti-platelet potential than the leaf extract or aronia extract.

A novel protease-activated receptor 1 inhibitor from the leech Whitmania pigra.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.

A novel anti-platelet aggregation target of chinensinaphthol methyl ether and neojusticin B obtained from Rostellularia procumbens (L.) Nees.

This study explored the possible bioactive ingredients and target protein of Rostellularia procumbens (L.) Nees. The results of optical turbidimetry revealed that the ethyl acetate extraction obtained from R. procumbens (L.) Nees could inhibit platelet aggregation. Gene chip was used to investigate differentially expressed genes. According to the results of the gene chip, the targets of compounds isolated from the ethyl acetate extraction were predicted by network pharmacology. Computational studies revealed that chinensinaphthol methyl ether and neojusticin B may target the integrin αIIbß3 protein. The results of Prometheus NT.48 and microscale thermophoresis suggested that the molecular interactions between the two compounds with purified integrin αIIbß3 protein in the optimal test conditions were coherent with the docking results. To our best knowledge, this is the first report to state that chinensinaphthol methyl ether and neojusticin B target the integrin αIIbß3 protein.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

In vitro anti-platelet aggregation effects of fourteen fruits and vegetables.

In the present study, the anti-platelet aggregation activity of 14 vegetables and fruits was tested in vitro. The aqueous, 90% ethanol and ethyl acetate extracts, as well as concentrated juices of 14 foods (fruits and vegetables) were prepared, and the anti-platelet aggregation activity of those extracts was analyzed on a platelet aggregation analyzer in vitro with adenosine 5'-diphosphate (ADP), bovine thrombin (THR) and arachidonic acid (AA) as aggregation inducers, respectively. Aspirin (ASP) was used as the positive control. A number of the tested foods had inhibitory effects in concentration-dependent manner on platelet aggregations induced by various agonists. Especially, some foods such as lemon, leek, garlic, scallion, ginger, tomato and grapefruit showed good anti-platelet aggregation effect similar or higher than that of positive control group i.e. aspirin (ASP). The results of present study provide scientific reference for reasonable selection of daily dietary with supplementary curative effects or prevention of cardiovascular diseases (CVD).