Use of Amphipathic Helical Peptides as An Anticoagulant

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4100-4100
Author(s):  
Sophie Charbonneau ◽  
Henry Peng ◽  
Pang N Shek ◽  
Mark Blostein
Keyword(s):  
Coagulation Factors ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Factor X ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Active Partial Thromboplastin Time ◽  
Direct Binding ◽  
High Concentrations

Abstract We have previously shown that an ideal amphipathic helical peptide of K7L15 composition (IAP) accelerates factor IXa-mediated factor X turnover and factor Xamediated prothrombin turnover in a phospholipid free system (Biochem J., 2008, 412:545). Under these conditions, IAP behaves as a phospholipid membrane allowing coagulation factors to bind and exert their actions. However, when IAP is used with in vitro assays that employ phospholipids such as an active partial thromboplastin time (aPTT), IAP paradoxically behaves as an anticoagulant by prolonging clotting times. We hypothesize that this anticoagulant effect occurs by blocking binding sites for coagulation factors on phospholipids membranes. To test this hypothesis, we employed three phopholipid-dependant coagulation assays, the aPTT, dilute PT and dilute RVV, with both low and high concentrations of phospholipids. We show that these coagulation times are prolonged by IAP in a concentration dependent manner and that this prolongation is abrogated by adding excess phospholipid, demonstrating phospholpid dependence for this inhibition. In purified tenase and prothrombinase assays, in the presence of phospholipids, IAP inhibits substrate turnover consistent with our hypothesis. To show direct binding between IAP and phospholipids, we conducted fluorescence spectroscopy experiments and show direct binding between IAP and phospholipid membranes. In summary, the above data demonstrate that IAP acts as an anticoagulant by blocking the interaction of coagulation factors with phospholipids membranes.

Characterization of an ideal amphipathic peptide as a procoagulant agent

2008 ◽  
Vol 412 (3) ◽  
pp. 545-551 ◽  
Author(s):  
Jorge G. Ganopolsky ◽  
Sophie Charbonneau ◽  
Henry T. Peng ◽  
Pang N. Shek ◽  
Mark D. Blostein
Keyword(s):  
Factor Xa ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Factor X ◽  
Concentration Dependent Manner ◽  
Amphipathic Peptide ◽  
Rich Domain ◽  
Factor Ixa ◽  
Direct Binding

On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000–12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys7Leu15 composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (γ-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.

scholarly journals Impact of Phytomediated Zinc Oxide Nanoparticles on Growth and Oxidative Stress Response of In Vitro Raised Shoots of Ochradenus arabicus

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Fahad Al-Qurainy ◽  
Salim Khan ◽  
Saleh Alansi ◽  
Mohammad Nadeem ◽  
Aref Alshameri ◽  
...  
Keyword(s):  
Zinc Oxide ◽  
Zinc Oxide Nanoparticles ◽  
Shoot Growth ◽  
System Response ◽  
Oxide Nanoparticles ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Shoot Number ◽  
High Concentrations

Biogenic nanoparticles have potential roles in the growth and development of plants and animals as they are ecofriendly and free of chemical contaminants. In this study, we assessed the effects of phytomediated zinc oxide nanoparticles (ZnONPs) on shoot growth, biochemical markers, and antioxidant system response in Ochradenus arabicus, which is a medicinal plant. The shoot length and fresh and dry weights were found to be higher in groups with 5 and 10 mg/L ZnONPs than in the control. At high concentrations of ZnONPs (50, 100, and 300 mg/L), biomass was decreased in a concentration-dependent manner. The shoot number was observed to be highest at 50 mg/L among all applied concentrations of ZnONPs. The levels of the stress markers proline and TBARS were found to be higher in shoots treated with 100 and 300 mg/L ZnONPs than in the control as well as NP-treated shoots. The levels of antioxidant enzymes were significantly increased at high concentrations of nanoparticles compared with the control. Thus, synthesized phytomediated ZnONPs from shoots of O. arabicus and their application to the same organ of O. arabicus in vitro were found to be effective as a low concentration of nanoparticles promoted shoot growth, resulting in high biomass accumulation. Thus, using green nanotechnology, such endemic plants could be conserved in vitro and multiple shoots could be produced by reducing the phytohormone concentration for multiple uses, such as the production of potential secondary metabolites.

scholarly journals In vitro Activity of Ozoroa pulcherrima Schweinf. Extracts and Fractions on Schistosoma mansoni Cercariae and Adult Worms

2020 ◽  
pp. 17-30
Author(s):  
Nestor Gipwe Feussom ◽  
Hermine Boukeng Jatsa ◽  
Mérimé Christian Kenfack ◽  
Emilienne Tienga Nkondo ◽  
Ulrich Membe Femoe ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Methanolic Extract ◽  
Lower Abdominal Pain ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
In Vitro Activity ◽  
Concentration Dependent Manner ◽  
Methanolic Extracts ◽  
Effective Drugs

Aims: Continuous attempts are being made to develop new and more effective drugs for the treatment of schistosomiasis. Ozoroa pulcherrima Schweinf. is a medicinal plant used in Africa for the treatment of dysmenorrhea, lower abdominal pain, dystocia and intestinal helminthiasis. This study provides findings on the cercaricidal and schistosomicidal activity of extracts and fractions of Ozoroa pulcherrima in in vitro assays. Methodology: The aqueous and methanolic extracts from Ozoroa pulcherrima root parts (62.5 – 2000 µg/mL), as well as the methanol derived fractions (n-hexane and ethyl acetate: 31.25 – 1000 µg/mL) were tested on cercariae and adult worms of Schistosoma mansoni. Niclosamide-olamine 5% (1 µg/mL) and praziquantel (10 µg/mL) were respectively used as reference drugs. During the assays, the mortality of cercariae after 2 hours, and adult worms’ mobility and mortality after 48 hours of incubation were evaluated. Results: Ozoroa pulcherrima extracts and fractions significantly increased cercariae and worm mortality in a concentration-dependent manner. The methanolic extract was the most active on cercariae with a LC50of 20.65 µg/mL after 30 minutes, while the n-hexane fraction was the most active on worm with a LC50 of 79.54 μg/mL (65.58 – 96.47 μg/mL) after 48 hours. Significant reduction of motor activity (18.47 to 100%) was recorded for surviving worms incubated in different concentrations of the extracts and fractions. Conclusion: This study proves that Ozoroa pulcherrima extracts and fractions have cercaricidal and schistosomicidal activities. Ozoroa pulcherrima may have great potential as an anti-schistosomal agent for further research.

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1925-1933 ◽  
Author(s):  
Baohe Shen ◽  
James L. Manley
Keyword(s):  
Dependent Manner ◽  
Loss Of Function ◽  
Concentration Dependent Manner ◽  
Downstream Target ◽  
Toll Signaling ◽  
Early Embryos ◽  
High Concentrations ◽  
L2 Cells

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll10b), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll10b, and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

1996 ◽  
Vol 76 (05) ◽  
pp. 786-790 ◽  
Author(s):  
Atsuhiro Sugidachi ◽  
Norbert Breiter ◽  
Taketoshi Ogawa ◽  
Fumitoshi Asai ◽  
Hiroyuki Koike
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Anticoagulant Activity ◽  
Acid Amide ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

scholarly journals In vitro antioxidant activity of stem bark of Trichilia catigua Adr. Juss

2012 ◽  
Vol 62 (3) ◽  
pp. 371-382 ◽  
Author(s):  
Jean Paul Kamdem ◽  
Sílvio Terra Stefanello ◽  
Aline Augusti Boligon ◽  
Caroline Wagner ◽  
Ige Joseph Kade ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Antioxidant Activities ◽  
Ethanolic Extract ◽  
Stem Bark ◽  
Total Phenolic ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
High Concentrations ◽  
Dpph Scavenging

Antioxidant activity of the ethanolic extract and fractions from the stem bark of T. catigua was investigated. IC50 (for DPPH scavenging) by T. catigua varied from 9.17 ± 0.63 to 76.42 ± 5.87 mg mL-1 and total phenolic content varied from 345.63 ± 41.08 to 601.27 ± 42.59 mg GAE g-1 of dry extract. Fe2+-induced lipid peroxidation was significantly reduced by the ethanolic extract and fractions. Mitochondrial Ca2+-induced dichlorofluorescein oxidation was significantly reduced by the ethanolic extract in a concentration-dependent manner. Ethanolic extract reduced mitochondrial Dym only at high concentrations (40-100 mg mL-1), which indicates that its toxicity does not overlap with its antioxidant effects. Results suggest involvement of antioxidant activities of T. catigua in its pharmacological properties.

scholarly journals 7-Acetoxycoumarin Inhibits LPS-Induced Inflammatory Cytokine Synthesis by IκBα Degradation and MAPK Activation in Macrophage Cells

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3124 ◽  
Author(s):  
Taejin Park ◽  
Jin-Soo Park ◽  
Ji Han Sim ◽  
Seung-Young Kim
Keyword(s):  
Inflammatory Cytokines ◽  
Mitogen Activated Protein Kinase ◽  
In Vitro Assays ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cells ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

Acetylation involves the chemical introduction of an acetyl group in place of an active hydrogen group into a compound. In this study, we synthesized 7-acetoxycoumarin (7AC) from acetylation of umbelliferone (UMB). We examined the anti-inflammatory properties of 7AC in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. The anti-inflammatory activity of 7AC on viability of treated cells was assessed by measuring the level of expression of NO, PGE2 and pro-inflammatory cytokines, namely interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in 7AC-treated RAW 264.7 macrophages. The 7AC was nontoxic to cells and inhibited the production of cytokines in a concentration-dependent manner. In addition, its treatment suppressed the production of pro-inflammatory cytokines in a dose-dependent manner and concomitantly decreased the protein and mRNA expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the levels of the phosphorylation of mitogen-activated protein kinase (MAPK) family proteins such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 and nuclear factor kappa B (NF-κB) were reduced by 7AC. In conclusion, we generated an anti-inflammatory compound through acetylation and demonstrated its efficacy in cell-based in vitro assays.

scholarly journals Effects of Methylprednisolone on Intracellular Bacterial Growth

2001 ◽  
Vol 8 (6) ◽  
pp. 1156-1163 ◽  
Author(s):  
G. Umberto Meduri ◽  
Siva Kanangat ◽  
Michael Bronze ◽  
David R. Patterson ◽  
Christopher U. Meduri ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Clinical Studies ◽  
Bacterial Infections ◽  
Dependent Manner ◽  
U937 Cells ◽  
Phagocytic Cells ◽  
Concentration Dependent Manner ◽  
Tnf Α ◽  
High Concentrations

ABSTRACT Clinical studies have shown positive associations among sustained and intense inflammatory responses and the incidence of bacterial infections. Patients presenting with acute respiratory distress syndrome (ARDS) and high levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6, have increased risk for developing nosocomial infections attributable to organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, andAcinetobacter spp., compared to those patients with lower levels. Our previous in vitro studies have demonstrated that these bacterial strains exhibit enhanced growth extracellularly when supplemented with high concentrations of pure recombinant TNF-α, IL-1β, or IL-6. In addition, we have shown that the intracellular milieu of phagocytic cells that are exposed to supraoptimal concentrations of TNF-α, IL-1β, and IL-6 or lipopolysaccharide (LPS) favors survival and replication of ingested bacteria. Therefore, we hypothesized that under conditions of intense inflammation the host's micromilieu favors bacterial infections by exposing phagocytic cells to protracted high levels of inflammatory cytokines. Our clinical studies have shown that methylprednisolone is capable of reducing the levels of TNF-α, IL-1β, and IL-6 in ARDS patients. Hence, we designed a series of in vitro experiments to test whether human monocytic cells (U937 cells) that are activated with high concentrations of LPS, which upregulate the release of proinflammatory cytokines from these phagocytic cells, would effectively kill or restrict bacterial survival and replication after exposure to methylprednisolone. Fresh isolates of S. aureus, P. aeruginosa, and Acinetobacter were used in our studies. Our results indicate that, compared with the control, stimulation of U937 cells with 100-ng/ml, 1.0-μg/ml, 5.0-μg/ml, or 10.0-μg/ml concentrations of LPS enhanced the intracellular survival and replication of all three species of bacteria significantly (for all, P = 0.0001). Stimulation with ≤10.0 ng of LPS generally resulted in efficient killing of the ingested bacteria. Interestingly, when exposed to graded concentrations of methylprednisolone, U937 cells that had been stimulated with 10.0 μg of LPS were able to suppress bacterial replication efficiently in a concentration-dependent manner. Significant reduction in numbers of CFU was observed at ≥150 μg of methylprednisolone per ml (Pvalues were 0.032, 0.008, and 0.009 for S. aureus, P. aeruginosa, and Acinetobacter, respectively). We have also shown that steady-state mRNA levels of TNF-α, IL-1β, and IL-6 in LPS-activated cells were reduced by treatment of such cells with methylprednisolone, in a concentration-dependent manner. The effective dose of methylprednisolone was 175 mg, a value that appeared to be independent of priming level of LPS and type of mRNA. We therefore postulate that a U-shaped relationship exists between the level of expression of TNF-α, IL-1β, and IL-6 within the phagocytic cells and their abilities to suppress active survival and replication of phagocytized bacteria.

EP receptor subtypes implicated in the PGE2-induced sensitization of polymodal receptors in response to bradykinin and heat

1996 ◽  
Vol 75 (6) ◽  
pp. 2361-2368 ◽  
Author(s):  
T. Kumazawa ◽  
K. Mizumura ◽  
H. Koda ◽  
H. Fukusako
Keyword(s):  
Receptor Subtypes ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ep Receptors ◽  
Ep1 Receptor ◽  
Ep Receptor ◽  
High Concentrations ◽  
Ep2 Receptor ◽  
Ep3 Receptor

1. Our previous studies, in which we used in vitro canine testispermatic nerve preparations, showed that prostaglandin E2 (PGE2) augments both bradykinin (BK)- and heat-induced discharges of polymodal receptors. However, the PGE2 concentration required to augment the BK responses were 100 times lower than those necessary for the heat responses, suggesting that different receptors are involved in these phenomena. We studied which receptors for E series of prostaglandins (EP receptors) were responsible, using the antagonist and agonists for three subtypes of EP receptors. 2. PGE2-induced augmentation of the BK responses was unaffected when treated with an antagonist for the EP1 receptor, AH6809. 3. An agonist for the EP3 receptor, M&B28767, at > or = 10 nM, significantly augmented the BK responses in a concentration-dependent manner that mimics the PGE2-induced effect. An agonist for the EP1 receptor, 17-phenyl trinor PGE2 (17-phen PGE2), at the high concentrations of 0.1 and 1 microM, augmented the BK responses in two and four of nine cases tested, respectively. However, this augmentation was not suppressed by the antagonist for the EP1 receptor, AH6809. In addition, an agonist for the EP2 receptor, butaprost, did not affect the BK responses even when applied at 10 microM. 4. In contrast, butaprost at > or = 10 nM significantly augmented the heat responses in a concentration-dependent manner. M&B28767 and 17-phen PGE2, respectively, augmented the heat responses at higher concentrations of 100 nM and 1 microM. 5. These results indicate that the EP3 and EP2 receptor subtypesx are differentially implicated in the respective PGE2-induced augmentation of BK responses and heat responses of polymodal receptors.

scholarly journals Antibacterial Activity of Licochalcone A against Spore-Forming Bacteria

2002 ◽  
Vol 46 (5) ◽  
pp. 1226-1230 ◽  
Author(s):  
Ryo-Ichi Tsukiyama ◽  
Harumi Katsura ◽  
Nozomu Tokuriki ◽  
Makio Kobayashi
Keyword(s):  
Antibacterial Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Licochalcone A ◽  
Heat Treated ◽  
Pharmaceutical Industries ◽  
High Concentrations ◽  
Glycyrrhiza Inflata ◽  
High Level

ABSTRACT Licochalcone A was isolated from the roots of licorice, Glycyrrhiza inflata, which has various uses in the food and pharmaceutical industries; isolation was followed by extraction with ethanol and column chromatography with silica gel. In this study, the activities of licochalcone A against some food contaminant microorganisms were evaluated in vitro. The vegetative cell growth of Bacillus subtilis was inhibited in a licochalcone A concentration-dependent manner and was completely prevented by 3 μg of licochalcone A/ml. Licochalcone A showed a high level of resistance to heating at 80 to 121°C for 15 min. Licochalcone A did not inhibit the germination of heat-treated spores of B. subtilis induced by l-alanine. Licochalcone A showed effects against all gram-positive bacteria tested and especially was effective against all Bacillus spp. tested, with MICs of 2 to 3 μg/ml, but was not effective against gram-negative bacteria or eukaryotes at 50 μg/ml. Although the cationic antimicrobial peptides protamine and ε-poly-l-lysine resulted in the loss of antimicrobial activity in the presence of either 3% (wt/vol) NaCl or protease at 20 μg/ml, the antibacterial activity of licochalcone A was resistant to these conditions. Thus, licochalcone A could be a useful compound for the development of antibacterial agents for the preservation of foods containing high concentrations of salts and proteases, in which cationic peptides might be less effective.

Sign in / Sign up

Export Citation Format

Share Document