Detection of Heparin in the Salivary Gland and Midgut of Aedes togoi

Mosquitoes secrete saliva that contains biological substances, including anticoagulants that counteract a host's hemostatic response and prevent blood clotting during blood feeding. This study aimed to detect heparin, an anticoagulant in Aedes togoi using an immunohistochemical detection method, in the salivary canal, salivary gland, and midgut of male and female mosquitoes. Comparisons showed that female mosquitoes contained higher concentrations of heparin than male mosquitoes. On average, the level of heparin was higher in blood-fed female mosquitoes than in non-blood-fed female mosquitoes. Heparin concentrations were higher in the midgut than in the salivary gland. This indicates presence of heparin in tissues of A. togoi.

Anticoagulant activity in salivary gland homogenates of Thyrsopelma guianense (Diptera: Simuliidae), the primary vector of onchocerciasis in the Brazilian Amazon

In this study, anticoagulant activity was detected in salivary gland homogenates (SGHs) of Thyrsopelma guianense (Diptera: Simuliidae). The SGH yielded 1.07 ìg ± 0.03 (n = 15) of total soluble protein per pair of glands. In addition, following SDS-PAGE (12.5 percent gel) and silver nitrate staining, 12 polypeptides with molecular weights ranging from 14-69 kDa were detected in all physiological ages analyzed (12 h, 24 h, 48 h and 72 h following emergence). Coagulation bioassays showed that the SGHs had activities that interacted at all levels of coagulation (the intrinsic, extrinsic and common pathways), by extending the plasma recalcification time, prothrombin time, thrombin time. This is the first report on the activity of salivary gland proteins from the main vector of onchocerciasis in Brazil. We also suggest detailed studies on the morphology and function of the salivary glands in order to understand the role of these proteins in host/vector interactions.

Identification and partial purification of an anticoagulant factor from the venom of the Iranian snake Agkistrodon halys

An anticoagulant factor was purified from the venom of the Iranian snake Agkistrodon halys by gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sepharose. In the final stage of purification, the percentage recovery of purified anticoagulant factor was found to be 83 percent. The purified anticoagulant factor revealed a single protein band in SDS-polyacrylamide electrophoresis under reducing conditions and its molecular weight was about 22 kDa. The purified peptide did not show any effect on casein, BApNA or plasma.(AU)

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10 percent of mammalian heparin and about 5 percent as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5 percent (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4 percent, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13 percent without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Targeting exosites on blood coagulation proteases

A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os exosítios das enzimas da coagulação. Dois exemplos são discutidos nesta curta revisão. A Botrojaracina é uma proteína derivada de veneno de serpente que se liga aos exosítios 1 e 2 da trombina. A formação do complexo impede várias atividades da trombina dependentes do exosítio 1 incluindo a clivagem do fibrinogênio e a ativação plaquetária. A Botrojaracina também interage com o proexosítio 1 da protrombina diminuindo a ativação do zimogênio pelo complexo protrombinase (FXa/FVa). O ixolaris é um inibidor com dois domínios Kunitz obtido da glândula salivar de carrapato, homólogo ao inibidor da via do fator tecidual. Recentemente foi demonstrado que o ixolaris se liga ao exosítio de ligação à heparina do FXa, impedindo o reconhecimento da protrombina pela enzima. Além disso, o ixolaris interage com o FX, possivelmente através do proexosítio de ligação à heparina. Diferentemente do FX, o complexo ixolaris-FX não é reconhecido como substrato pelo complexo tenase intrínseco (FIXa/FVIIIa). Nós concluimos que esses inibidores podem servir como ferramentas para o estudo dos exosítios da coagulação, assim como protótipos para novas drogas anticoagulantes.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50 percent acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Seperation and determination of anticoagulant cumarins which have different therapeutical effects by HPLC

The separation and determination of some anticoagulants drugs like cumarins derivatives were done directly by high performance liquid chromatography reverse phase.We got two separate mobile phases in different percentages, the first one is water 5%: methanol 95%, and the second is water 20%: acetonitril 80% with PH = 4 for both mobile phases. The steady phase was a column of octadecyle 5 micrometre [250mm X 46mm] Nucleosil, then we have got the best results of separation and retention time and determination of the concentrations using different wave lengths of U. V as a detector, that there was a special maximum wave length for each cumarin derivative, where coefficient variation was between 0.04%-3.9% and the limit of detection was between 0.3-1.9 microgram/ml for studied cumarins depending on the mobile phase and cumarin derivative. The assay method was used to qualify the purity of studied cumarin compounds especially used in human uses and quantify and separate it of other compounds in addition to toxicity cases. This method is accurate and sensitive based on quantitative studies

Lectinas, agentes antihemostáticos y compuestos positivos al reactivo de Dragendorff en algas marinas de Venezuela y Túnez / Lectins, antihaemostatic agents and dragendorff positive compounds in marine algae from Venezuela and Tunibia

Las algas marinas recolectadas en Túnez y Venezuela han sido analizadas buscando la presencia de lectinas, agentes antihemostáticos y compuestos Dragendorff positivo. De 34 especies estudiadas, la actividad de las lectinas fue demostrada en los extractos de 14 de ellas. De éstas, las especies consideradas de más valor para estudios posteriores fueron Halimeda tuna, H.opuntia, Bryotamnion triquetum, B. seaforthii, Codium isthmocladum y Centroceras clavulatum. La actividad anticoagulante fue detectada en extractos de 26 de las especies analizadas. El extracto con más actividad de potencia inhibidora fue el de Centroceras clavulatum, pero el de Codium isthmocladum también se consideró interesante. Once especies fueron estudiadas por su contenido de compuestos Drangendorff positivo. Tanto las betainas como el compuesto de sulfonio terciario del tipo 3-dimetilsulfoniopropinato (DMSP) o una mezcla de DMSP y una betaina fueron aisladas y caracterizadas en todas las especies analizadas