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.

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea)

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 ñ SEM = 3.44 ñ 0.84 mg/ml). The <500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68 per cent with 500 mug/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66 per cent of the control value after 24-h incubation with 100 mug/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 ñ SEM = 1.12 ñ 0.02 mglml for a 0.5 per cent erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 mug/ml) increased the EC50 by twofold to 2.86 ñ 0.04 mg/ml, but phosphatidylcholine (80 mug/ml) did not modify hemolysis.