New class of heparin derivatives with a potent antithrombotic effect and a very limited hemorrhagic activity.

Eight heparin derivatives (HD1 to HD8) were prepared by mixing various doses of protamine with a fixed amount of heparin. After centrifugation and elimination of the formed complex, the supernatant was lyophilized and titrated. A dose of 5 mg (dry weight)/kg of these heparin derivatives was injected subcutaneously to rats in classical thrombosis model induced by stasis. Antithrombotic, hemorrhagic and anticoagulant activities are investigated and compared to those of Unfractionated Heparin (UFH) and LMWH (Enoxaparin). Seven rats in each group were studied. Significant antithrombic effect was exhibited by HD1, HD2, HD3, and HD4 which decreased progressively. Only HD1, HD2, and HD3 augmented hemorrhagic activity but to a lower degree than UFH and LMWH. No change was observed by coagulation assays; Activated Partial Thromboplastin Time (APTT) and Diluted Thrombin Time (dTT) and there was no effect on platelet aggregation except for UFH. These heparin derivatives might present advantages over UFH and LMWH in the treatment of thrombosis.

[Antithrombotic effect of piavit--a substance from the medicinal leech (Hirudo medicinalis), containing prostanoid and destabilase fractions]. / Protivotromboticheskii éffekt piiavita--preparata iz meditsinskoi piiavki (Hirudo medicinalis), soderzhashcheisia v nem fraktsii prostanoidov i fermenta destabilazy.

The protective antithrombotic effects of piyavit, a novel pharmacological preparation from the medicinal leech, its water extract concentrated and diluted 1600-fold, the piyavit fraction containing leech prostanoids and highly purified destabilase, epsilon-(gamma-Glu)-Lys-isopeptidase isolated from the medicinal leech (Hirudo medicinalis), have been studied. All the preparations under study inhibited clot formation in rat mesanterium arterioles stimulated by laser energy, contained leech prostanoids immunoenzymatically identified as 6-keto-PgF1 alpha and inhibited platelet aggregation induced by ADP. The putative mechanism of their antithrombotic effect is discussed.

[Experimental models of venous thrombosis]. / Les modèles expérimentaux de thrombose veineuse.

Various experimental models have been developed in order to more clearly understand deep vein thrombosis, the mechanisms involved and its treatment. These models are based on venous stasis, either alone or combined with the injection of thrombogenic substances or endothelial lesions. Other models only use endothelial lesions. Thrombogenic substances are mostly composed of activated factor X or thrombin, which raises the problem of purity of the substances and determination of the antithrombotic activities of the substance tested, especially heparin and hirudin and their derivatives, and consequently their efficacy. Endothelial lesions can be induced by chemical, physical or electrical agents or by repeated application of clamps, or cellular crushing. These models result in the formation of various forms of venous thrombus. The development and improvement of experimental models is very important in every case. Experimental models of thrombosis constitute the best tool for the study of thrombosis, in which many points remain to be elucidated. They also allow the study and development of various antithrombotic substances the improvement of their efficacy. These models must be validated, standardized, reproductible and in agreement with local legislation in each country.

Arterial antithrombotic effect of piyavit, the novel pharmacological preparation from the medicinal leech, and of its components, prostanoids and enzyme destabilase.

Piyavit, the novel pharmacological preparation from the medicinal leech (Hirudo medicinalis) contained the leech saliva, produces the potent arterial antithrombotic effect examined on experimental of Laser Induced Thrombus formation. Administrated orally into rats or injected subcutaneously as water extract, non-diluted or in 1600 times diluted, it inhibits statistically significant comparing with control platelet thrombus stimulated by laser beams. Its components, prostanoid fraction and purified enzyme destabilase, endo-epsilon-(gamma-Glu)-Lys-isopeptidase, also inhibit thrombus formation in statistically different manner, comparing with control. All the tested preparations inhibit platelet aggregation induced by ADP. The dependence of arterial antithrombotic effect on the leech prostanoid is discussed.

Thrombogenicity of ionic and nonionic contrast media tested in a laser induced rat thrombosis model.

Contrast media are used as substances for visualization of vascular system. But, their administration is often associated with thromboembolic complications. The purpose of this study is to evaluate the thrombogenic action of ionic and non-ionic contrast media on thrombus formation. The experimental destruction of endothelial cells by Laser injury leads to thrombus and emboli formation. Two ionic and two non-ionic contrast media were injected intravenously via penis vein and tested at various dosages (1.0 and 2.5 ml/kg) 5, 30, 45 and 65 minutes after injection. The administration of these contrast media decreases the number of Laser injuries required to induce thrombus formation, increases the number of emboli which detached from thrombus and prolongs duration of embolization (p < or = 0.05). These experimental results suggest that ionic and non-ionic contrast media induce thrombogenic effects. This thrombogenicity was the greatest for non-ionic contrast media. It was observed the decrease of the white cells, red cells and platelets.

Prevention of laser arterial thrombus formation with unfractionated heparin.

A method to induce microthrombi in small mesenteric arteries (30-40 microns) has been developed to study platelet reactions and to investigate antithrombotic drugs. This model was used to evaluate the effect of Ca-heparin. Mesenteric lesions are induced in the vascular system of Wistar rats with an argon laser. The laser induced the formation of vessel wall lesion with damage of endothelial cells. Thrombi formed within seconds after the laser lesion and grew rapidly. Embolization began within a minute following the laser flash. Thrombus formation and embolization were repetitive phenomena. The administration of Ca-heparin at different dosages (0.5, 1.0 and 2.0 mg/kg) increases the number of laser injuries required to induce thrombus formation, and dose-dependently decreases the number of emboli and the duration of embolization. The highest dose injected (2.0 mg/kg) induced the strongest reduction in the number of emboli and duration of embolization.

Effects of aprotinin on heparin activities and heparin neutralization with protamine.

In order to investigate the new situation in which aprotinin is proposed as a novel approach to reducing post operative bleeding, specially in cardiopulmonary bypass (CPB) surgery during which heparin and protamine are commonly used, preliminary in vitro and in vivo studies have been performed. Aprotinin increases the anticoagulant heparin effects in vitro, and the hemorrhage time in vivo. But in addition to protamine, there are no statistically significant differences with heparin-protamine situation, indicating aprotinin does not disturb the neutralizing activities of protamine on heparin.