INTRODUCTION:

Thrombosis is an intravital blockage of a blood vessel (artery or vein) by coagulated blood. Among the many reasons contributing to the occurrence of vascular thrombosis, an increase in the coagulation properties of blood is essential. Thrombosis is the result of increased thrombin formation as an excessive response of the hemostatic system to damage to the vascular wall or any other pathological process in the body^1,²^,³.

An important role in the development of thrombosis is also played by damage to the vascular wall due to various reasons(infectious process, atherosclerosis, etc.), as well as a slowdown in blood flow. The therapeutic value of anticoagulants is that, used prophylactically in persons with increased blood clotting, they prevent the formation of blood clots (by normalizing the ratio between the coagulating and anticoagulating properties of blood). When a thrombus is formed, they limit its further spread, which in some cases prevents the complete closure of the vessel lumen and thereby reduces the risk of a necrotic process in the organ or tissues that receive blood supply from this artery⁴.

Anticoagulants also contribute to the resorption of the formed blood clots. The search for substances for the emergency stop of bleeding of various etiologies is urgently needed for modern medicine, because the hemostasis system is one of the first to react to any interference with the human body and is one of the reasons for the death of a person if it is not timely normalized⁵.

It is known that modified sulfated polysaccharides (MSPs) are a large and complex group of macromolecules that possess a wide range of important biological properties. They have antihypoxic, anti-inflammatory, immunotropic, antitumor, antiviral, antimicrobial, sorption, anticoagulant properties⁶^,7.

At present, interest in polysaccharides of plant origin, as compounds with low toxicity, is largely considered as direct anticoagulants⁸. The search for compounds that would reproduce the therapeutic effect of heparin, but have no side effects and are less toxic, still continues.

The mechanism of action of sulfated polysaccharides differs from that of heparin. Sulfated polysaccharides (SP) directly inhibit thrombin without the involvement of plasma inhibitors of serine proteinases compared to heparin. No direct inhibition of factor Xa was found; SP inhibit the internal coagulation pathway in low doses, and in high doses, the external coagulation pathway. It was revealed that sulfated polysaccharides do not have acute toxicity, but they have a broad therapeutic effect. However, the anticoagulant activity of the natural and synthetic sulfated polysaccharides obtained on the basis of cellulose, dextrans, and pullulans^9,10,11 is lower than that of heparin. Probably, the anticoagulant activity of sulfated polysaccharides is due not only to the presence of sulfate groups and their distribution, but also to the structure of carbohydrate chains of polysaccharides, the presence of other functional substituents, their position and distribution. Thus, sulfated polysaccharides exhibit anticoagulant properties associated with the inhibition of fibrinogen clotting and amidolytic activity of thrombin and factor aXa by antithrombin. The specific anticoagulant activity of the synthesized sulfated derivatives depends on the number of sulfate groups.

The aim of this work is to study the anticoagulant and antithrombotic effects of new sulfated polysaccharides in laboratory animals and on a model of thromboplastin-induced thrombosis.

MATERIALS AND METHODS:

Anticoagulant activity in in vitro experiments was investigated by the Lee White method "Blood clotting time"¹². The sulfated polysaccharides GSC-14, GSC-63 and BOS-122 were tested at 1 mg/mL; 5 mg/mL; 12 mg/mL; 25 mg/mL concentrations (Fig. 1). To do this, 1 mL of blood was added dropwise from the rabbit's ear to tubes with 0.1 mL of each sample (the experiments were repeated 2 times). The blood was mixed, placed in a water bath with a temperature of 37⁰C. until the time of complete blood coagulation. The stopwatch was turned on immediately after the first drop. The controls were supplemented with 0,1 mL of 0.9% sodium chloride solution¹³^,14. The obtained research results, which have a numerical expression, were analyzed by the method of variation statistics for small series of observations. To assess the significance of differences, the Student's confidence coefficient (t) and the degree of probability (p) were calculated. Differences were considered significant at p <0.05.

Model experiments were carried out on white mice of both sexes weighing 20±2g, in vivo. As a thrombotic agent, a suspension of thromboplastin in 0.9% sodium chloride solution (25 mg/kg) was used, which was injected into the tail vein of the animal in a volume of 0,02 mL. Sulfated polysaccharides BOS-122, GSC-63, GSC-14 at a dose of 2,5 mg/kg and 5 mg/kg and heparin (SIGMA 5000 IU/mL) firm at a dose of 130 IU / kg (1 mg / kg) were injected intraperitoneally for 10 minutes before the introduction of a thrombotic agent. As a criterion for the formation of blood clots, the number of dead animals and a macroscopic examination of the lungs of dead and surviving mice were recorded one day after the administration of the thrombotic agent and the compounds under study.

The following model experiments were carried out on white rats of both sexes weighing 180-200 g. Thromboplastin was injected intravenously at a dose of 10 mg/kg in the background of preliminary intraperitoneal administration of the compounds GSC-14, GSC-63 and BOS-122 in a dose of 5 mg/kg and heparin from SIGMA 5000 IU/mL at a dose of 130 U/kg for 10 minutes before the introduction of the thrombotic agent thromboplastin. Control rats were injected with 0.9% NaCl solution into the same vein instead of a suspension of thromboplastin.

10 and 30 minutes after the introduction of thromboprlastin, the blood taken from the rat gums was centrifuged on a coagulant (3.8% sodium citrate solution) 9:1 at 3000 r/pm for 10 minutes (Centrifuge OPN-8 (rotor RU180 L, 8,000 rpm) 2007-2008. Russia). The resulting plasma was examined in the activated partial thromboplastin time (APTT) test. The influence of the studied samples of compounds was evaluated using conventional tests using an optical-mechanical coagulometer (CYANCoag, Belgium.CY003, SN:5400439).

RESULTS AND DISCUSSION:

Studies have shown that when 0,1 mL of physiological sodium chloride solution was added to 1 ml of rabbit blood, the blood coagulation time was 4 minutes. Experiments carried out in test tubes with citrated plasma and whole blood showed that sulfated polysaccharides GSC-14 and BOS-122 with a concentration of 25 mg/mL proved to be the most effective, where the time of clot formation was 50 and 60 minutes, respectively (Fig. 1).

Fig. 1: Time of blood coagulation under the action of: 1-Control; 2-GSC-14; 3-GSC-63; 4-BOS-122; 5-Heparin. *- Р<0.05, **- Р<0.01. (n=6).

Experiments have shown that various modified sulfated polysaccharides, mainly with an anticoagulant effect MSP (BOS-122, GSC-63, GSC-14), to varying degrees, affect various links of the hemostasis system and exhibit anticoagulant properties on rabbit blood.

Further, we reviewed the effect of the studied drugs on pulmonary thrombosis caused by the introduction of thromboplastin. As can be seen from the data shown in Table 1, after the introduction of a thrombotic agent, 3 out of 4 mice died in the control group of animals, which amounted to 75% death. When thromboplastin was administered intravenously in the background of heparin (1 mg/kg), no death of the animals was observed.

With the introduction of thromboplastin in the background of the studied compounds (BOS-122, GSC-63 and GSC-14) at a dose of 5 mg/kg, there was also no death of animals (table 1).

With the introduction of thromboplastin in the background of the studied compounds (BOS-122, GSC-63 and GSC-14) at a dose of 2,5 mg/kg, 1 mouse out of 4, or 25% died (Table 2). The death of animals was noted after 15 minutes, as well as in other periods of the first day of observation.

Table 1: Effect of BOS-122, GSC-63 and GSC-14 at a dose of 5 mg/kg on animal death in a model of pulmonary thrombosis caused by the introduction of thromboplastin.

Drugs	Doses, mg/kg	Animal weight	Number of animals in a group	Total number of animal death	% death
Control		20±0,2	4	3	75%
Heparin	1 mg/kg		4	-	0%
BOS-122	5 mg/kg		4	-	0%
GSC-63	5 mg/kg		4	-	0%
GSC-14	5 mg/kg		4	-	0%

Table 2: The effect of BOS-122, GSC-63 and GSC-14 at a dose of 2,5 mg/kg on the death of animals in the model of pulmonary thrombosis caused by the introduction of thromboplastin.

Drugs	Doses, mg/kg	Animal weight	Number of animals in a group	Total number of animal death	% death
Control		20±0,2	4	3	75%
Heparin	1 mg/kg		4	-	0%
BOS-122	2,5 mg/kg		4	1	25%
GSC-63	2,5 mg/kg		4	1	25%
GSC-14	2,5 mg/kg		4	1	25%

Thus, it has been shown that intraperitoneal administration of BOS-122, GSC-63 and GSC-14 increases the survival rate of animals with exogenous thromboplasthenemia, limiting destructive changes in internal organs.

The following model experiments were carried out on white rats of both sexes weighing 180-200 g. Thromboplastin was injected intravenously at a dose of 10mg/kg in the background of preliminary intraperitoneal administration of compounds GSC-14, GSC-63 and BOS-122 in a dose of 5mg/kg and heparin from SIGMA 5000IU/mL at a dose of 130U/kg for 10 minutes before the introduction of the thrombotic agent thromboplastin.

In this experiment, it was revealed that 10 minutes after intravenous of thromboplastin to experimental rats in the background of preliminary administration of isotonic sodium solution, APTT sharply decreases compared with intact by 40%. After 10 minutes, under the conditions of the thromboplasthenemia model in the background of preliminary administration of heparin (1mg/kg), an increase in APTT was observed by 3,6 times compared with the control group of animals. In the background of preliminary administration of polysaccharides to experimental rats, an increase in APTT was observed at a dose of 5mg/mL GSC-14 3,6 times, GSC-63 1,6 times and BOS-122 4,3 times (Fig. 2).

Fig. 2: In the conditions of the thromboplastinemia model in the background of preliminary administration of thromboplastin, the coagulation time is lengthened. 1. Intact; 2. Thromboplastin; 3. Heparin; 4. GSC-63; 5. GSC-14; 6. BOS-122. **- Р<0.01. (n=6).

60 minutes after the introduction of thromboplastin, in the background of preliminary administration of polysaccharides to the experimental rats, an extension of APTT was observed with the introduction of heparin at a dose of 130 U/kg up to 126 sec., GSC-63 up to 274 sec., with the introduction of GSC-14 and BOS-122 plasma clotting was not observed.

These results indicate that the compounds, having an anticoagulant effect, may also have an effect on Ca²⁺ - dependent factors on the external pathway of blood coagulation. Studies have shown that when polysaccharides are added to the studied plasma, factors VIII, IX, XI and XII are completely inactivated. This indicates that the anticoagulant effect of these compounds is carried out through the blockade of factors - internal - the blood coagulation pathway. It is known that these sulfated polysaccharides affect factor Xa than factor IIa (thrombin), which results in a more pronounced suppression of thrombin formation: inactivation of one molecule of factor Xa can prevent the synthesis of about 50 molecules of factor IIa. Polysaccharides inhibit the release of von Willebrand factor, which prevents its acute phase increase. Polysaccharides have a stronger effect on the release of the plasminogen activator inhibitor, which expands the complex antithrombotic effect. SCs are less associated with platelets and platelet factor IV, so the likelihood of developing autoimmune thrombocytopenia becomes lower¹⁵^,16,17. Heparin has a weaker effect on vascular permeability, which is associated with a smaller number of bleeding when using them¹^8,19,20.

Output. The results obtained show that sulfated polysaccharides, being direct anticoagulants, when administered to experimental animals, minimize the effects of thromboplastin and, in the background of thromboplastinemia, limit the degree of plasma coagulation activation and accelerate the normalization of hemostasis parameters. These data once again proves the effective action of the investigated compounds, anticoagulant properties, in contrast to heparin.

Their thrombolytic activity is of interest in terms of its use as an anticoagulant in medicine.

ACKNOWLEDGEMENT:

The work was supported by the Applied Research Program of the Ministry of Innovation of the Republic of Uzbekistan (project PZ-2017092060 - “Development of a heparin-like anticoagulant based on sulfated polysaccharides”).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 03.02.2021 Modified on 13.03.2021

Research J. Pharm. and Tech 2021; 14(11):6085-6088.

DOI: 10.52711/0974-360X.2021.01057