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The hemocompatibility of arabinogalactan, betulin and its derivatives was evaluated in vitro and samples suitable for creation of nanostructures or materials in contact with blood were selected. The prospects of arabinogalactan as a component of the construct (nanostructure) for drug delivery are due to the fact that it did not affect blood/plasma coagulation (at concentrations of 0.0033-3.333 mg/ml and 0.00465-4.65 mg/ml, respectively), platelet aggregation (0.00182-0.182 mg/ml), and demonstrated the degree of erythrocyte hemolysis less than 3%. Sodium salt of betulin monosulfate, diarginine salt of betulin disulfate (up to 0.465 mg/ml), and especially betulin and allobetulin formate with procoagulant properties (degree of hemolysis less than 2%) can be used to create a material, for example, sponge, gel, active against blood coagulation., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Uncontrolled bleeding that occurs during surgery, trauma, and in combat conditions is critical and require immediate action. Chitosan is a polysaccharide, obtained from natural sources with unique biological properties. It is often used as basis for local hemostatic agents (LHA). We summarized the data on hemostatic properties of chitosan, commercially available chitosan-based products with focus in the field of chemical modification of chitosan. Various approaches are used to enhance hemostatic activity of chitosan-based materials. The approach with chemical modification of chitosan allows changing the properties of the polymer in order to obtain an active macromolecule that contributes to hemostasis. Ongoing research on the mechanism of interaction with blood components in the case of different chitosan derivatives will make it possible to identify promising directions for chemical modification to obtain an effective LHA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alexey Lunkov reports financial support was provided by Ministry of Science and Higher Education of the Russian Federation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Intravenous injection of cationic starch conjugated with sterically hindered phenol (terpenophenol) to guinea pigs did not increase hemorrhagic activity (in doses of 2 and 4 mg/kg) and plasma clotting time in activated partial thromboplastin time and prothrombin time tests (in a dose of 4 mg/kg) in comparison with administration of physiological saline. Intravenous injection of the cationic starch conjugate with the highest content of terpenophenol fragments (4.1%wt) in a dose of 2 mg/kg to guinea pigs leads to a decrease in hemorrhagic activity by 4 times in comparison with the control., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Polysaccharides are important structural components of all plant species. Gel-like polysaccharides have found wide application in various fields, including medicine, construction, and the food industry. In the present work, galactomannan and galactoglucomannan gel-like polysaccharides were modified with sulfate groups and their anticoagulant activity was studied. Sulfation with chlorosulfonic acid in pyridine and with sulfamic acid in pyridine and a sulfamic acid-urea deep eutectic solvent were used as synthesis routes. The resulting gel-like polysaccharide sulfates were studied by elemental analysis, Fourier-transform infrared spectroscopy, and gel permeation chromatography. It was established that the anticoagulant effect of sulfated galactoglucomannan (SGGM) and galactomannan (SGM-1 and SGM-2) is related to an independent antithrombin-independent decrease in the amidolytic activity of thrombin and factor Xa. It is shown that the inhibitory activity of SGGM and SGM-2 against the collagen-induced platelet aggregation can be an additional factor in selecting compounds that are most promising for modifying polymer surfaces to ensure resistance to blood clotting.

We identified compounds that do not independently provoke aggregation of human platelets and do not affect hemolysis of human erythrocytes in vitro: lacking anticoagulant activity sulfated galactoglucomannan (polydispersity 1.43; degree of sulfation 0.66) in concentrations ≤0.2 mg/ml; exhibiting anticoagulant activity (in concentrations up to 0.002 mg/ml) sulfated galactoglucomannan (polydispersity 1.5; degree of sulfation 1.81) and galactomannan obtained by sulfation with the sulfamic acid-urea complex (polydispersity 2.75; degree of sulfation 1.25) and galactomannans obtained by sulfation with chlorosulfonic acid in 1,4-dioxane (polydispersity 1.61/22.27; degree of sulfation 1.00/0.74)., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Despite the introduction of direct oral anticoagulants, the search for new oral anticoagulants remains an urgent task., Objective: By using docking and scoring, based on physical methods, simple chemical rules, methods of synthesis, and activity measurement, develop new low-molecular-weight inhibitors of factor Xa, which are potential anticoagulants., Methods: The development of leads was based on chemical synthesis and structure-based drug design methods. The basic idea was to combine the two approaches: one based on predictive modeling and the other based on the experimental data., Results: In this study, we developed some nanomolar leads. Further chemical modification improved the inhibition constant by more than one order., Discussion: The method proposed in this paper, as well as other methods, includes virtual screening, chemical synthesis, and activity measurement. However, the most time-consuming process in this method (chemical synthesis) was simplified, and the cost was reduced to the extent that was allowed; a very simple chemical reaction was chosen, i.e., the formation of an amide bond., Conclusion: In this work, we demonstrated how using simple chemical rules based on the structurebased drug design, substances with a nanomolar concentration of activity can be developed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Oligochitosan Сh10/85 with a molecular weight of 10 kDa and a deacetylation degree of 85% prevented the development of experimental venous thrombosis in guinea pigs after intravenous administration in a dose of 30 mg/kg. In a concentration of 0.005-0.5 mg/ml, oligochitosan Ch10/85 did not provoke hemolysis of human red blood cells in in vitro experiments. The antithrombotic effect of oligochitosan Ch10/85 that exhibits low anticoagulant activity (by two orders of magnitude lower than that of unfractionated heparin) can be associated with inhibition of platelet aggregation., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Promotion of promising cellulose nanocrystals (CNC) is largely dependent on the relationship between their morphology, surface chemical composition, and supramolecular structure with toxicity, hemocompatibility, and biodegradability. This paper outlines comparative and integrated analysis of the mentioned biocompatibility aspects of partially acetylated rod-, and disc-lake morphology of CNC with crystalline cellulose allomorphs I and II. These data have also included the study of CNC obtained from the sulfuric acid solutions. The aqueous solution of all types of tested CNC has not been toxic to mice after oral administration. Morphology of internal organs has not changed. However, in case of disc-like particles, the kidney mass coefficient noticeably changed. CNC have neither triggered platelet aggregation nor destroyed the red cell membrane. Intravenous administration to rabbits has not affected the plasma clotting time. Rod-like CNC are more resistant, and the disc-like particles are more susceptible to degradation under the influence of cellulases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The effects of sulfated organosolv lignins derived from fir (Abies sibirica) and larch (Larix sibirica) (SLf and SLl; 4-3-7.5% sulfur, median-weight molecular mass 2960-4888 Da), on human blood/plasma clotting, platelet aggregation, and erythrocyte hemolysis were studied in vitro. Antithrombin activities of the samples were below 2 U/mg. Specimens of SLf (sulfur content 6.5, 6.6, and 7.5%, molecular weights 3503, 3487, and 3580 Da, respectively) and SLl (4.3 and 6.3%, 2960 and 3497 Da) in a concentration of 0.01 mg/ml did not prolong the blood clotting time, did not provoke human platelet aggregation, did not destroy erythrocyte membranes, and could be used for construction of drug delivery systems. The SLf sample (6.5%, sulfur, 3503 Da) in concentrations from 0.09 to 1.82 mg/ml did not stimulate platelet aggregation, reduced ADP-induced platelet aggregation, and 2-fold prolonged the blood/plasma clotting time 2-fold in comparison with control and could be used for creation of biomaterial with clot-resistant surface.

Cationic polymers are of interest as the basis for obtaining various biomaterials. Hydrophilic biopolymers and their modification products are of main interest. Biocompatibility is the prime criterion that makes this material usable for said purposes. In this research, hydroxyethyl starch (HES) was used as a basis for synthesis of aminodeoxy derivatives, containing n-butylamin (BA) and dibutylamin (DBA) fragments. Bromodeoxy HES was an intermediate compound. The structure of synthesized polymers was confirmed with NMR, elemental analysis and FTIR methods. The derivatives with 0.6 and 0.9 degree of substitution were tested for compatibility with blood. The research showed that HES base does not have an anticoagulation activity, does not affect human platelet aggregation and in concentrations up to 10 mg/mL of cell suspension in a buffer solution does not destroy red blood cell membrane, and therefore can be used as a component of drug delivery systems. Addition of aminodeoxy derivatives of HES hindered development of ADP-induced platelet aggregation. Derivatives of HES-DBA and HES-BA0.9 may also be of interest, but their concentration must not exceed 1 * 10 -5 mg/mL of blood. Biodegradation of HES cationic derivatives were analyzed through identification of reducing sugars after treatment with amylase and pancreatin., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest in regard to this research or its funding., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We studied hemocompatibility of silver nanoparticles synthesized on the basis of a conjugate of quaternized chitosan with gallic acid (QChit-Gal). For the three variants of silver particles (Nos. 1, 2, and 3), the QChit-Gal:AgNO 3 ratio was 5:1, 5:3, and 1:1, respectively. Anticoagulant activity of all samples of silver nanoparticles was lower than that of the conjugate. Samples of nanoparticles Nos. 1 and 2 in a concentration of 0.0233 mg/ml did not affect plasma clotting time and can be used for intravenous administration. However, their concentration in the blood should not exceed 0.01 mg/ml, because in this concentration they do not affect erythrocyte membrane, do not induce platelet aggregation, and do not affect platelet aggregation induced by ADP.

Quaternized derivatives of chitosan with a substitution degree of 85-98% (highly substituted) synthesized from chitosans with a molecular weight of 5, 10, 20 kDa, with a degree of deacetylation of 89-98%, and the code numbers of QChit 5, QChit 10, QChit 20, respectively, completely neutralize antithrombin activity of unfractionated heparin and partially neutralize aXa activity of low-molecular-weight heparin (clexane), similar to protamine sulfate. The advantages of QChit 5 and QChit 10 over QChit 20 and protamine sulphate are the follows: the effect is achieved at lower concentrations and in greater concentration range; they do not promote platelet aggregation; in a concentration of 0.0072 mg/ml they do not destroy the erythrocyte membranes.

Nanocrystalline particles of chitin in the form of hydrosol in a concentration of 0.63 mg/ml have no effect on aggregation of human platelets and clotting time of platelet-poor plasma in coagulation tests. ADP-induced aggregation of human platelets was inhibited by these nanoparticles in concentrations of 0.63 and 1.00 mg/ml in comparison with the control. Intravenous administration of nanoparticles in a dose of 1 mg/kg to guinea pigs produced no anticoagulant effect. The nanocrystalline particles of chitin can be of interest as potential drug delivery systems.

Background: Factor Xa (FXa) is known to play a central role in blood coagulation cascade and considered to be one of the most attractive targets for oral anticoagulants of new generation., Objective: Our approach for the development of directly acting oral anticoagulants (DOAC), FXa inhibitors was demonstrated in this work., Method: Chemical synthesis is the base of our approach for the development of potential inhibitors. In this work, the substances like R1-(CONH)-R2-(CONH)-R3 are being developed, using previously described docking and screening methods, where R1, R2 and R3 are some chemical groups and (CONH) are amide bonds connecting R1, R2 and R3. The direction of amide bond (CONH) could be arbitrary for R1, R2 and R2, R3., Results: Chemical modifications were made in the frame of the results, taking into account the structure of FXa, chemical synthesis capabilities, as well as patentability of the target compounds. Subnanomolar potency of several developed compounds was achieved. Several analyzers and various testing-suites have been used to measure the concentration that doubled the prothrombin time (PTx2). Moreover, in human plasma the PTx2 concentration of the compound 217 (DD217) turned out to be 80±20 nM. The compound efficacy has proved by in vivo assays including oral administrations in rats, rabbits and monkeys., Conclusion: The pharmacodynamic profile of DD217 for oral administration in cynomolgus monkeys proves the efficacy of the compound, which makes it promising for the future preclinical trials., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Sulfated derivatives of xylan (isolated from Bétula pubéscens wood) with average molecular weight ~34 kDa, sulfur content of 11.3-17.5%, a degree of substitution of 0.74-1.64 are anticoagulants of direct type of action. Antithrombin and antifactor Xa activities in three tested xylan samples did not differ and reached 30.8-31.8 and 13.5-14.3 U/mg, respectively.

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

Sulfation (to 2.8) of dextrans with molecular weight of 150 and 20 kDa was followed by the appearance of anticoagulant activity that increased with decreasing their molecular weight and did not depend on antithrombin, plasma inhibitor of serine proteases of the blood coagulation system. Antithrombin activity of dextran sulfate with a molecular weight of 20 kDa reached 12.6-15.3 U/mg. Dextran sulfates with molecular weights of 20 and 150 kDa did not potentiate ADP-induced human platelet aggregation.

Alkylated derivatives of low molecular weight chitosan with different substitution degrees of 98, 40, and 9% (I, II, and III respectively) have been synthesized. The structure of the obtained derivatives was defined by spectral assays (IR-spectroscopy and proton magnetic resonance). Chitosan derivatives were characterized with positive zeta-potential (33–51 mV) and solubility from 2 to 100 mg/mL in pH 7.4 and 25°C. It was shown that, at a concentration of 0.0014–0.0029 mg/mL, derivative I, as well as protamine sulfate, could be used to neutralize the anticoagulant activity of unfractionated or low molecular weight heparin. At a concentration of 0.0029–0.58 mg/mL, derivative I enhanced platelet aggregation, which would be necessary when hemostatic compounds or materials were used. Derivatives II and III enhanced platelet aggregation to a lesser extent.

Experiments on rabbits showed that increasing the dose of intravenously administered cellulose sulfate from wheat straw (dynamic viscosity 3.4 cP, sulfur content 14.1%) increased plasma clotting time in some coagulation tests and plasma anticoagulant activity. When cellulose sulfate was administered in the dose of 1 mg/kg, plasma clotting time in the presence of the anticoagulant (5 min after administration) was ~3-fold higher than after saline administration.

RA36 DNA aptamer is a direct anticoagulant prolonging clotting time of human, rabbit, and rat plasma in the thrombin time test. Anticoagulant activity of RA36 is lower than that of recombinant hirudin. During inhibition of human plasma clotting activated with echitox (coagulase from Echis multisquamatus venom), the aptamer presumably binds to meisothrombin exosite I. The sensitivity of human plasma to the aptamer 5-fold surpasses that of rat plasma. Analysis of RA36 binding to coagulase of Agkistrodon halys venom (ancistron) is required for proving the effect of aptamer on polymerization of human fibrinogen.

Polyanions (in an amount within 1.5 - 6.0 mg), including cellulose sulfates (excreted from Gossipium hirsutum L., molecular weight 22.0 kDa, degree of sulfation within 0.8 - 1.8), inulin sulfates (excreted from Helianthus tuberosus, molecular weight 8.0 kDa, degree of sulfation within 0.6 - 1.6), pectin sulfates (excreted from Abies sibirica L., molecular weight 24.0 kDa, degree of sulfation within 0.8 - 1.1), give rise to peaks of precipitation with polycations of protamine sulfate. Only cellulose sulfates (in amount within 0.38 - 6.00 mg) give the peaks of precipitation with chitosan polycations (molecular weight 10 kDa, degree of deacetylation 85%) during horizontal biospecific electrophoresis. The height of the peak of precipitation with protamin sulfate was found to grow with increasing antithrombin activity of cellulose sulfates and pectin sulfate (for polyanions in an amount within 1.5 - 6 mg). The size of the area of precipitation with chitosan was found to decrease with increasing antithrombin activity of cellulose sulfates.

With fucoidan from Fucus evanescens dose increase from 1 to 5 mg/kg plasma coagulation time in test A(see symbol)TB increases. Sulphate protamin in final concentration 0.67-1.35 mkg/ml will neutralise antithrombin activity of fucoidans from brown seaweed Fucus evanescens and Laminaria cichorioides. The gravimetrichesky relation for the investigated samples makes an antidot/anticoagulant 1.

We have studied a relationship between the degree of sulfonation and anticoagulant activity of starch from Solanum tuberosum (molecular weight, 25000-30000 Da; sulfonation degree, 0.4-2.5) and inulin from Helianthus tuberosus (molecular weight, 7000-8000 Da; sulfonation degree, 0.6-1.6). Starch and inulin sulfates (i) increased the time of appearance of fibrin clots in plasma in coagulometric tests and (ii) reduced (via antithrombin) the rate of thrombin-induced hydrolysis of a chromogen substrate. The antithrombin (aIIa) activity of starch sulfates reached 16.8-70.0 IU/mg and the activity against factor Xa (aXa activity) was 2.3-16.6 IU/mg. The antithrombin activity of inulin sulfates was within 5.5-11.4 IU/mg and the activity against factor Xa (aXa activity) was within 0-1.4 IU/mg. An increase in the degree of sulfonation led to a growth in the anticoagulant activity of starch sulfates. The anticoagulant activity of starch sulfates and inulin sulfate with sulfonation degree 1.0 is mediated by antithrombin, which is the plasma inhibitor of serine proteases.

DNA aptamer RA36 with a molecular weight of 10000 is direct-acting anticoagulant whose efficacy is lower than that of recombinant hirudin and unfractionated heparin (UFH) in blood clotting time (BCT), activated blood recalcification time (ABRT), recalcification time (RT), prothrombin time (PT), and activated partial thromboplastin time (APTT) tests. The anticoagulant effect of RA36 is comparable with that of UFH in the thrombin time (TT) test, but is lower than the effect of recombinant hirudin. Analysis of the blood and plasma anticoagulant activity during intravenous bolus administration of aptamer RA36 in rabbits and rats is based on the use ABRT (in blood case) and APTT/RT (in plasma case) tests. The range of doses for evaluation of pharmacodynamic parameters of RA36 during intravenous bolus administration in rabbits and rats is 3 - 34 mg/kg and 1 - 27 mg/kg, respectively. Accordingly, designed dose range for humans is 1 -29 mg/kg.

Sulfated derivatives based on powdered cellulose were obtained, including those containing additional (carboxymethyl, ethyl amide or phosphate) groups, and their activity against blood clotting factors (thrombin and Xa) was studied. Maximum antithrombin activity of the test compounds, measured using a coagulation test, was 144±11 U/mg.

The antithrombotic and hemorrhagic activities of fucoidan with molecular weight within 20 - 40 kD isolation from Fucus evanescens seaweed have been investigated. The antithrombin activity of fucoidan is 41 +/- 9 aIIa IU/mg and the anti-factor Oà activity is 38 +/- 8 aOà IU/mg. The antithrombin and anti-factor Oà activities of plasma, antithrombotic activity (100% prevention of formation of a blood clot observed at a dose of 10 mg/kg), and hemorrhagic activity (to a lesser degree, than that of unfractionated heparin in comparable antithrombotic activity doses) increase as the doses of fucoidan increases from 2.5 to 10 mg/kg at intravenous injection in rats.

We have investigated in vitro the anticoagulant (AC) activity of proanthocyanidins from the bark of birch, cedar, spruce, pine, and larch; sulfated arabinogalantan and dihydroquercetin from larch wood; extracts from birch, cedar, and spruce; microcrystalline cellulose (MCC) from aspen and fir wood and wheat straw; and methylcellulose (MC) from aspen wood. The AC properties of the investigated substances are related mostly to their antithrombin activity. The AC activity increases with the content of sulfur in MCC of wheat straw, MC of aspen wood, and arabinogalañtan of larch wood. The maximum AC activity was observed in samples of sulfated MCC from fir wood and wheat straw. Their antithrombin activity (134 +/- 8 and 96 +/- 6, respectively) is worth of carrying out model tests in vivo.

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.

Specimens of fucoidan extracted from Fucus evanescens were purified from protein and polyphenols, deacetylated and depolymerized by fucoidanase for evaluation of their biological activity. Deacetylation did not modify the capacity of fucoidan to inhibit thrombin and factor Xa, while purification from protein and polyphenols reduced this capacity. Depolymerization of fucoidan increased its capacity to inhibit thrombin mainly through heparin cofactor II. All the studied specimens formed complexes with protamine sulfate.

We have studied in vitro the ability of the Siberian cedar crust (SCC) extract (Pinus sibirica Du Tour) and arabinogalactan sulphate (AGS) extracted from wood of Siberiam pine-tree (Larix sibirica Ledeb.) to increase the human blood plasma coagulation time and also to inhibit the amydolytic activity of thrombin (aIIa) and the coagulation factor Xa (aXa). A method has been developed by means of which SCC increases the aXa activity by a factor of 3.7 and the aIIà activity by a factor of 2.5. The AGS preparation increased the blood plasma coagulation time in the test for activated partial thromboplastin time. An effective concentration, at which the time of plasma coagulation was increased by a factor of 2 (in comparison to the control) was 2.94 +/- 0.33 mg/ml. AGS did not exhibit the ability to inhibit the Xa activity.

We studied anticoagulant activity in vitro and in vivo of sulfate depolymerisation galactomannan guar with the following characteristics: Man:Gal 1.64, molecular mass 127 kDa, sulfation degree 1.46. We found the ability of galactomannan-HSO3Na (GM) to increase the time of blood coagulation in the test aPTT with an increase of its concentration and to decrease velocity of chromogenic substrate on the factor Xa hydrolysis. Specific antithrombin and anti-factor Xa activities of GM were 35.8 +/- 1.8 IU/mg and 6.6 +/- 0.5 IU/mg, respectively. In biospecific electrophoresis complexes arise between GM and protamine sulfate. Intravenous injection of GM to rats prolonged plasma coagulation time in the aPTT test with dose reduction from 1 to 3 mg/kg. In rat thrombosis model a dose of 3 Mg/kg produced a 100% inhibition of blood clot.

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3(-) groups was 48.05% +/- 2.31. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests--aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65-87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.

Original synthetic peptide derivatives exhibit anticoagulant activity in vitro and in vivo. They delayed fibrin clot formation from human blood plasma in tests for the intrinsic coagulation pathway (activated partial thromboplastin time) and final stage of plasma coagulation (thrombin time) and inhibited amidolytic activity of thrombin. We determined the minimum effective dose of the most active compound providing a 2-fold lengthening of blood clotting time (activated partial thromboplastin time test and thrombin time test), which persisted for 2-3 h.

The anticoagulant activity of low-molecular weight heparins (LMWH-PC) with average distribution of molecular weights within 3.4-5.8 kD was investigated. The samples of LMWH-PC were obtained from unfractionated heparin using immobilized enzyme complex of protease C. The LMWH-PC derivatives inhibited the activity of blood coagulation factors IIa (thrombin) and Xa. The LMWH-PC derivatives had an anti-factor-Xa activity up to 131-208 IU/mg and anti-factor-IIa activity up to 81-175 IU/mg. All LMWH-PC derivatives form complexes with protamine sulfate during electrophoresis in agarose gel. The anticoagulant activity of rabbit plasma exhibits a doze-dependent increase upon the intravenous or subcutaneous injection of LMWH-PC with a molecular weight of 5.4 kD.

The anticoagulant activity of low-molecular-weight heparin with an average molecular weight of 4.7 kD (LMWH-4.7) has been studied. This derivative was prepared from unfractionated heparin with the help of chitinolytic enzyme complex from Streptomyces kurssanovii. The antithrombin activity of LMWH-4.7 (aIla activity) was 72 +/- 9 IU/mg and the activity with respect to the blood coagulation factor Xa (aXa activity) was 200 +/- 33 IU/mg, which corresponded to an aXa/aIIa ratio of 2.8 (necessary for effective antithrombotic drugs). The aIIa and aXa activity exhibited a dose-dependent variation upon intravenous and subcutaneous injections in rabbits, so that a high aIIa/aXa ratio was retained: 5 min after the intravenous injection of a minimum dose (0.3 mg/kg), this ratio was 2, 7, and for a greater dose (3.0 mg/kg) it reached 3.8. Subcutaneous injections were followed by slow elimination of the anticoagulant within 24 h. LMWH-4.7 upon intraperitoneal injections produced a dose-dependent inhibition of a model thrombosis in rats. Complete inhibition was observed for a dose of 3 mg/kg. Thus, it is possible to obtain active LMW heparin with the help of chitinases.

The paper reviews published data on the chemical structure of sulfated polysaccharides of animal, plant, and microbial origin as well as synthetic or semi-synthetic derivatives, and on the anticoagulant activity of these compounds in vitro and in vivo. The influence of sulfated polysaccharides on the plasma and cell hemostasis links is considered. Relationships between the structure and activity and the mechanisms of action are discussed.

Fucoidans isolated from Fucus evanescens and Laminaria cichorioides kelp can inhibit thrombin and factor Xa of the blood coagulation system. In rats, intravenous injection of fucoidans dose-dependently increased anticoagulant activity of the plasma. Fucoidans can form complexes with protamine sulfate. The observed quantitative differences in the action of fucoidans can result from different sulfation degree and the presence of various types of glycoside bonds in polysaccharide molecules.

Antithrombotic activity of Russian preparation Antithrombin III was studied on rat model of induced venous thrombosis. Optimal doses of antithrombin and heparin preventing thrombus growth were determined.

It is possible to successfully determine both the activity against activated factor Xa in animals plasma after heparins administration and specific activity against activated factor Xa of new anticoagulants by means of the domestic diagnostic kit "Reachrom-Heparin" developed by the company "Renam". It was compared to the similar kit "Berichrom heparin" (Dade Behring). No statistically significant differences were detected.

The possibility of obtaining low-molecular-weight heparins using the chitinolytic enzymatic complex immobilized on Silochrom has been demonstrated. The optimal conditions of this process (sodium acetate buffer, pH 7.0-7.5; temperature, 40-45 degrees C; and duration of hydrolysis, 3 h) were determined. Depending on the ratio between heparin and the immobilized enzymatic complex, samples with molecular weight varying from 1.7 to 4.7 kDa, were obtained. These complexes inhibited factor Xa in 2.0-3.7 times more effectively than original heparin.

In order to choose the proper method for evaluating the antithrombin activity in samples of chitosan polysulphate (CP) with different polymerization degrees and sulphation degrees, we estimated the ability of direct anticoagulants to depress the coagulability of recalcified sheep blood using the third international heparin standard (A1 - in vitro system) and determined such activity on pharmacodynamic curve (A2 - in vivo system). The curve admits the kinetics of CP elimination to be nonlinear in case of intravenous injection to rabbits, as it is observed in heparin: Ct = C(o)exp(-K(e)lt), where Ct is the CP concentration at the time moment t; C(o) is the CP concentration at the injection moment; Kel is the elimination constant. Besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific activity A2: T = KTCt+T(in), where T is the time of clot formation at different time intervals after CP injection; T(in) is the time of clot formation prior to CP injection. T value was assessed in two tests: blood coagulation time (BCT) and activated partial thromboplastin time (APTT). No correlation was observed between A1 and A2. At the same time, the values of Kel and the period of semi-elimination, with the use of the biospecific cetylpyridinium chloride electrophores for the quantitative determination of CP in rabbit's blood taken at different time intervals after injection, showed a close correlation (r = .94, P < .05) between the same parameters, obtained with the help of the rectilinear pharmacodynamic plot in BCT test. Thus, experimentally, it was proven that the assumption of the CP nonlinear elimination and the CP effect-dose dependence was true, which is necessary for A2 calculation. Relatively low molecular weights (MW 61-82 kDa, polymerization degree 188-252 ) and high sulphation patterns (sulphur amounts 15.6-16.9%, sulphation degree 1.58-1.86) were slowly cleared and there was more antithrombin activity (30-52 IU/mg). We recommend the use of in vivo system for evaluating the antithrombin activity of the CP derivatives.

Mechanisms of the anticoagulant activity of p-aminobenzoic acid (PABA) were studied. The specific antithrombin activity of PABA is aIIa = 7.00 +/- 0.32 IU/mg and the specific antiactivated factor Xa activity is aXa = 6.70 +/- 0.12 IU/mg. Study of the antithrombotic activity of PABA in rats with a model of venous stasis showed that intravenous injection of PABA at a dose of 1.5 mg/kg prevented the experimental thrombosis development 1.5 h after injection. The activity exhibited a peak 3 h after drug injection and ceased by the 5th hour. Equal antithrombotic activity in the rat blood plasma was observed for fraxiparin at a dose of 40 aXa IU/kg and PABA at 25 aXa IE/kg (1.5 mg/kg). At the same time, PABA affected neither the number of thrombocytes nor their response to the thrombocyte aggregation factors (ADP or adrenaline).

It was shown for the first time that p-aminobenzoic acid (PABA), in addition to the previously described fibrinolytic activity, exerts the properties of a direct anticoagulant both in vitro and in vivo. PABA not only displays antithrombin activity, but also inhibits activated factor X and, upon intravenous injection to rats and rabbits, shows the antithrombotic effect. The most pronounced antithrombotic affect was observed at a dose of 1.5 mg/kg. PABA at 0.5 mg/kg has insignificant efficacy and at 3 mg/kg, a high efficacy, but induces hemolysis of erythrocytes in about a half of cases. Equally efficient antithrombotic activity of blood plasma of rats was noted after intravenous injection of low molecular weight heparin "Fraxiparin" at 40 anti-Xa U/kg and PABA at 25 anti-Xa U/kg (1.5 mg/kg). Unlike "Fraxiparin", which exerts an immediate effect, the effect of PABA was expressed within 1.5 to 5 h after injection with a peak of antithrombotic activity at 3 h (which correlates with anti-IIa and anti-Xa activities of plasma) and terminated by 5 h after injection. For PABA, the ratio of anti-Xa to anti-IIa activities (an important parameter, which determines the antithrombotic potential of drugs) equals 2.4. PABA at 0.5 or 1.5 mg/kg did not affect the number of thrombocytes, while at 3 mg/kg, it decreased the number of thrombocytes by 20%. Thus PABA at 1.5 mg/kg, which has a high anticoagulant activity and does not cause side effects, is most interesting for further studies.

We studied anticoagulant effects of combined administration of heparin (H) and chitosan sulfate ether (CS) (specific activity 20 UE/mg) in the ratio 1 : 1. CS enhanced anticoagulant activity of heparin in rabbits by a factor of 1.95 +/- 0.15. The intravenous injection of the mixture in a dose of 0.5 mg(H)/kg + 0.5 mg(CS)/kg and heparin injection in a dose of 1mg/kg induced the same effect. Haemorrhagic effect of this mixture was less pronounced compared to heparin, anticoagulant and antithrombotic activities remained the same. The mixture was found to decrease a number of platelets, however, this was also less pronounced compared to heparin. Thus, the use of the mixture CS + H (1 : 1) instead of double heparin dose resulted in the same effect.