Mantskava Maya (Principal Investigator), Assoc.Prof., I.Beritashvili Center of Experimental Biomedicine, Georgia, Tbilisi, 0160, Gotua St., 14, mantskavamaka@rambler.ru; biomedicine.net@gmail.com;

Momtselidze Nana (Investigator), Assoc.Prof., Beritashvili Center of Experimental Biomedicine, Georgia, Tbilisi, 0160, Gotua St., 14;

Davlianidze Lena (Investigator), Assoc.Prof., Beritashvili Center of Experimental Biomedicine, Georgia, Tbilisi, 0160, Gotua St., 14;

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Two different systems that provide motion and movement of blood are involved simultaneously in human body (Kamensky A. and Kamkin A., 2004, ISBN 5-7695-1675-5). This is coagulation/anticoagulation and rheological. Blood is not a homogeneous (Newtonian) liquid and it is a suspension having suspended particles, it has exceptional features and it is studied by coagulation and hemorheology. Coagulation and hemorheology play a significant role in blood circulation and ensure its trophic function. (Hoffman M., Monroe D., 2006). Changes in coagulation and rheological properties of blood may cause a slowdown in the flow, establishment of stasis, thrombus, which is accompanied by multi-disease.[/su_animate]​

           Coagulation involves the formation of various substances which are produced as a genetically determined body components (primary anticoagulants) or arise in blood clotting and fibrinolysis (secondary anticoagulant).

         They distinguish physiological anticoagulant – a primary anticoagulants, constantly contained in the blood, the synthesis of which does not depend on the activity of the coagulation system in the body at the moment. These substances are released into the blood stream at a constant rate, where they interact with the active coagulation factors, causing their neutralization. On the basis of the above mentioned the blood remains in the form of liquid. They act solely on active forms of coagulation factors, whereas the inactive form factors (proenzymes or procoagulants) are not subjects for inactivation.[su_spoiler title=”Develop”]

           Heparin belongs to the first anticoagulants. This is polysaccharide, which are synthesized by mast cells associated with endothelium that are main repository of heparin and vasoactive substances (histamine and serotonin), and granulocytes. A significant number is defined in the liver, heart, muscle, lung, and mast cells and basophiles. It does not have excess to the placenta. It is a polyvalent of inhibitor action that restricts all phases of coagulation (Goljan E., 2013, ISBN9780323084383).

           Heparin also activates fibrinolysis, which contributes to dissolution of blood clots to form complexes with the plasma, fibrinogen and epinephrine, which have anticoagulant and fibrinolytic action. Activation of anticoagulation system is accomplished along with activation of the coagulation system at the same time, in fact, self-locking of the hemostatic system is marked at the stage of activation of the coagulation system. The point application physiological anticoagulants are only active form of the blood coagulation factors. (Xu Y., Masuko S., Takieddin M. Et al., 2011; doi :10.1126/science.1207478. PMC 3425363. PMID 22034431). It is important that anticoagulation system is being depleted much faster than clotting. The pace development physiological anticoagulants in pathological conditions lag far behind the pace of consumption, which dictates the need to fill their deficit in terms of pathology.

           The physiological role of anticoagulation system is to keep the blood in the liquid state and limitation of thrombus formation process. Anticoagulants block a reactive plasma blood coagulation factors. Self-locking system of a hemostasis is observed at all stages of coagulation. (Weitz D., Weitz J., at al., 2010, doi:10.1007/s11239-009-0411-6. PMID19882363).

           Normal blood flow as well as anticoagulant system is necessary to ensure adequate blood flow that is provided by rheological properties of the organism. All abnormal processes are caused by violations of the macro- and microcirculation in blood flow. The effectiveness of blood flow depends on vascular content and vascular reactions. The vascular reactions and rheological properties of blood determine the intensity of microcirculation, which in turn provides an adequate blood supply needs of the tissue (Mchedlishvili G. and Maeda N., 2001). Rheological properties of blood flow characteristic parameters (local hematocrit, erythrocytes aggregation, red blood cells and plasma viscosity deformation) and their changes play a leading role in the slowing down or stopping the flow and therefore in tissue hypoxia, which is accompanied by many diseases (Mchedlishvili G., 2004).

           According to a modern concept it is impossible to develop physiological process if coagulation and blood rheology systems are not within the normal range. In the development of all pat-physiological processes coagulation and hemorheology play compensatory role, which is directed to adequacy and improvement of the tropic function. Therefore it is very important the normalization of the coagulation system in physiological and especially in pathological conditions as well as the improvement of both coagulation and hemorheology systems.

Background of the theme of the project and justification of its scientific and practical interest

           In today’s world of pharmacology, medicine, biotechnology, the problem of production, use, testing of anticoagulants, and especially heparin, take an important place. This is due to a large proportion of the exogenous heparin use in virtually all areas of therapeutic medicine. If we consider that in modern medicine surgery is an important part of therapy and heparin therapy is present in a set of standard assignments for pre- and post-operative actions, the importance of studies of the effect of aspirin on the course of physiological and pathological processes, the effectiveness of anticoagulant, is gaining special scientific and practical sense.

           The rising scientific and practical interest is due to the fact that anticoagulation treatment is assigned to a large part of the total population. This determines the importance of the issue. Great scientific interest is due to the fact that the disorders coagulation system is the background of many diseases and during disease management antithrombotic treatment is necessary. Anticoagulation is often one or the only solution. Sometimes inadequate or excessive anticoagulation therapy is a barrier for curing the disease. It is therefore very important to select the correct therapeutic dose. We hope that the antithrombotic measures should promote the reduction of mortality and disability (E.g. thrombosis, embolism, heart attacks, etc.). Taking into consideration the fact that anticoagulant treatment is prescribed and important therapeutic, post and preoperative segments and also very important in prevention activities, all the studies, which will be dedicated to anticoagulants, will be very timely and important for scientific and practical biomedicine. In addition, the appearance of a new drug to market needs the refinement and modification of the coagulation treatment algorithm.

           Taking into consideration that annual sales of the antithrombotic drugs along with all the above mentioned represent according to the pharmaceutical company Portola Pharmaceuticals (www.portola.com) market size of anticoagulants in the Western community ~ $ 10-15mlrd. per year. (marketing agency DSM Group (http://www.dfm.ru) up to 750 billion. (analytical agency IMS Health, (www.ims health.com), except scientific and practical significance of anticoagulant studies dedicated to anticoagulants  gains  special importance in financial terms.

Main and secondary objectives of the research t

           The ultimate goal of our research is the study the action of low molecular bemiparin in normal blood circulation and in blood flow. It serves as the main reason of planning our original designed research. In addition, within the frames of our project the comparative effectiveness of low molecular bemiparin and high molecular heparin will be made.

           A number of secondary goals may be arise during the working process. In order to reach the main goal of our experiment we will study bemiparin action on coagulation and rheology; effective doses of bemiparin will be determined not only to improve the coagulation system, but also to examine the effective doses for operating on the rheological parameters. We will study the effect of bemiparin during gestation.

Materials and methods

           Research studies are underway in the various systems: in vitro, in vivo, in situ. The study involved 140 rats of both sexes. Among them 10 rats (1:1) are in the control group. The average starting weight of the rats are 200±23 grams. At the same time, a separate group is allocated pregnant rats and female rats control group. In blood samples of the rats the coagulation research and/or rheological studies are made. Coagulation research is made in Coatron M1 (Germany).

           The Coatron M1 is a single-channel optical coagulometer is to determine the basic parameters of the second stage of Hemostasis (coagulation cascade) in human citrated plasma, it is designed for in-vitro coagulation testing in the clinical laboratory. Clotting assays with fibrin formation as the end point may be run on the instrument, as well as immunoturbidimetric test (like quantitative D-Dimer) or chromogenic test (eg. Antithrombin, protein C, Ecarin Chromogenic Assay).

Following tests and features are available on the instrument:

PT (Prothrombin Time). The PT is expressed in seconds (100ms sampling rate and automatically normalized into INR (International Normalized Ratio). A normal PT-value (100%) and the Thromboplastin ISI – value (International Sensitivity index) can be stored on board. Additionally the results can be converted into %-Activity, when the 100% value and the 25% value are stored in the memory.

APTT (Activated Partial Prothrombin Time). The APTT is expressed in seconds and automatically normalized into Ratio. The normal APTT-value can be stored on board.

TT (Thrombin Time). The TT is expressed in seconds and automatically normalized into Ratio. The normal TT-value can be stored on board.

FIB (Fibrinogen). The FIB is expressed in seconds and automatically converted into mg/dL. Three calibration points can be stored on board.

FAC (Factors). The coagulation factors are expressed in seconds and automatically converted into activity % in plasma. A calibration is necessary to obtain the results in %. Three calibration points can be stored on board.

DD (D-Dimer).The D-Dimer is measured immunoturbidimetric and expressed in milli optical density per minutes (mOD/min) and converted into ng/mL concentration in plasma. A calibration is necessary to obtain the results in ng/mL. Three calibration points can be stored on board.

AT3 (Anti-thrombin)

PC (Protein C).The tests are measured chromogenic and expressed in milli optical density per minutes (mOD) and converted into % plasma activity.  Up to three calibration points can be stored on boardto % plasma activity.

ECAT (Ecarin Chromogenic Assay for Thrombin)

ECAH (Ecarin Chromogenic Assay for Hirudin) ECA tests are used to monitor the direct Thrombon inhibitors (such as Hirudin). ECA tests are equivalent to Ecarin Clotting Time, but the performance is much advanced by chromonic method. The result is expressed in milli optical density (MOD) and converted into % plasma activity. Up to three calibration points can be stored on board.

           Reserved for OEM partner. Not installed on standard configuration. The Coatron M1 supports an indirectional RS232 interface (fixed setting at 2400, 8, 1, No). All results are printed automatically, if the interface is set to print mode. The RS 232 can also set to LIS mode. Then the results including their reaction curves are sent in RECAM SMART format to the PC. TECAM SMART is a easy using coagulation data management system which fulfills LIS demands (laboratory information system).

Incubation area for 6 sample and 2 reagent positions. The Coatron M1 needs 3-5 min to warm up to 37.0. A green signal light indicates the correct temperature.

           Rheological research (1. Erythrocyte concentration-hematocrite; 2. Erythrocytes deformability; 3. Erythrocytes aggregability; 4. Viscosity) is held by following methodologies:

The index of erythrocytes aggregability, which represents aggregated erythrocytes area ratio against whole area of the erythrocytes. To this end we used the so-called “Georgian technique” (Mchedlishvili G., 2004). Erythrocyte aggregation was evaluated by using the “Georgian technique”. Blood samples (4ml) were centrifuged and about 0.1 ml blood was diluted 1:200 in own plasma in the Thoma pipettes preliminary rinsed with 5% sodium citrate solution without addition of any other anticoagulants to the blood under study. Following standard mixing the diluted blood was placed into a glass chamber 0.1 mm high. The quantitative index of erythrocyte aggregation, which was assessed with a special program at the Texture Analysis System (TAS-plus, “Leitz, Germany), represented itself the relationship of the aggregated and unaggregated red cells.

           Evaluation of Erythrocytes Deformability Index: Evaluation of erythrocytes deformability was performed with an aid of the nucleopore membrane filter method, which is based on assessing velocity of the erythrocytes passage through the very small pores (5 μm, which is a diameter of the smallest capillary) of the filter, at constant pressure (10 cm of water column) and temperature (37°C). Obtaining the pure erythrocytes was performed by centrifuging the blood sample at 3000 rpm, for 15 min. Resulting plasma was aspirated with micropipette and the remaining blood cells were added with bovine serum albumin (0.2 mg per 5 ml) dissolved in the phosphate buffer. Then the blood was centrifuged second time at 1000 rpm for 5 min. The precipitated erythrocytes, as well as thin layer of leukocytes and thrombocytes, were separated from the phosphate buffer. This procedure was repeated three times. Purified erythrocyte mass was diluted in the phosphate buffer, with hematocrit of 10%. Evaluation of the deformability index implied measuring a velocity of the erythrocyte passage through the filter (mm/min) was recorded. The high quality polycarbonate filters (with 5 μm diameter pores) were used in measuring procedures.

           Blood plasma viscosity: Blood plasma viscosity was examined in the capillary viscometer at 37º C. Diameter of the capillary was about 1.8 mm. Displacement of plasma samples was induced by the gravity force related to the difference of niveaux of the plasma under study – about 65 (without application of an additional pressure). For evaluation of the plasma viscosity in centipoises (cp) we determined the calibration factor (F). Blood plasma viscosity was calculated by multiplying the time for plasma displacement through the capillary by the instrument calibration factor.

           Evaluation of systemic hematocrit: Systemic hematocrit was assessed with centrifuging the blood sample in the standard hematocrit centrifuge, at 8000 rpm, for 10 min.

For the acute trials the rats of the both sexes will be used. We will cause experimental thrombosis and stasis on the animals being under narcosis. For the above mentioned 10% solution of a half ferric chloride and sodium chloride crystals will be needed.

           The anesthesia was performed with chloral hydrate (1 ml of a 4% solution per 100g body weight). Before the anesthesia the animal should be pierced pro-Medolit solution (0.02 ml of a 2% solution of 100 g). We will carry out surgery and isolate mesentery and post the mesentery on a warm table (37°C – approximate conditions in situ). We observed in the blood flow of rats’ microscope “Ortoplan“ and lens 170/- Plapo (×6.3) (“Leitz”, Germany). Special camera “Vario-Ortomat” made micrographs of normal and pathological blood flow. The subject of the research – Bemiparin (Tsibor) 2500. Bemiparin – Low molecular weight heparin obtained by depolymerization of heparin sodium isolated from porcine intestinal mucosa. At the same time, we should bear in mind that the anticoagulant activity of heparin is determined by the biological method and standardization activities in international units (UI). During UI 1 taken an amount of heparin that prevents coagulation of blood in 4 ml for 24 hours.

Bemiparin has the lowest meaning of molecular weight (3600 Da), the longest half-life (5.3 h) and the largest anti-factor Xa: antifactor IIa ratio (8:1) of all LMWHs. Bemiparin promotes a greater release of tissue factor pathway inhibitor than unfractionated heparin (UFH) or dalteparin. These properties could result in a more favorable efficacy: safety ratio than the currently marketed LMWHs.

           We use a concentration of 0.01; 1 and 0.1 UI / ml, which covers the therapeutic range of doses used in clinical practice and selected from scientific papers (Vignoli A., Marchetti M., Russo L. 2011).

           For the comparison of the action of bemiparin we will chose macromolecular heparin (the study will be blind and therefore successful brands, Trade Marks and accurate molecular weight cannot be anonymous).

Research plan

Table1. List of tasks with the indication of period and performers

           The main goal of our research is to study the effect of bemiparin blood flow. For this reason studies are planned in different directions. For the achievement of the main goal the planned tasks and sub tasks are give in the table by periods.

Task N1. Preparatory works. Preparation of materials and equipment. the Measurement of coagulation and hemorheology in control animals (local hematocrit, aggregation of erythrocytes, deformation of erythrocytes and plasma viscosity). (n=10).

Task N2. Study of the different concentration bemiparine effect on coagulation in the experiment.

2.1. In vitro. coagulation will be measured in blood samples before and after adding bemiparin (0,01; 0,1 и 1 UI / ml), in order to  determine which concentration of bemiparine causes the maximum improvement of coagulation parameters in comparison with control value. Bemiparin’s solution, which will cause maximum improvement of coagulation, will be recognized as an effective dose by us and in further objectives only this dose will be used. (n=30; (10-10-10)).

2.2. We will study impact of bemiparin’s effective dose on coagulation in the in situ system. Subcutaneous injections will be made on a daily basis. Coagulation evaluation will be done on a daily basis. Trial will continue for 10 days. The analysis of the variables on bemiparin’s impact on coagulation in time respect and the impact of subcutaneous injection on each individual coagulation parameters will be determined. (n=10).

2.3. We will cause experimental thrombosis in the mesentery of the animals being under in vivo narcosis and study the coagulation actions of the bemiparin dose: we will conduct visual observation and measure the specific weight of thrombi vessels and bemiparin’s lysis effect on thrombus (n=5).

2.4. On the basis of statistical analysis we will determine bemiparin’s impact on coagulation parameters in time respect.

Task N3. We will study bemiparins effective dose impact on rheology in this experiment (based on 2.1).

3.1. We will study bemiparins effective dose impact on rheology in the in vitro system (n=5).

3.2. We will study bemiparins effective dose impact on hemorheology in the in situ system. The injections will be made in daily basis. Evaluation of rheological status will be made on daily basis as well. Trail will last for 10 says. The analysis of the variables on bemiparin’s impact on rheology in time respect and the impact of subcutaneous injection on each individual rheology parameters will be determined. (n=50 (5-5-5-5-5-5-5-5-5-5)). (According to the methodology and recommendations and permition of the Ethics Commission in each sub group five animals will be placed).

3.3. In vivo. We will cause stasis in the rat mesentery by sodium chloride and evaluate anti-aggregative action of bemiparin, calculate linear speed of blood and intensity of the microcirculation (number of blind capillary opening) n=5).

Task N4. We will be comparing bemiparins’ and unknown macromolecular anticoagulants action on coagulation.

4.1. In vitro. We will study the impact of high molecular heparin (unknown to the researchers, blind study) on coagulation (n=10).

4.2. In vivo. We will study the impact of high molecular heparin (unknown to the researchers, blind study) on coagulation (n=5).

4.3. Statistical comparison between the unknown effects of heparin and bemiparin shall be made (based on 2.1).

Task N5. We will study bemiparins effective dose impact on pregnant rats in this experiment (based on 2.1).

5.1. Coagulation examination will be made on rats every day from 15th day of gestation until delivery.  (n=5).

5.2. On the 14th day of gestation in vivo rats will be injected bemiparin’s effective dose and from the 15th day till the delivery coagulation examinations will be conducted on daily basis.

5.3. We will examine coagulation data of the rats both with and without bemiparin in comparison with the control group’s average coagulation data.

5.4. Time-dependent coagulation changes will be made in pregnant rats (based on 4.2)

Task N6. The comparison of anticoagulation and rheological features shall be made based on a statistical analysis

6.1. The confirmation of the impact of bemiparin shall be done based on the statistically obtained variables from the previous sub-tasks. The obtained data shall be processed with various statistical programs by the professional programmer. The data must be analyzed in different directions.

Task N7. Final works

7.1. Publication of the data, presentation at conferences.

Management of  research

           Our project is theoretical and experimental. The our project will last 18 mounts (3 periods x 6 months). The conditions for conducting the work on animals corresponded to the European Convention on the protection of experimental animals, adopted in 1986 in Strasbourg (http://www.ncbi.nlm.nih.gov/pubmed/24502054) and for the conducting the experiment the consent of Ethics Committee was obtained. All researchers are experienced in the field of the project. They have skills and analytical thinking. There also are a technician and a programmer participating in the project.  Our results are discussed in conferences, and published in science articles. Material-technical basis necessary for the implementation of the project 60 sq.m. surgical rooms, 30 sq. m laboratory room, 30 sq. m procedure room. Tas plus (Leitz) teqsture analysizator, Plasma analizator, apparats of hematocrit, aparats of depormability, blood viscosity measurement, Coatron M1. To be purchased new statistic programs and pc. Budget see in Table 2. The principal investigator will must forward to ROVI at every six months (VI month, ХII month, XVIII month) a status report of the project. The progect’ investigators will collaborate in procedures leading to recognition of Rovi as the owner of these inventions.

Table 2. Budget of project

Discussions

           The scientific innovation will be identified by us as a result of the planned tasks and objectives.

Our research will prove bemiparin’s impact on coagulation system. Despite the studies and information on bemiparin, bemiparin affects on blood flow is insufficiently described for clinicians (Lorenzo De F., Newberry D., et al., 2002; Rodríguez-Mañas L., Gómez-Huelgas R., et al., 2010;  Ciccone M.M., Cortese F., et al., 2014 ).

Makin bemiparin injections will cause coagulation picture changes in animals. We will show how the best parameters of coagulation may be reached and the number and the specific concentration of bemiparin.  We will statistically clarify certain coagulation parameters and the functional impact of bemiparins subcutaneous injection’s frequency and quantity (Mantskava M. and Momtselidze N., 2015). This will determine that the daily injections of bemiparin will be effective. This data will help us to determine bemiparin’s features for preventive measures based on the fundamental literature. Side effects may be discussed indirectly based on this data. Generalization of this data may serve as a basis for preventing any complication during number of diseases. This will help us in choosing treatment tactics. Achievement of final goal has applicable value as well as fundamental nature for biomedicine. 

           It will be for the first time that we will study bemiparin impact on blood coagulation and rheological features in experiments complexly, as well as the optimization of blood flow intensity (experimental stopping blood flow restoration). If we take into consideration that the impairment of intensiveness of blood flow may equally be caused by changes in blood coagulation and hemorheology system, and become the basis for many diseases (Bokariov I., Popova L.,2014, http://cyberleninka.ru/article/n/sovremennye-problemy-trombozov-arteriy-i-ven#ixzz3b4AuGkx0l), modeling of thrombosis and stasis (Mchedlishvili G., Gobejishvili L. et al., 1999; Nebilicin A.,2007;) is very important and evaluation of behiparin at the same time. Blood flow disruption is often a central ring in formation of a number of pathologies and their complication (Sherwood L., 2011 ISBN 978-1-133-10893-1). Vascular factor is out of doubt (Jonson L., 2003, ISBN 978-0123875846), but platelets, red erythrocytes and blood plasma also have an important role. Hypertension, strokes, heart attacks and cancer diseases are subjects of World Health Organization’s interest. Development and management of these diseases normal circulation of blood plays an important role. Diabetic angiopathy, atherosclerotic events, as well as Raynaud’s syndrome prevention requires the timely regulation flow system (Bhatt DL. et al., 2006; Gayraud M., 2007; Negrean V., Suciu I., et al., 2004). Normalization of rheological factors play positive role.

           Rheology characteristics is determined by the flow of blood: aggregation of erythrocytes, deformation, their number (Hct) and plasma viscosity.

           These features describe rheological status and have significant role in accomplishment in blood trophic function. By the concept of our laboratory normalization of blood clotting status is primary and most important to stimulate microcirculation and blood circulation in overall. It is also very important that it will be for the first time when we will evaluate bemiparin impact rheological properties of blood, as the improvement of rheological status will be contributing factor for transition of the diseases in plateau phase and their rapid cure.

           Various molecular weight endogenous heparin is used in practical medicine. Therefore it is necessary to study bemiparin action on the blood coagulation system and evaluation analysis to be done in comparison with other anticoagulant drugs in vivo and in vitro conditions.

           For the first time we will evaluate bemiparin not only as an anticoagulant remedy, but we will examine what impact bemiparin has on blood’s rheological features as well.

           At first we will study the features of the blood complexly in in vivo experiment, which provide blood flow. We will compare efficiency of bemiparin and high molecular heparin on coagulation and rheology for the first time. We hope that all this will shed light on the effectiveness of bemiparin on the diseases progressed by the changes of coagulation and hemorheology. This will help clinicians for correction of treatment tactics and for establishment of preventive measures.

           It will be the first time that the research of bemiparin will be conducted on rats, by us, which will allow us to generalize the study and provide recommendations for obstetrician-gynecologist.

           Our research team has extensive experience in the study of the mechanisms of blood rheology and microcirculation. It was for the first time that the changes in blood flow during different types of diseases had been studied by us (Mantskava M., Momstelidze N., Pargalava N., 2004; 2006;   Momtselidze N., Mantskava M., Mchedlishvili G. 2006). [/su_spoiler]​

           On the basis of the researches carried out in previous years different postulates have been presented for discussion by our research group. Clinicians and scientists represented the audience. Big number of our proposals gained practical responses and some clinics are guided by some of our recommendations at present.

           The conduction of the researches described in the project is very important and significant, in order to reveal the previously unknown features of bemiparin.