M. Mantskava, N. Momtselidze,
L. Davlianidze, T. Urdulashvili.

Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia, 0160, Gotua St., 14, biomedicine.net@gmail.com;
Medical Centre “Medisoni”, Tbilisi, Georgia, 0182, Kaloubani St., 12
t.urdulashvili@yahoo.com.

Abstract

Coagulation and hemorheology play a significant role in blood circulation and ensure its trophic function. 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. To study bemiparin we made research in vivo and in vitro systems. And found that bemiparin has a positive effect not only on the coagulation, but also on the rheological properties of blood.

Keywords: Bemiparin, Erythrocyte, Rheology, Microcirculation

1. Introduction

    Two different systems that provide motion and movement of blood are involved simultaneously in human body [1]. 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 [2]. 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. 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. 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 [3].

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    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 [4]. 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 [5]. 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 [6]. 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 [7].

    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. We cause stasis in the rat mesentery by Dexstran and sodium chloride and evaluate anti-aggregative action of bemiparin, calculate linear speed of blood and intensity of the microcirculation and rheological status.

2.Methods

2.1 Investigation of Microcirculation

The study involved 30 rats of both sexes. Among them 10 rats (1:1) are in the control group. Rats’ weight – 200±23g. 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. 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) [8]. The anesthesia was performed with chloral hydrate (1 ml of a 4% solution per 100g body weight). Under anesthesia (0,02 ml a 2% solution of promedol per 100 g body weight). We will be surgery and isolated mesentery and post the mesentery on a warm table (37 ° C – approximate conditions in situ). We observed in the blood flow of rats’ by microscope “Ortoplan, 170 / -Plapo (× 6.3)”, special camera “Vario-Ortomat”«Leitz», Germany.

2.2. Investigation of Blood Rheology

Blood plazma viscosity was examined in the capillary viscometer at 37̊ C (Diameter – 1.8 mm). Displacement of plazma samples was induced by the gravity force related to the difference of niveaux of the plazma under study. For evaluation of the plazma viscosity in centipoises (cp) we determined the calibration factor (F). Blood plazma viscosity was calculated by multiplying the time for plazma displacement through the capillary by the instrument calibration factor. Hct was assessed with centrifuging the blood sample in the standard hematocrit centrifuge, at 8000rpm, for 10 min. To this end we used the so-called “Georgian technique”. Blood samples (4ml) were centrifuged and about 0.1 ml blood was diluted 1:200 in own plazma in the 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
TAS-plus, (“Leitz”,Germany), represented itself the relationship of the aggregated and unaggregated red cells [7].

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) [9]. Blood plazma viscosity was examined in the capillary viscometer at 37º C. Diameter of the capillary was about 1.8 mm. Displacement of plazma samples was induced by the gravity force related to the difference of niveaux of the plazma under study – about 65 (without application of an additional pressure). For evaluation of the plazma viscosity in centipoises (cp) we determined the calibration factor (F). Blood plazma viscosity was calculated by multiplying the time for plasma displacement through the capillary by the instrument calibration factor. Systemic hematocrit was assessed with centrifuging the blood sample in the standard hematocrit centrifuge, at 8000 rpm, for 10 min.

3. Results

Results of the microcirculation study showed that blood flow restores, when bemiparin (microdroplet) applique on the capillary with stasis (NaCl crystal did stasis). in vivo. See Fig.1. Changes of rheological properties in the systemic blood see in Fig.2, Fig.3, Fig.4, Fig.5.

Fig.1. Impact of bemiparin on the microcirculation

Fig.2. Тhe dependence of the RBC aggregation on bemiparin concentration (in vitro)

Fig.3.Тhe dependence of the RBC deformability on bemiparin concentration (in vitro)

Fig.4.Тhe dependence of the blood plasma viscosity on bemiparin concentration (in vitro)

Fig.5.Тhe dependence of the hematocrit (Hct) on bemiparin concentration (in vitro)

4. Discussion.

    Despite the studies and information on bemiparin, bemiparin affects on blood flow is insufficiently described for clinicians [10,11,12]. Makin bemiparin injections cause coagulation picture changes in animals. We show how the best parameters of coagulation may be reached and the number and the specific concentration of bemiparin.  We statistically clarify certain coagulation parameters and the functional impact of bemiparins subcutaneous injection’s frequency and quantity. This determine that the daily injections of bemiparin will be effective. This data 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.

We 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 [13], modeling of thrombosis and stasis  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 [14]. Vascular factor is out of doubt [15], but platelets, red erythrocytes and blood plazma 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 [16,17,18]. Normalization of rheological factors play positive role. Rheology characteristics are determined by the blood flow: aggregation of erythrocytes, deformation, their number (Hct) and plazma 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 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 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 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.[/su_spoiler]

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 [19, 20, 21].  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.

References

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[17]  Gayraud M., 2007. Raynaud’s phenomenon. Joint Bone Spine  Jan; 74 (1), 1-8. PMID:17218139.

[18] Negrean V., Suciu I., et al., 2004. Rheological changes in diabetic microangiopathy. Rom J Intern Med.  42(2), 407-13.

[19] Mantskava M., Pargalava N., Mchedlishvili G., 2004. Direct beneficial effect of insulin on blood rheological disorders in the microcirculation. Clinical Hemorheology and Microcirculation. 30, 431-433.[/su_spoiler]

[20] Momtselidze N., Mantskava M., Mchedlishvili G., 2006. Hemorheological disorders during ishcemic brain infarcts in patients with and without diabetes mellitus. Clinical Hemorheology and Microcirculation. 35, 261-265.

[21] Mantskava M., .Momtselidze N., Pargalava N., Mchedlishvili G., 2006. Hemorheological disorders during the 1st and 2nd types of diabetes mellitus in patients with foot gangrenes. Clinical Hemorheology and Microcirculation. 35,  307-311.