POSITIVE FEEDBACK LOOP OF ADHESION DISEASE: FIBRIN AND OTHERS

Summary. Adhesion disease is a common complication of abdominal operations, which is often manifested by acute adhesion obstruction of the intestine with a high rate of recurrence on surgical treatment. Mechanical damage to the peritoneum, which leads to uncontrolled


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between the coagulation and fibrinolytic links of the hemostasis system causes various functional complications.The formation of adhesions can be considered as an extreme example of such an imbalance.This leads to the search for the reasons for the inefficiency of the fibrinolytic system in relation to fibrin deposits in adhesions.The aim of the work: study of the structure of fibrin deposits in adhesions using histological methods and infrared spectroscopy.Results and discussion.It is shown that adhesions of the peritoneum are a complex structure formed by protein and cellular components.The protein component is formed by fibrin and collagen, and the difference in the structure of these proteins from the native one with a pronounced content of β-structured aggregates is noted.The cellular component is mainly represented by fibroblasts -the main cells of connective tissue that synthesize collagen, elastin, proteoglycans and glycoproteins.Such composition ensures resistance of adhesions to fibrinolysis and their ability to regenerate.Key words: adhesion disease, hemostatic system, histology, infrared spectroscopy.

State of the problem.
Adhesion disease (AD) is the most common complication of abdominal operations, which is often accompanied by acute adhesion obstruction of the intestine with a high rate of recurrence during surgical treatment [1].Mechanical damage to the peritoneum, which leads to uncontrolled leakage of blood from the damaged vessels and the formation of blood clots with the loss of fibrin at the site of damage, is recognized as the leading trigger of adhesion disease.The adhesion process in the abdominal cavity accompanies 67-93% of abdominal operations [2,3].The stability of the formed adhesions, their ability to grow and regenerate are in conflict with the known data on the mechanisms of the hemostasis system.The latter, as is known, consists of two multidirectional activation cascades, which ensure the blocking of hemorrhage due to vascular damage due to the formation of a fibrin clot with its subsequent splitting into large soluble blocks.An imbalance between the coagulation and fibrinolytic links of the hemostasis system causes various functional complications [4].The formation of adhesions can be considered as an extreme example of such an imbalance.This leads to the search for the reasons for the inefficiency of the fibrinolytic system in relation to the fibrin deposits of adhesions.There is an opinion that insufficient fibrinolysis is caused by insufficient blood supply and, as a result, a deficiency of plasminogen activators [1].Such a point of view has the right to exist, but it seems somewhat one-sided.As a rule, when considering functional disorders caused by certain enzymatic processes, the most attention is paid to the enzymatic component.However, the substrate component deserves no less attention, the properties of which can undergo significant changes [5].A typical example of a highly organized and labile substrate is fibrin.As already mentioned, maintenance of the liquid state of the blood and prevention of hemorrhage due to vascular damage are the key functions of the hemostasis system.Vascular damage initiates an activation cascade of the blood coagulation system, largely mediated by endothelial cells and their expressed substances.Instead, after the restoration of the damaged vessel, the spent fibrin clot undergoes enzymatic cleavage into soluble fragments, which is provided by the activation cascade of the fibrinolytic system.The activation of each of the links of the agglutinating and fibrinolytic cascades is mediated by highly specific proteinases of the trypsin series.Both a lack and an Авторські права захищені | Creative Commons Attribution-ShareAlike 4.0 International License 529 СЕКЦІЯ XXVIII.МЕДИЧНІ НАУКИ ТА ГРОМАДСЬКЕ ЗДОРОВ'Я excess of functional activity of any of these components disrupts the hemostatic balance and leads to severe complications [4].Excessive blood coagulation plays a significant role in the development of atherosclerosis and its complications, myocardial infarction, impaired cerebral circulation, diabetes, malignant neoplasms, complications of pregnancy, septicemia and septic shock, hereditary thrombophilic disorders, postoperative complications and many other diseases.On the other hand, insufficient blood coagulation or excessive fibrinolysis cause various bleedings, which are a serious complication during injuries and surgical interventions.The dynamic balance between the coagulation and fibrinolytic cascades of the hemostatic system is ensured by the regularity and strict limitation of the course of each of their stages.The formation of a fibrin clot is a highly ordered process that includes the enzymatic cleavage of fibrin peptides A and B from the fibrin molecule by thrombin, the lateral association of fibrin-monomer molecules, and the covalent insertion of the formed associate with transaminase XIIIa (Fig. 1).

Fig. 1. Simplified scheme of fibrin coagulation and fibrinolysis
That is, it is a regular and highly organized process.No less strict are the requirements for the regularity of the fibrinolytic process.The effectiveness of the fibrinolysis process is due to the ability of plasmin (E,C.3.4.21.7) to split the fibrin clot into large soluble blocks.The targeting of the hydrolytic center of the enzyme to certain bonds of fibrin and its ability to migrate from one site of cleavage to another is ensured by an ensemble of binding sites of the enzyme [6].This gives special importance to the regularity of the fibrin structure.Due to its violation, the block disassembly mediated by plasmin is significantly complicated, and the resistance of the clot to fibrinolysis increases.[7].In AD, there is a pronounced functional insufficiency of the fibrinolytic system.This can be a consequence of both a local lack of certain components of the fibrinolytic system and a violation of the regular structure of fibrin.It is known that the imbalance of the protein structure creates prerequisites for the development of aggregation processes, among which the most energetically beneficial is the formation of β-structured protein aggregates [8,9].The latter are characterized by high stability, 530 SECTION XXVIII.MEDICAL SCIENCES AND PUBLIC HEALTH insolubility, immunogenicity and the ability to sorb and rearrange according to their own pattern of soluble proteins.The presence of such deposits in the composition of a fibrin clot can increase its resistance to the action of the fibrinolytic system and provides the possibility of self-assembly of a structure based on irregular fibrin.Thus, systemic disruption of the endothelium of blood vessels leads to excessive and uncontrolled activation of blood clotting and the formation of fibrin deposits with a disturbed regularity of the structure [10].For the above reasons, it seemed appropriate to conduct a comprehensive study of the structure of adhesions using the methods of histological staining and infrared spectroscopy as the most informative regarding the protein structure.
Materials and methods.
The material was collected by an incisional method without the use of electric tools to exclude electrodenaturation of protein and degradation of cellular structures.The drugs were withdrawn with the consent of the patients during laparoscopic and open operations.In all cases, adhesions were the result of previous surgical interventions, and it is characteristic that the number and prevalence of adhesions in the abdominal cavity after open operations significantly exceeded those during laparoscopic ones.We believe that this is due to the less traumatic nature and greater accuracy of laparoscopic interventions.For histological studies, the tissues of freshly isolated adhesions were fixed in 30% formalin as a medium that blocks conformational rearrangements of the protein structure and excludes obtaining false-positive results when stained with Congo red dye specific for β-folded structures.Paraffin sections were stained with hematoxylin and eosin, Van Gieson's picrate fuchsin, and Congo red.During spectroscopic studies, the tissue of freshly isolated adhesions was stored in a 0.9% sodium chloride solution and stored in a frozen state at -20°C.
Results and discussion.
As can be seen from the image shown in Fig. 2, when stained with hematoxylin and eosin, strands formed by fibrin and collagen are observed.The high content of cells that can be identified as fibroblasts by the characteristic shape of the nuclei catches the eye.When staining the preparation with collagen-specific picrate fuchsin according to Van Gieson, a weak pink color is observed throughout the entire field of the preparation (Fig. 3).In this context, it should be emphasized that collagen is a contact inducer of activation of the blood coagulation system [4].In case of a systemic violation of the endothelium of vessels, this contact causes the formation of a fibrin clot with a disturbed regularity of the structure [5].
When staining Congo red in a light microscope, an intense red color of the drug is observed (Fig. 4, left), which acquires an apple-green color in a polarizing microscope (Fig. 4, right).That is, a typical color characteristic of β-structured protein aggregates is observed.Neither the collagen of healthy tissues nor fibrin, which is normally formed mainly by α-spiralized structures with a small content of β-folded sheets (Fig. 5), does not undergo Congo red staining.Fig. 5. Model of fibrin protofibril obtained from the crystal structure of bovine fibrinogen.[11].

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Instead, the pronounced Congo red coloring in light and polarization microscopy indicates the presence of β-structured aggregates, which exceed the Abbe diffraction limit in terms of size.
Infrared spectra of adhesion preparations have a complex character, which is due to their multicomponent composition (Fig. 6).The analysis of infrared absorption spectra of abdominal adhesion tissues shows the presence of β-folded antiparallel layers in the tissue structure.Marker bands of βfolds were obtained by the authors for lysozyme protein samples under the influence of nanoparticles [12,13].Such spectra agree well with the infrared spectra of fibrin with normal and artificially increased content of β-folded structures [12].
Discussion and conclusions.
As follows from the given data, the structure of adhesions is much more complex than a fibrin clot and contains at least three different components.These include fibrin, collagen and cellular elements represented mainly by fibroblasts.At the same time, the structure of the first two components is significantly different from the native one and contains a certain proportion of β-structured protein aggregates.In favor of the latter assumption, the data obtained during the microscopic study of preparations stained with Congo red testify.This dye binds specifically and quantitatively to β-structured protein aggregates.Similar preparations are observed in light microscopy as colored brick-red, and in polarizing microscopy -as apple-green.This dye does not interact either with the collagen of healthy tissues or with the fibrin formed mainly by α-spiralized domains.The СЕКЦІЯ XXVIII.МЕДИЧНІ НАУКИ ТА ГРОМАДСЬКЕ ЗДОРОВ'Я selectivity of Congo red sorption is the basis of generally accepted methods for detecting amyloidosis both invitro and invivo.At the same time, amyloidosis is an extreme and most pronounced case of the formation of β-structured protein aggregates, which are observed to a lesser extent in other pathological conditions caused by a violation of protein metabolism [4].As can be seen from those shown in Fig. 4 images, a certain proportion of β-structured protein aggregates is present in the adhesion.It should be emphasized that although such structures are a kind of "energy bottom" of the variety of denatured proteins, there are also many rather stable irregular aggregates [8].The presence of a certain proportion of β-structured protein aggregates in the composition of mature adhesions is a kind of tip of the iceberg, which indicates a high degree of disruption of the regularity of fibrin and collagen.This, in turn, can be the reason for the resistance of adhesions to fibrinolysis and their propensity for self-reproduction and growth.
From the given data, it follows that AD is a consequence of the autochthonous formation of a complex multicomponent association of cellular and protein components.This is in good agreement with data on the lability of the fibrin structure, its propensity to form β-folded structures upon denaturation [14], as well as on the propensity of fibrin to contact denaturation [15].All this conditions the process of structuring adhesions [16].Uncontrolled activation of the blood clotting process leads to the formation of structurally damaged fibrin, which is prone to the formation of β-folded aggregates.The latter provide not only resistance to fibrinolysis, but also sorption of fibroblasts [17].Synthesis of collagen by the latter creates conditions for uncontrolled contact activation of the blood coagulation cascade.The formation of such positive feedback creates prerequisites for the resistance of mature adhesions to fibrinolysis, their ability to spontaneous growth and regeneration.

Fig. 6 .
Fig. 6.Spectra of tissue absorption of abdominal adhesions in the infrared range (top); decomposition and components of the amide I band of the IR absorption spectrum of abdominal adhesion tissues (bottom).