Hemostasis

The system of hemostasis (hemostasis) is a set of functional-morphological and biochemical mechanisms ensuring the preservation of the liquid state of the blood, preventing and stopping bleeding, and the integrity of blood vessels.

In a complete organism, in the absence of any pathological effects, the liquid state of the blood is a consequence of the equilibrium of factors conditioning the processes

Coagulation and impeding their development. Violation of such a balance can be caused by many factors, but regardless of etiological reasons, thrombogenesis in the body occurs according to unified laws with the inclusion of certain cellular elements, enzymes and substrates in the process.

There are two links in blood clotting: cellular (vascular-platelet) and plasma (coagulation) hemostasis.

• Cellular hemostasis means the adhesion of cells (that is, the interaction of cells with a foreign surface, including cells of a different kind), aggregation (gluing the same blood cells among themselves), as well as the release of elements that activate plasma hemostasis.
• Plasma (coagulation) hemostasis is a cascade of reactions in which clotting factors occur, resulting in the formation of fibrin. The resulting fibrin is further destroyed by plasmin (fibrinolysis).

It is important to note that the division of haemostatic reactions into cellular and plasma is conditional, however, it is valid in the in vitro system and significantly facilitates the selection of adequate techniques and the interpretation of the results of laboratory diagnostics of the pathology of hemostasis. In the body, these two links of the coagulating blood system are closely related and can not function separately.

A very important role in the implementation of hemostasis reactions is played by the vascular wall. Endothelial vascular cells are capable of synthesizing and / or expressing on their surfaces various biologically active substances that modulate thrombus formation. These include von Willebrand factor, endothelial relaxation factor (nitric oxide), prostacyclin, thrombomodulin, endothelin, tissue type plasminogen activator, tissue type plasminogen activator inhibitor, tissue factor (thromboplastin), tissue factor pathway inhibitor, and several others. In addition, membranes of endotheliocytes carry receptors, which under certain conditions mediate binding to molecular ligands and cells freely circulating in the bloodstream.

In the absence of any damage, the lining vessels of the endothelial cells have thrombolytic properties, which helps maintain the liquid state of the blood. Endothelial thrombose resistance provides:

• contact inertness of the inner (turned into the lumen of the vessel) of the surface of these cells;
• synthesis of a powerful platelet aggregation inhibitor - prostacyclin;
• the presence on the membrane of endothelial cells thrombomodulin, which binds thrombin; while the latter loses the ability to cause blood clotting, but retains the activating effect on the system of two most important physiological anticoagulants - proteins C and S;
• high content of mucopolysaccharides on the internal surface of the vessels and fixation of the heparin-antithrombin III (ATIII) complex on the endothelium;
• the ability to secrete and synthesize a tissue plasminogen activator that provides fibrinolysis;
• the ability to stimulate fibrinolysis through a system of proteins C and S.

Violation of the integrity of the vascular wall and / or changes in the functional properties of endotheliocytes can contribute to the development of prothrombotic reactions - the antithrombotic potential of the endothelium is transformed into a thrombogenic one. The causes leading to vascular trauma are very diverse and include both exogenous (mechanical damage, ionizing radiation, hyper and hypothermia, toxic substances, including drugs, etc.), and endogenous factors. The latter include biologically active substances (thrombin, cyclic nucleotides, a number of cytokines, etc.), capable of exhibiting membrane-aggressive properties under certain conditions. Such a mechanism of involvement of the vascular wall is typical for many diseases, accompanied by a tendency to thrombosis.

All cellular elements of the blood take part in thrombogenesis, but for platelets (in contrast to erythrocytes and leukocytes) the procoagulant function is the main one. Platelets not only act as the main participants in the process of thrombus formation, but also have a significant effect on other links of hemocoagulation, providing activated phospholipid surfaces necessary for the realization of plasma hemostasis processes, releasing a number of coagulation factors into the blood, modulating fibrinolysis and disrupting hemodynamic constants both by transient vasoconstriction , caused by the generation of thromboxane A ₂, and by the formation and isolation of mitogenic factors contributing to hyperplasia of the vascular wall. When thrombogenesis is initiated, platelets are activated (i.e., activation of platelet glycoproteins and phospholipases, metabolism of phospholipids, formation of secondary mediators, protein phosphorylation, arachidonic acid metabolism, actin and myosin interaction, Na ⁺ / H ⁺ exchange, fibrinogen receptor expression and calcium ion redistribution) and the induction of their adhesion, release and aggregation reactions; the adhesion precedes the release and platelet aggregation reaction and is the first step in the haemostatic process.

When the endothelial lining is broken, the subendothelial components of the vascular wall (fibrillar and nonfibrillar collagen, elastin, proteoglycans, etc.) come in contact with blood and form a surface for binding the von Willebrand factor, which not only stabilizes factor VIII in plasma, but also plays a key role in process of platelet adhesion, binding subendothelial structures to cell receptors.

Adhesion of platelets to the thrombogenic surface is accompanied by their spreading. This process is necessary for more complete interaction of platelet receptors with fixed ligands, which contributes to the further progression of thrombus formation, since, on the one hand, it provides a stronger bond of adherent cells to the vascular wall, and on the other hand, immobilized fibrinogen and von Willebrand factor are able to act as platelet agonists, promoting further activation of these cells.

In addition to interacting with an alien (including damaged vascular) surface, platelets are able to adhere to each other, that is, aggregate. Aggregation of platelets is caused by substances of different nature, for example, thrombin, collagen, ADP, arachidonic acid, thromboxane A ₂, prostaglandins G ₂ and H ₂, serotonin, adrenaline, platelet activation factor, and others. Proagregantami can be exogenous substances (not in the body), such as latex.

Both adhesion and aggregation of platelets can lead to the development of a release reaction - a specific Ca ²⁺ -dependent secretory process in which platelets release a number of substances into the extracellular space. Induced release reaction of ADP, adrenaline, subendothelial connective tissue and thrombin. First, the contents of dense granules are released: ADP, serotonin, Ca ²⁺; to release the contents of α-granules (platelet factor 4, β-thromboglobulin, platelet-derived growth factor, von Willebrand factor, fibrinogen and fibronectin) requires more intensive platelet stimulation. Liposomal granules containing acid hydrolases are released only in the presence of collagen or thrombin. It should be noted that the released from the platelet factors contribute to the closure of the vascular wall defect and the development of the hemostatic plug, but with a sufficiently pronounced vessel damage, further activation of platelets and their adhesion to the injured portion of the vascular surface forms the basis for the development of a widespread thrombotic process followed by vessel occlusion.

In any case, the result of damage to endotheliocytes is the acquisition of intimal vessels with procoagulant properties, which is accompanied by the synthesis and expression of tissue factor (thromboplastin), the main initiator of the blood clotting process. Thromboplastin itself does not possess enzymatic activity, but can act as a cofactor of activated factor VII. The thromboplastin / factor VII complex is able to activate both factor X and factor XI, thereby generating thrombin generation, which in turn induces further progression of the reactions of both cellular and plasma hemostasis.

[1], [2], [3], [4]

Mechanisms of hemostasis regulation

A number of inhibitory mechanisms prevent uncontrolled activation of coagulation reactions, which can lead to local thrombosis or disseminated intravascular coagulation. These mechanisms include inactivation of procoagulant enzymes, fibrinolysis and cleavage of activated clotting factors, mainly in the liver.

Inactivation of clotting factors

Inhibitors of plasma proteases (antithrombin, tissue factor inhibitor, and _2- macroglobulin, heparin cofactor II) inactivate coagulation enzymes. Antithrombin inhibits thrombin, factor Xa, factor Xla and factor IXa. Heparin increases the activity of antithrombin.

Two vitamin K-dependent proteins, protein C and protein S form a complex that proteolytically inactivates factors VIlla and Va. Thrombin, connecting with the receptor on endothelial cells, called thrombomodulin, activates protein C. Activated protein C together with protein S and phospholipids as cofactors subject factors VIIIa and Va to proteolysis.

Fibrinolysis

The deposition of fibrin and fibrinolysis should be balanced to maintain and limit the haemostatic clot when restoring a damaged vascular wall. The fibrinolytic system dissolves fibrin with plasmin, a proteolytic enzyme. Fibrinolysis is activated by plasminogen activators released from vascular endothelial cells. Plasminogen activators and plasminogen plasma are attached to fibrin. Plasminogen activators catalytically cleave plasminogen to form plasmin. Plasmin forms soluble degradation products of fibrin, which are released into circulation.

Activators of plasminogen are divided into several types. The tissue activator of plasminogen (tAP) of endothelial cells has a low activity, being in free form in solution, but its effectiveness increases with its interaction with fibrin in close proximity to plasminogen. The second type, urokinase, exists in single-stranded and double-stranded forms with different functional properties. Single-stranded urokinase is not capable of activating free plasminogen, but like a tPA it is able to activate plasminogen when interacting with fibrin. The trace concentrations of plasmin split the single-stranded into two-chain urokinase, which activates plasminogen in dissolved form, as well as that associated with fibrin. Epithelial cells in excretory ducts (for example, renal canalis, mammary ducts) secrete urokinase, which in these channels is a physiological activator of fibrinolysis. Streptokinase, a bacterial product that is not normal in the body, is another potential activator of plasminogen. Streptokinase, urokinase and recombinant tap (alteplase) are used in therapeutic practice to induce fibrinolysis in patients with acute thrombotic diseases.

[5], [6], [7], [8], [9], [10], [11], [12]

Regulation of fibrinolysis

Fibrinolysis is regulated by inhibitors of the plasminogen activator (PAI) and plasmin inhibitors, which slow down fibrinolysis. PAI-1 is the most important PAI, is released from vascular endothelial cells, inactivates TPA, urokinase and activates platelets. The most important inhibitor of plasmin is a-antiplasmin, which inactivates the free plasmin released from the clot. Part of the a-antiplasmin can bind to the fibrin clot with factor XIII, which prevents excessive plasmin activity within the clot. Urokinase and TPA are rapidly excreted by the liver, which is another mechanism to prevent excessive fibrinolysis.

Hemostatic reactions, a combination of which is commonly called plasma (coagulation) hemostasis, eventually lead to the formation of fibrin; these reactions are mainly realized by proteins called plasma factors.

International nomenclature of factors of blood coagulation

Factors	Synonyms	Half-life, h
I	Fibrinogen *	72-120
II	Prothrombin*	48-96
III	Tissue thromboplastin, tissue factor	-
IV	Calcium ions	-
V	Proaccelerin *, Ac-globulin	15-18
VI	Accelerin (excluded from use)
VII	Proconvertin *	4-6
VIII	Antigemophilic globulin A	7-8
IX	The Christmas factor, the plasma thromboplastin component,	15-30
	The antihemophilic factor B *
X	The Stewart-Power Factor *	30-70
XI	Antigemophilic factor C	30-70
XII	Hageman factor, contact factor *	50-70
XIII	Fibrinase, fibrin-stabilizing factor Additional:	72
	Von Willebrand factor	18-30
	Fletcher factor, plasma precalicyrein	-
	Fitzgerald factor, high molecular weight kininogen	-

* Synthesized in the liver.

Phases of hemostasis

The process of plasma hemostasis can be conditionally divided into 3 phases.

I phase - the formation of prothrombinase or contact-kallikrein-kinin-cascade activation. Phase I is a multi-step process, as a result of which a complex of factors capable of converting prothrombin to thrombin accumulates in the blood, therefore this complex is called prothrombinase. There are internal and external ways of protrombinase formation. On the internal pathway, the coagulation of the blood is initiated without the involvement of tissue thromboplastin; Plasma factors (XII, XI, IX, VIII, X), kallikrein-kinin system and platelets take part in the formation of prothrombinase. As a result of the initiation of reactions of the internal pathway, a complex of factors Xa and V is formed on the phospholipid surface (platelet factor 3) in the presence of ionized calcium. This entire complex acts as a prothrombinase, converting prothrombin to thrombin. The triggering factor of this mechanism is XII, which is activated either by the contact of blood with a foreign surface, or by the contact of blood with subendothelium (collagen) and other components of connective tissue in damage to the walls of the vessels; or factor XII is activated by enzymatic cleavage (kallikreinom, plasmin, other proteases). In the external pathway for the formation of prothrombinase, the main factor is the tissue factor (factor III), which is expressed on cell surfaces with tissue damage and forms a complex with factor VIIa and calcium ions capable of translating factor X into factor Xa, which activates prothrombin. In addition, factor Xa retrogradely activates the complex of tissue factor and factor VIIa. Thus, the inner and outer paths are connected on the coagulation factors. The so-called "bridges" between these paths are realized through the mutual activation of factors XII, VII and IX. This phase lasts from 4 minutes 50 seconds to 6 minutes 50 seconds.

II phase - the formation of thrombin. In this phase, prothrombinase, together with coagulation factors V, VII, X and IV, transfers the inactive factor II (prothrombin) to the active factor IIa-thrombin. This phase lasts 2-5 s.

Phase III - formation of fibrin. Thrombin cleaves two peptides A and B from the fibrinogen molecule, converting it to fibrin monomer. The molecules of the latter are polymerized first into dimers, then into still soluble, especially acidic, oligomers, and eventually into fibrin-polymer. In addition, thrombin promotes the conversion of factor XIII to factor XIIIa. The latter in the presence of Ca ²⁺ changes fibrin-polymer from a labile, readily soluble fibrinolysin (plasmin) form into a slowly and boundedly soluble form, which forms the basis of the blood clot. This phase lasts 2-5 s.

In the process of formation of the hemostatic thrombus of thrombus formation from the site of damage to the vessel wall along the vascular bed does not occur, as it is impeded by the rapidly increasing anticoagulant potential of blood after activation and the activation of the fibrinolytic system.

Preservation of blood in a liquid state and regulation of the rates of interaction of factors in all phases of coagulation are largely determined by the presence of natural substances with anticoagulant activity in the bloodstream. The liquid state of blood provides a balance between the factors that induce blood clotting and the factors that prevent its development, the latter not being allocated to a separate functional system, since the realization of their effects is most often impossible without the participation of procoagulation factors. Therefore, the allocation of anticoagulants, preventing the activation of clotting factors and neutralizing active forms, is very arbitrary. Substances that have anticoagulant activity are constantly synthesized in the body and released into the bloodstream at a certain rate. These include ATIII, heparin, proteins C and S, the recently discovered inhibitor of the tissue clotting pathway - TFPI (inhibitor of the tissue factor factor VIIa-Ca ²⁺ complex ), α ₂ -macroglobulin, antitrypsin, etc. In the process of blood clotting, fibrinolysis from coagulation factors and other proteins, substances with anticoagulant activity are also formed. Anticoagulants have a pronounced effect on all phases of blood coagulation, so the study of their activity in the case of blood clotting disorders is very important.

After stabilization of fibrin, together with the shaped elements of the primary red blood clot forming, two main processes of the postcoagulable phase begin: spontaneous fibrinolysis and retraction, resulting in the formation of a hemostatically complete final thrombus. Normally, these two processes proceed in parallel. Physiological spontaneous fibrinolysis and retraction contribute to tightening the thrombus and performing hemostatic functions. In this process, an active part is taken by the plasmin (fibrinolytic) system and fibrinase (factor XIIIa). Spontaneous (natural) fibrinolysis reflects a complex reaction between the components of the plasmin system and fibrin. The plasmin system consists of four main components: plasminogen, plasmin (fibrinolysin), activators of fibrinolysis proenzymes and its inhibitors. Violation of the ratios of the components of the plasmin system leads to pathological activation of fibrinolysis.

In clinical practice, the study of the hemostasis system has the following objectives:

• Diagnosis of hemostasis system disorders;
• elucidation of the admissibility of surgical intervention with revealed violations in the hemostasis system;
• monitoring of anticoagulant treatment of direct and indirect action, as well as thrombolytic therapy.

Vascular-platelet (primary) hemostasis

Vascular-platelet, or primary, hemostasis is disturbed by changes in the vascular wall (dystrophic, immunoallergic, neoplastic and traumatic capillaropathies); thrombocytopenia; thrombocytopathy, a combination of capillaropathies and thrombocytopenia.

Vascular component of hemostasis

There are the following indicators characterizing the vascular component of hemostasis.

• Sample pinch. Collect the skin under the clavicle in the crease and make a pinch. In healthy people, no changes occur on the skin either immediately after the pinch, or after 24 hours. If the resistance of the capillaries is impaired, the petechiae or bruising appear on the place of the pinch, especially clearly visible after 24 hours.
• The sample is harnessed. Leaving 1.5-2 cm down from the fossa of the ulnar vein, draw a circle about 2.5 cm in diameter. On the shoulder, put a cuff of the tonometer and create a pressure of 80 mm Hg. The pressure is kept strictly at the same level for 5 minutes. In the circumscribed circle, all the petechiae appeared. In healthy individuals petechiae are not formed or there are no more than 10 (negative test of the tourniquet). When the resistance of the wall of the capillaries is impaired, the amount of petechiae increases sharply after the test.

Platelet component of hemostasis

The parameters characterizing the platelet component of hemostasis:

• Determination of the duration of bleeding by Duke.
• Counting the number of platelets in the blood.
• Determination of platelet aggregation with ADP.
• Determination of platelet aggregation with collagen.
• Determination of platelet aggregation with adrenaline.
• Determination of platelet aggregation with ristocetin (determination of von Willebrand factor activity).