Hemostasis

The hemostasis system (hemostasis) is a set of functional, morphological and biochemical mechanisms that ensure the maintenance of the liquid state of the blood, the prevention and stopping of bleeding, as well as the integrity of blood vessels.

In a whole organism, in the absence of any pathological effects, the liquid state of the blood is a consequence of the balance of factors that determine the processes

Coagulation and preventing their development. Violation of such a balance can be caused by many factors, however, regardless of the etiological causes, thrombus formation in the body occurs according to uniform laws with the inclusion of certain cellular elements, enzymes and substrates in the process.

In blood coagulation, two links are distinguished: cellular (vascular-platelet) and plasma (coagulation) hemostasis.

• Cellular hemostasis is understood as cell adhesion (i.e. the interaction of cells with a foreign surface, including cells of a different type), aggregation (the gluing of the same blood cells together), as well as the release of substances from formed elements that activate plasma hemostasis.
• Plasma (coagulation) hemostasis is a cascade of reactions involving blood coagulation factors, ending with the process of fibrin formation. The resulting fibrin is further destroyed by plasmin (fibrinolysis).

It is important to note that the division of hemostatic reactions into cellular and plasma is conditional, but it is valid in the in vitro system and significantly simplifies the choice of adequate methods and interpretation of the results of laboratory diagnostics of hemostasis pathology. In the body, these two links of the blood coagulation system are closely related and cannot function separately.

The vascular wall plays a very important role in the implementation of hemostasis reactions. Endothelial cells of blood vessels are capable of synthesizing and/or expressing on their surface various biologically active substances that modulate thrombus formation. These include von Willebrand factor, endothelial relaxing factor (nitric oxide), prostacyclin, thrombomodulin, endothelin, tissue-type plasminogen activator, tissue-type plasminogen activator inhibitor, tissue factor (thromboplastin), tissue factor pathway inhibitor, and some others. In addition, endothelial cell membranes carry receptors that, under certain conditions, mediate binding to molecular ligands and cells freely circulating in the bloodstream.

In the absence of any damage, the endothelial cells lining the vessel have thromboresistant properties, which helps maintain the liquid state of the blood. The thromboresistance of the endothelium is ensured by:

• contact inertia of the internal (facing the lumen of the vessel) surface of these cells;
• synthesis of a powerful inhibitor of platelet aggregation - prostacyclin;
• the presence of thrombomodulin on the endothelial cell membrane, which binds thrombin; in this case, 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 inner surface of blood vessels and fixation of the heparin-antithrombin III (ATIII) complex on the endothelium;
• the ability to secrete and synthesize tissue plasminogen activator, which ensures fibrinolysis;
• the ability to stimulate fibrinolysis through the protein C and S system.

Violation of the integrity of the vascular wall and/or changes in the functional properties of endothelial cells can contribute to the development of prothrombotic reactions - the antithrombotic potential of the endothelium is transformed into thrombogenic. The causes leading to vascular injury 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.), which under certain conditions can exhibit membrane-aggressive properties. Such a mechanism of vascular wall damage is characteristic of many diseases accompanied by a tendency to thrombus formation.

All cellular elements of the blood participate in thrombogenesis, but for platelets (unlike 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 implementation 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 release of mitogenic factors that promote hyperplasia of the vascular wall. When thrombogenesis is initiated, platelet activation occurs (i.e. activation of platelet glycoproteins and phospholipases, phospholipid metabolism, formation of secondary messengers, protein phosphorylation, arachidonic acid metabolism, actin and myosin interaction, Na ⁺ /H ⁺ exchange, expression of fibrinogen receptors and redistribution of calcium ions) and the induction of their adhesion processes, release and aggregation reactions; adhesion precedes the release and aggregation reaction of platelets and is the first step in the hemostatic process.

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

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

In addition to interaction with a foreign (including damaged vascular) surface, platelets are able to stick to each other, i.e. aggregate. Platelet aggregation is caused by substances of various natures, such as thrombin, collagen, ADP, arachidonic acid, thromboxane A ₂, prostaglandins G ₂ and H ₂, serotonin, adrenaline, platelet activating factor, and others. Exogenous substances (absent in the body), such as latex, can also act as proaggregants.

Both platelet adhesion and aggregation 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. The release reaction is induced by ADP, adrenaline, subendothelial connective tissue and thrombin. Initially, the contents of dense granules are released: ADP, serotonin, Ca ²⁺; more intense stimulation of platelets is necessary for the release of the contents of α-granules (platelet factor 4, β-thromboglobulin, platelet growth factor, von Willebrand factor, fibrinogen and fibronectin). Liposomal granules containing acid hydrolases are released only in the presence of collagen or thrombin. It should be noted that the factors released from platelets contribute to the closure of the vascular wall defect and the development of a hemostatic plug, however, with sufficiently pronounced vascular damage, further activation of platelets and their adhesion to the injured area of the vascular surface forms the basis for the development of a widespread thrombotic process with subsequent vascular occlusion.

In any case, the result of endothelial cell damage is the acquisition of procoagulant properties by the vascular intima, which is accompanied by the synthesis and expression of tissue factor (thromboplastin), the main initiator of the blood coagulation process. Thromboplastin itself does not have enzymatic activity, but can act as a cofactor of activated factor VII. The thromboplastin/factor VII complex is capable of activating both factor X and factor XI, thereby causing the generation of thrombin, which in turn induces further progression of both cellular and plasma hemostasis reactions.

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Mechanisms of hemostasis regulation

A number of inhibitory mechanisms prevent uncontrolled activation of coagulation reactions that could lead to local thrombosis or disseminated intravascular coagulation. These mechanisms include inactivation of procoagulant enzymes, fibrinolysis, and degradation of activated coagulation factors, primarily in the liver.

Inactivation of coagulation factors

Plasma protease inhibitors (antithrombin, tissue factor pathway inhibitor, a ₂ -macroglobulin, heparin cofactor II) inactivate coagulation enzymes. Antithrombin inhibits thrombin, factor Xa, factor Xla, and factor IXa. Heparin enhances 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, by binding to a receptor on endothelial cells called thrombomodulin, activates protein C. Activated protein C, together with protein S and phospholipids as cofactors, proteolyzes factors VIIIa and Va.

Fibrinolysis

Fibrin deposition and fibrinolysis must be balanced to maintain and limit the hemostatic clot during repair of the damaged vessel wall. The fibrinolytic system dissolves fibrin using plasmin, a proteolytic enzyme. Fibrinolysis is activated by plasminogen activators released from vascular endothelial cells. Plasminogen activators and plasma plasminogen bind to fibrin. Plasminogen activators catalytically cleave plasminogen, forming plasmin. Plasmin forms soluble fibrin degradation products, which are released into the circulation.

Plasminogen activators are divided into several types. Tissue plasminogen activator (tPA) of endothelial cells has low activity when free in solution, but its effectiveness increases when it interacts with fibrin in close proximity to plasminogen. The second type, urokinase, exists in single-chain and double-chain forms with different functional properties. Single-chain urokinase is unable to activate free plasminogen, but like tPA, it can activate plasminogen when interacting with fibrin. Trace concentrations of plasmin cleave single-chain into double-chain urokinase, which activates plasminogen in solution as well as bound to fibrin. Epithelial cells in excretory ducts (e.g., renal tubules, mammary ducts) secrete urokinase, which is a physiological activator of fibrinolysis in these channels. Streptokinase, a bacterial product not normally found in the body, is another potential plasminogen activator. Streptokinase, urokinase, and recombinant tPA (alteplase) are used therapeutically to induce fibrinolysis in patients with acute thrombotic diseases.

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Regulation of fibrinolysis

Fibrinolysis is regulated by plasminogen activator inhibitors (PAIs) and plasmin inhibitors, which slow fibrinolysis. PAI-1 is the most important PAI, released from vascular endothelial cells, inactivates tPA, urokinase, and activates platelets. The most important plasmin inhibitor is α-antiplasmin, which inactivates free plasmin released from the clot. Some α-antiplasmin can bind to the fibrin clot via factor XIII, preventing excessive plasmin activity within the clot. Urokinase and tPA are rapidly cleared by the liver, which is another mechanism to prevent excessive fibrinolysis.

Hemostatic reactions, the totality of which is commonly referred to as plasma (coagulation) hemostasis, ultimately lead to the formation of fibrin; these reactions are primarily realized by proteins called plasma factors.

International Nomenclature of Coagulation Factors

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 (withdrawn from use)
VII	Proconvertin*	4-6
VIII	Antihemophilic globulin A	7-8
IX	Christmas factor, plasma thromboplastin component,	15-30
	Antihemophilic factor B*
X	Stewart-Prower factor*	30-70
XI	Antihemophilic 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 prekallikrein	-
	Fitzgerald factor, high molecular weight kininogen	-

*Synthesized in the liver.

Phases of plasma hemostasis

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

Phase I - formation of prothrombinase or contact-kallikrein-kinin-cascade activation. Phase I is a multi-stage process resulting in accumulation of a complex of factors in the blood that can convert prothrombin into thrombin, which is why this complex is called prothrombinase. There are intrinsic and extrinsic pathways for prothrombinase formation. In the intrinsic pathway, blood clotting is initiated without the participation of tissue thromboplastin; plasma factors (XII, XI, IX, VIII, X), the kallikrein-kinin system, and platelets participate in the formation of prothrombinase. As a result of initiation of reactions of the intrinsic pathway, a complex of factors Xa with V is formed on the phospholipid surface (platelet factor 3) in the presence of ionized calcium. This entire complex acts as prothrombinase, converting prothrombin into thrombin. The trigger factor of this mechanism is XII, which is activated either as a result of blood contact with a foreign surface, or upon blood contact with subendothelium (collagen) and other components of connective tissue upon damage to the vessel walls; or factor XII is activated by enzymatic cleavage (by kallikrein, plasmin, other proteases). In the extrinsic pathway of prothrombinase formation, the main role is played by tissue factor (factor III), which is expressed on cell surfaces upon tissue damage and forms a complex with factor VIIa and calcium ions capable of converting factor X into factor Xa, which activates prothrombin. In addition, factor Xa retrogradely activates the complex of tissue factor and factor VIIa. Thus, the intrinsic and extrinsic pathways are connected at the coagulation factors. The so-called "bridges" between these pathways are realized through mutual activation of factors XII, VII and IX. This phase lasts from 4 min 50 sec to 6 min 50 sec.

Phase II - thrombin formation. In this phase, prothrombinase together with coagulation factors V, VII, X and IV converts inactive factor II (prothrombin) into active factor IIa - thrombin. This phase lasts 2-5 sec.

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

During the formation of a hemostatic thrombus, the spread of thrombus formation from the site of damage to the vessel wall along the vascular bed does not occur, as this is prevented by the rapidly increasing anticoagulant potential of the blood following coagulation and the activation of the fibrinolytic system.

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

After fibrin is stabilized, together with the formed elements that form the primary red thrombus, two main processes of the postcoagulation phase begin - spontaneous fibrinolysis and retraction, which ultimately lead to the formation of a hemostatically complete final thrombus. Normally, these two processes occur in parallel. Physiological spontaneous fibrinolysis and retraction contribute to the compaction of the thrombus and the performance of its hemostatic functions. The plasmin (fibrinolytic) system and fibrinase (factor XIIIa) take an active part in this process. 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 ratio of the components of the plasmin system leads to pathological activation of fibrinolysis.

In clinical practice, the study of the hemostasis system pursues the following goals:

• diagnostics of hemostasis system disorders;
• determining the admissibility of surgical intervention in the event of identified disorders in the hemostasis system;
• monitoring treatment with direct and indirect anticoagulants, as well as thrombolytic therapy.

Vascular-platelet (primary) hemostasis

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

Vascular component of hemostasis

There are the following indicators that characterize the vascular component of hemostasis.

• Pinch test. The skin is gathered under the collarbone into a fold and pinched. In healthy people, no changes occur on the skin either immediately after the pinch or after 24 hours. If the capillary resistance is impaired, petechiae or bruises appear at the site of the pinch, which are especially clearly visible after 24 hours.
• Tourniquet test. Stepping back 1.5-2 cm down from the fossa of the cubital vein, draw a circle approximately 2.5 cm in diameter. Place the cuff of the tonometer on the shoulder and create a pressure of 80 mm Hg. Maintain the pressure strictly at one level for 5 minutes. All petechiae that appear in the outlined circle are counted. In healthy individuals, petechiae do not form or there are no more than 10 of them (negative tourniquet test). If the resistance of the capillary wall is impaired, the number of petechiae increases sharply after the test.

Platelet component of hemostasis

Indicators characterizing the platelet component of hemostasis:

• Determination of bleeding duration according to 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).