BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 14. BIOCHEMISTRY OF BLOOD

III. Blood Clotting System

When a Blood vessel is injured, a cascade of reactions is triggered, resulting in The formation of a blood clot (thrombus) that prevents bleeding. Platelets and a series of Plasma Proteins play the primary role in Blood Coagulation.

There are 3 stages in arresting blood loss. The First stage involves the constriction of the blood vessel. Then, platelets adhere to the site of injury and, layering upon one another, form a platelet plug (white thrombus). The white thrombus is fragile and can only seal a small blood vessel. In the Third Stage, the soluble plasma protein fibrinogen is converted into the insoluble protein fibrin, which is deposited between the platelets to form a sturdy fibrin thrombus. Such a thrombus incorporates erythrocytes and is therefore called a red thrombus.

The formation of a fibrin thrombus is preceded by a cascade of proteolytic reactions leading to the activation of the enzyme Thrombin, which in turn converts fibrinogen into fibrin. All proteins involved in blood clotting are called clotting factors. They are synthesized primarily in The Liver and Blood Cells as inactive precursors; they are designated by Roman numerals but also have trivial names (Table 14-1). Most of these proteins are activated within the enzymatic cascade of blood coagulation. The active forms of these proteins are designated by the same Roman numerals with The addition of the letter "a".

Class="center">Table 14-1. Main Functions and plasma concentrations of blood coagulation factors

* Contains carboxyglutamic acid residues required for the formation of membrane enzyme complexes in the procoagulant blood clotting pathway.

A. Formation of the fibrin thrombus

The formation of a fibrin thrombus begins with The conversion of the soluble plasma protein fibrinogen into insoluble fibrin.

Fibrinogen (factor I) — a glycoprotein with a Molecular Weight of 340 kD. It is synthesized in the liver and is present in Blood Plasma at a concentration of 8.02 — 12.9 µmol/L (2 — 4 g/L). The fibrinogen molecule consists of six polypeptide chains linked together by Disulfide Bonds. The polypeptide chain COMPOSITION OF THE fibrinogen molecule is designated as Aα₂, Bβ₂, y₂. Capital letters correspond to the regions that are cleaved by thrombin during the conversion of fibrinogen to fibrin. Fragments A in the Aα chains and B in the Bβ chains contain A large number of aspartate and glutamate residues. This creates a strong negati

ve charge at the N-termini of fibrinogen molecules and prevents their aggregation.

The fibrinogen molecule consists of three globular domains: one at each end of the molecule (D domains) and one in the middle (E domain). The domains are separated from each other by rod-shaped segments of polypeptide chains. The N-terminal fragments A and B of the Aα and Bβ chains protrude from the central E domain (Fig. 14-8).

Fig. 14-8. Structure of fibrinogen. Fibrinogen consists of six polypeptide chains: Aα₂, Bβ₂, and y₂. A, B — negatively charged fragments that prevent fibrinogen molecules from aggregating. D, E — globular domains of the fibrinogen molecule. The domains are separated by rod-shaped polypeptide chain segments. The N-terminal regions of fragments A and B of the Aα₂ and Bβ₂ chains protrude from the central globular domain E.

Four stages can be distinguished in the formation of a fibrin thrombus.

1. Conversion of fibrinogen to fibrin monomer.

First, fibrinogen molecules are stripped of the negatively charged fragments A and B, resulting in the formation of fibrin monomers. The conversion of fibrinogen (factor I) to fibrin (factor Ia) is catalyzed by the enzyme thrombin (factor IIa). In each fibrinogen molecule, thrombin hydrolyzes four arginyl-glycyl peptide bonds, two of which connect fragments A to the α-chain, and the other two connect fragments B to the β-chain in the Aα₂ and Bβ₂ chains of fibrinogen. The fibrin monomer formed from fibrinogen has the composition (α, β, y)₂.

2. Formation of an insoluble fibrin gel.

In the second stage, an insoluble polymeric fibrin clot — the fibrin gel — is formed. As a result of the conversion of fibrinogen to fibrin monomer, binding sites for the D domains are exposed in the E domain. Furthermore, the E domain contains aggregation sites that form only after partial proteolysis of fibrinogen by thrombin, whereas the D domain bears constitutive aggregation sites. Primary aggregation of fibrin molecules occurs through the interaction of binding sites on the E domain of one molecule with complementary regions on the D domains of other molecules. Thus, non-covalent bonds are formed between the domains of fibrin monomer molecules. During the "self-assembly" of the fibrin gel, double-stranded protofibrils are initially formed, in which the fibrin molecules are staggered relative to each other by 1/2 of their length. Once the protofibrils reach a certain critical length, their lateral association begins, leading to the formation of thick fibrin fibers (Fig. 14-9). The resulting fibrin gel is fragile because the fibrin molecules within it are linked by non-covalent bonds.

Fig. 14-9. Formation of fibrin gel. Fibrinogen, having its negatively charged fragments (fibrinopeptides 2A and 2B) cleaved by thrombin, is converted into fibrin monomer. Through the interaction of complementary Regions of the E and D domains of the fibrin monomer, linear followed by lateral polymerization of the molecules occurs, resulting in the formation of a fibrin gel.

3. Stabilization of the fibrin gel.

The fibrin gel is stabilized as a result of the formation of amide bonds between Lysine residues of one fibrin molecule and glutamine residues of another. The transamidation reaction is catalyzed by the enzyme transglutaminase (factor XIIIa) (Fig. 14-10). Factor XIII is activated by partial proteolysis via thrombin.

Fig. 14-10. Formation of the amide bond between fibrin molecules.

Transglutaminase also forms amide bonds between fibrin and fibronectin, a glycoprotein of the Extracellular matrix and blood plasma (see Chapter 15). This anchors the blood clot at the site of vascular injury.

4. Fibrin clot retraction.

Clot contraction (retraction) is driven by platelet Actomyosin, a contractile protein called thrombosthenin, which exhibits ATPase activity. Thrombosthenin also participates in platelet activation and aggregation. Clot retraction prevents complete vascular occlusion, thereby preserving blood flow.

The Mechanism of thrombus formation comprises three functionally distinct stages: the procoagulant pathway, the contact pathway, and the anticoagulant phase, which prevents the excessive spread of the clot.

B. Procoagulant pathway of blood coagulation

Stopping bleeding from capillaries and Blood Vessels requires the rapid formation of a robust hemostatic plug to prevent blood loss. This is achieved through an enzymatic cascade featuring signal Amplification mechanisms at multiple stages.

The procoagulant pathway occupies a central role in blood coagulation (Fig. 14-11).

Fig. 14-11. The procoagulant pathway of blood coagulation. activation of blood clotting factors; —> Fig. positive feedback activation of blood clotting factors;membrane phospholipid component of enzyme complexes. Activator proteins are enclosed in boxes. 1, 2 — factor VIIa of the VIIa-TF-Ca²⁺ membrane complex activates factors IX and X; 3 — factor IXa of the IXa-VIIIa-Ca²⁺ membrane complex activates factor X; 4, 5 — factor Xa of the Xa-Va-Ca²⁺ membrane complex converts prothrombin (factor II) into thrombin (factor IIa) and activates factor VII; 6–10 — thrombin (factor IIa) converts soluble fibrinogen into insoluble fibrin, and activates factors VII, VIII, V, and XIII.

Circulating blood contains zymogens of Proteolytic Enzymes: factor II (prothrombin), factor VII (proconvertin), factor IX (Christmas factor), and factor X (Stuart factor). Factors Va (accelerin) and VIIIa (antihemophilic factor), which are present in the blood, as well as the membrane protein tissue factor (TF, factor III), act as activator proteins for these enzymes (Table 14-1).

Upon vascular injury, the enzyme cascade is triggered, leading to the sequential formation of three Cell membrane-associated phospholipid enzyme complexes. Each complex consists of a proteolytic enzyme, an activator protein, and Ca²⁺ ions: VIIa-TF-Ca²⁺, IXa-VIIIa-Ca²⁺ (tenase), and Xa-Va-Ca²⁺ (prothrombinase) (Fig. 14-12). The Xa-Va-Ca²⁺ complex (prothrombinase complex) activates prothrombin (factor II). The enzymatic cascade culminates in the generation of fibrin monomers and the subsequent formation of a blood clot.

Fig. 14-12. Proteolytic activation of prothrombin by factor Xa of the prothrombinase complex. carboxyglutamic acid residues; dashed arrows indicate the positions of peptide bonds hydrolyzed within the prothrombin molecule. The prothrombin molecule consists of a single polypeptide chain, whereas thrombin, generated via partial proteolysis of prothrombin, consists of two polypeptide chains linked by a single disulfide bond.

Three core mechanisms drive the activation of the cascade enzymes: partial proteolysis, interaction with activator proteins, and interaction with modified cell membranes.

Activation by partial proteolysis. All Enzymes of the procoagulant pathway are Serine proteases synthesized in the liver as inactive zymogens, in which form they circulate in the blood. Upon triggering of the thrombogenic signal, these zymogens (factors VII, IX, X, and II) are converted into active enzymes via partial proteolysis.

Thrombin (factor IIa) is a glycoprotein with a molecular weight of 39 kDa. It is generated in the blood from its inactive precursor, prothrombin. Prothrombin is synthesized in the liver, has a molecular weight of 70 kDa, and contains y-Carboxyglutamic acid residues. The normal concentration of this protein in the blood is 0.1 g/L. It binds to the Xa-Va-Ca²⁺ membrane enzyme complex by interacting, on the one hand, through its y-carboxyglutamate residues with Ca²⁺ ions, and on the other hand, directly with the Va activator protein. This establishes optimal steric conditions for the enzymatic reaction. Factor Xa hydrolyzes two peptide bonds in the prothrombin molecule, yielding a thrombin molecule composed of two chains—light and heavy—linked by a single disulfide bond (Fig. 14-12). The thrombin molecule lacks y-carboxyglutamate residues and dissociates from the prothrombinase complex. Through partial proteolysis, thrombin converts fibrinogen into fibrin and activates factors VII, VIII, V, and XIII.

Thrombin performs several vital physiological functions: it acts as an enzyme in the procoagulant and contact pathways of blood coagulation, initiates the anticoagulant phase reactions, induces platelet aggregation, and exerts mitogenic effects involved in cell proliferation and repair.

Factors V and VIII are also activated by partial proteolysis, converting into factors Va and VIIIa, respectively. This activation alters their conformation and increases their affinity for membrane Phospholipids and the enzymes they activate.

Interaction of activator proteins with proteolytic enzymes. Tissue factor, factor Va, and factor VIIIa possess binding sites for membrane phospholipids and for the enzymes VIIa, IXa, and Xa, respectively. Binding to these activator proteins induces conformational changes that enhance the catalytic activity of the enzymes.

Tissue factor (factor III) is a complex composed of a protein and phosphatidylserine. The protein moiety of tissue factor (apoprotein III) is exposed On the surface of various cells (Brain, Lungs, liver, Spleen, etc.) and is associated with Plasma Membrane phosphatidylserine. However, apoprotein III appears on The surface of blood-contacting cells (endothelial cells and monocytes) only under specific conditions: upon vascular injury and/or disruption of the normal Asymmetry of their Plasma Membranes. Tissue factor does not require proteolytic activation.

Factor V and factor VIII are multidomain proteins circulating in the blood. Factor V is synthesized in the liver, whereas factor VIII is produced by endothelial cells. Both factors are activated by thrombin-mediated partial proteolysis. In blood plasma, factor VIII circulates in a complex with von Willebrand factor (a platelet factor). In this complex, von Willebrand factor stabilizes factor VIII, protecting it from degradation by protein C, a proteolytic enzyme of the anticoagulant phase.

Interaction of enzyme complexes with cell membranes occurs via Ca²⁺-dependent mechanisms. All procoagulant zymogens (II, VII, IX, X) contain y-carboxyglutamic acid residues, which are formed through post-translational modification of these proteins in The Endoplasmic reticulum of hepatocytes.

The y-carboxyglutamic acid residues in factors VIIa, IXa, and Xa facilitate the Ca²⁺-mediated binding of these enzymes to negatively charged phospholipids of cell membranes. In the absence of Ca²⁺ ions, blood coagulation cannot occur.

The Role of vitamin K in the carboxylation of glutamic acid residues in proenzymes of the procoagulant blood coagulation pathway. The carboxylation of glutamic acid residues in proenzymes of the procoagulant pathway is catalyzed by carboxylase, whose coenzyme is the reduced form of vitamin K (naphthoquinone)—vitamin K dihydroquinone (see Section 3).

Upon entering the body, vitamin K (naphthoquinone) is reduced in the liver by an NADPH-dependent vitamin K reductase to form vitamin K dihydroquinone. During the carboxylation of glutamic acid residues in the procoagulant pathway proenzymes, the dihydroquinone is oxidized and epoxidized to yield vitamin K 2,3-epoxide. The epoxide is regenerated back into vitamin K dihydroquinone as follows: first, vitamin K 2,3-epoxide is reduced to vitamin K by a thiol-dependent epoxide reductase, whose coenzyme is a thioredoxin-like protein. Next, the resulting vitamin K is reduced to vitamin K dihydroquinone by a thiol-dependent vitamin K reductase. The hydrogen donor in this reaction, as in the previous one, is a thioredoxin-like protein (Fig. 14-13).

Fig. 14-13. The role of vitamin K in the post-translational carboxylation of glutamic acid. 1 — reduction of exogenous vitamin K by NADPH-dependent reductase; 2 — y-carboxylation of glutamic acid residues in factors II, VII, IX, X, and protein C by vitamin K-dependent carboxylase, accompanied by The oxidation of dihydroquinone to form vitamin K 2,3-epoxide; 3 — reduction of 2,3-epoxide by thiol-dependent vitamin K reductase; 4 — reduction of vitamin K by thiol-dependent vitamin K reductase; a) and b) — reduced and oxidized forms of the thioredoxin-like protein.

Vitamin K deficiency leads to impaired carboxylation of procoagulant pathway proenzymes and is accompanied by a tendency to bleed, as well as subcutaneous and internal hemorrhages.

The structural analogs of vitamin K, dicumarol and warfarin, inhibit the thiol-dependent enzymes vitamin K 2,3-epoxide reductase and vitamin K reductase, thereby inhibiting blood coagulation (Fig. 14-14). These drugs are used clinically to prevent thrombosis.

Fig. 14-14. Structural analogs of vitamin K: dicumarol and warfarin.

Initiation of the procoagulant pathway reaction cascade. Enzymatic membrane complexes of the procoagulant pathway are formed only in the presence of tissue factor and negatively charged phospholipids on the outer surface of the cellular plasma membrane. The transverse asymmetry of plasma membranes is determined, in particular, by the predominance of neutral phospholipids (phosphatidylcholine and sphingomyelin) in the outer layer and negatively charged phospholipids (phosphatidylinositol bisphosphate and phosphatidylserine) in the inner layer (see Section 5). A specialized enzymatic system ensures transmembrane transport and a distribution of phospholipids in cell membranes such that, under normal conditions, the outer surface of cellular plasma membranes is uncharged (see Section 5).

When the transverse asymmetry of platelet and endothelial cell membranes is disrupted, negatively charged (thrombogenic) patches form on their surface, and tissue factor apoprotein III is exposed. Such disturbances can arise from physical trauma. In this case, tissue factor and the inner surface of The Cell membrane become accessible to plasma factors of the procoagulant pathway. Furthermore, the interaction of signaling molecules that trigger thrombogenesis with endothelial cell and platelet receptors activates Ca²⁺-dependent regulatory systems (see Section 5). Ultimately, this leads to an increase in cytoplasmic Ca²⁺, which inhibits the ATP-dependent aminophospholipid translocase. This enzyme plays a crucial role in maintaining membrane transverse asymmetry by transferring phosphatidylserine from the outer lipid layer to the inner one. A decrease in aminophospholipid translocase activity leads to an increased content of phosphatidylserine in the outer layer of the Cell Membrane and the formation of negatively charged areas required for the assembly of membrane enzyme complexes. In addition, as a result of this plasma membrane structural disruption, tissue factor is exposed on its outer surface, forming the first enzyme complex of the blood coagulation procoagulant pathway: VII-TF-Ca²⁺.

The activation of enzymes within each complex results from the interaction of all its components. While factors IX, X, and II require activation, factor VII possesses low proteolytic activity. Factor VII within the VII-TF-Ca²⁺ membrane complex partially proteolyzes and activates factors IX and X. The active factors IXa and Xa become incorporated into the formation of the IXa-VIIIa-Ca²⁺ and Xa-Va-Ca²⁺ membrane complexes. Here, factor Xa proteolytically activates factor V, while the prothrombinase complex not only converts prothrombin to thrombin but also activates factor VII, whose proteolytic activity within the VIIa-TF-Ca²⁺ complex is 10,000 times higher than in the VII-TF-Ca²⁺ complex.

The thrombin generated as a result of the reaction cascade catalyzes the partial proteolysis of fibrinogen and factor XIII and, via a positive feedback loop, proteolytically activates factors V, VII, and VIII.

Two signal amplification mechanisms operate during the coagulation process: a reaction cascade in which each enzymatic step provides signal amplification, and positive feedback loops.

B. The contact pathway of blood coagulation

The contact pathway of blood coagulation begins with the interaction of the factor XII proenzyme with the damaged endothelial surface of the vessel wall. This interaction activates factor XII and initiates the formation of the contact phase coagulation membrane complexes. These complexes contain the enzymes kallikrein, factors XIa (plasma thromboplastin antecedent) and XIIa (Hageman factor), as well as an activator protein—high-molecular-weight kininogen (HMWK) (Fig. 14-15).

Fig. 14-15. Scheme of the procoagulant ( extrinsic ) and contact ( intrinsic ) pathways of blood coagulation. Designations: HMWK — high-molecular-weight kininogen; TF — tissue factor; —> — activation of blood coagulation factors; activation of coagulation factors via a positive feedback loop; — membrane phospholipid component of enzyme complexes. All enzymes of the blood coagulation membrane complexes are proteases and are activated by partial proteolysis. 1 — factor XII, activated as a result of contact with the subendothelium, converts prekallikrein into kallikrein; 2 — kallikrein of the kallikrein-HMWK membrane complex activates factor XII; 3 — factor XIIa activates factor XI; 4 — factor XIIa, activated by partial proteolysis, converts prekallikrein into kallikrein via positive feedback; 5 — factor XIa of the XIa-HMWK membrane complex activates factor IX; 6 — factor IXa of the IXa-VIIIa-Ca²⁺ membrane complex activates factor X; 7, 8 — factor VIIa of the VIIa-TF-Ca²⁺ membrane complex activates factors IX and X; 9 — factor Xa of the prothrombinase complex activates factor II; 10, 11 — thrombin (factor II) converts fibrinogen to fibrin and activates factor XIII; 12 — factor XIIIa catalyzes the formation of amide bonds in the fibrin gel.

Factor XII is a proenzyme that circulates in the blood. It is sequentially activated in two ways: first, as a result of a conformational change upon interacting with the negatively charged surface of the damaged endothelium, and second, via partial proteolysis by the kallikrein-HMWK membrane complex.

High-molecular-weight kininogen is an activator protein in the XIIa-HMWK, XIa-HMWK, and kallikrein-HMWK membrane enzyme complexes. HMWK is a plasma glycoprotein synthesized in the liver with a molecular weight of 120 kDa. It mediates the interaction of the proteolytic enzymes of the contact coagulation phase with subendothelial Collagen and also serves as a component of the kallikrein-kinin system.

Kallikrein is a serine protease whose substrates, In addition to factor XII, include plasma proteins plasminogen (a proenzyme involved in fibrin dissolution) and kininogens of low (69 kDa) and high (120 kDa) molecular weight. Partial proteolysis of kininogens yields regulatory Peptides called kinins. Specifically, bradykinin, a potent vasodilator, increases vascular permeability and causes the destruction of endothelial cell membranes.

Upon contact with the vascular subendothelium, factor XII is activated. Active factor XIIa, in a complex with HMWK, proteolytically converts prekallikrein—bound to the membrane via HMWK—into kallikrein. The kallikrein-HMWK membrane complex, through positive feedback, activates factor XII via partial proteolysis. In doing so, factor XII acquires maximum enzymatic activity and, via positive feedback, activates HMWK-bound prekallikrein. Furthermore, factor XIIa generated by partial proteolysis proteolytically activates factor XI, and factor XIa within the XIa-HMWK enzyme complex activates factor IX. Factor IXa of the IXa-VIIIa-Ca²⁺ membrane complex activates factor X, which, as part of the prothrombinase complex, activates prothrombin.

The reaction cascade leading to the formation of thrombin can proceed via two pathways—the procoagulant (extrinsic) and the contact (intrinsic) pathways (Fig. 14-15). Initiating the extrinsic pathway requires the appearance of tissue factor on the outer surface of The Plasma Membrane of cells in contact with blood. The intrinsic pathway begins with the activation of factor XII upon its contact with the damaged vascular endothelial surface and the mutual activation of the prekallikrein and factor XII enzymes.

Thus, in both the procoagulant and contact pathways of blood coagulation, the sequential formation of membrane enzyme complexes leads to the activation of factor X and the formation of prothrombinase. The stages common to both coagulation pathways are referred to as the common pathway of blood coagulation. Currently, The concepts of intrinsic and extrinsic pathways are considered quite conventional, as it has become clear that the VIIa-TF-Ca²⁺ complex activates factor IX more efficiently than factor X, and factor VII is activated by factor IXa, albeit much slower compared to activation by factor Xa. Consequently, it can be suggested that the blood coagulation cascade proceeds predominantly in a linear sequence rather than via two relatively independent pathways. The contact pathway is clearly not strictly essential for initiating coagulation; it likely serves to couple the BLOOD COAGULATION SYSTEM with various bodily regulatory systems, such as the kallikrein-kinin system and the fibrinolytic enzyme system responsible for dissolving blood clots.

Healthy human blood clots in vitro within 5–10 min. The formation of the prothrombinase complex takes 5–8 min, prothrombin activation takes 2–5 s, and the conversion of fibrinogen to fibrin takes 2–5 s.

Decreased blood coagulability. Decreased blood coagulability is observed in disorders accompanied by recurring bleeding. Hemophilias are Hereditary diseases characterized by an increased bleeding tendency. The cause of these hemorrhages (whether spontaneous or trauma-induced) is a hereditary deficiency of blood coagulation proteins.

Hemophilia A (classic hemophilia) is caused by a mutation in the factor VIII Gene located on the X chromosome. Classic hemophilia accounts for 80% of all hemophilia cases. Hemophilia B is less common and results from a genetic defect in factor IX.

The factor VIII gene defect is inherited as a recessive trait, which is why this form of hemophilia affects exclusively males. The condition is characterized by subcutaneous, intramuscular, and intra-articular hemorrhages, which can sometimes be life-threatening. A factor VIII deficiency occurs in approximately one out of every 10,000 newborns. Patients are treated with factor VIII preparations derived from donor blood or produced via Introduction/32.html">Genetic Engineering.

G. The Anticoagulant Blood System

Physiological blood coagulation inhibitors play a vital role in maintaining hemostasis by keeping blood in a fluid state and preventing the thrombus from spreading beyond the injured site of the blood vessel.

Thrombin, generated during the procoagulant and contact pathways of blood coagulation, is washed out of the thrombus by the bloodstream. It can either be inactivated through interaction with coagulation Enzyme Inhibitors or trigger the anticoagulant phase, which suppresses thrombus formation.

Anticoagulant phase. Blood clotting must be limited not only spatially, but also temporally. The anticoagulant phase restricts the lifespan of active factors in the blood and is initiated by thrombin itself. Consequently, thrombin acts in a dual manner: as the final enzyme of the coagulation cascade, it accelerates blood clotting, while simultaneously inhibiting it by inducing the formation of anticoagulant enzyme complexes on the uninjured vascular endothelium. This stage represents a short cascade of reactions involving thrombin, the activating protein thrombomodulin (Tm), the vitamin K-dependent serine protease protein C, the activating protein S, and factors Va and VIIIa (Fig. 14-16).

Fig. 14-16. The anticoagulant phase. Tm — thrombomodulin; C — protein C; Ca — active protein C; S — protein S; bold lines — membrane-bound complex. 1 — thrombin (IIa) forms a membrane complex with the protein thrombomodulin (Tm); 2 — thrombin as part of the IIa-Tm-Ca²⁺ membrane complex activates protein C; 3 — activated protein C as part of the Ca-S-Ca²⁺ enzymatic membrane complex hydrolyzes 2 peptide bonds in factors Va and VIIIa, converting them into inactive peptides.

The cascade of reactions in the anticoagulant phase sequentially forms two membrane complexes: IIa-Tm-Ca²⁺ and Ca-S-Ca²⁺.

Thrombomodulin — an integral membrane protein of endothelial cells. It does not require proteolytic activation and functions as a thrombin cofactor. Thrombin acquires The ability to activate protein C only after interacting with thrombomodulin; moreover, thrombomodulin-bound thrombin loses its ability to convert fibrinogen into fibrin and no longer activates factor V or platelets.

Protein C — a zymogen containing y-carboxyglutamate residues. Thrombin within the IIa-Tm-Ca²⁺ membrane complex activates protein C via partial proteolysis. Activated protein C (Ca) forms a membrane-bound Ca-S-Ca²⁺ complex with the activating protein S. Within this complex, Ca hydrolyzes two peptide bonds in factors Va and VIIIa, thereby inactivating them. Under the action of the Ca-S-Ca²⁺ complex, 80% of The activity of factors VIIIa and Va is lost within 3 minutes. Thus, through positive feedback, thrombin not only accelerates its own production but also inhibits the blood clotting process by activating protein C.

Hereditary deficiencies of proteins C and S lead to a reduced rate of inactivation of factors VIIIa and Va and are associated with thrombotic disease. A mutation in the factor V gene that results in the synthesis of a protein C-resistant factor V variant also promotes thrombogenesis.

The anticoagulant phase suppresses the blood coagulation cascade, while coagulation enzyme inhibitors neutralize active enzymes within the bloodstream.

Blood coagulation enzyme inhibitors. Physiological inhibitors of coagulation enzymes restrict the spread of a thrombus to the site of vascular injury. The plasma protein antithrombin III — is the most potent inhibitor of blood coagulation, accounting for approximately 80–90% of the blood's anticoagulant activity. It inactivates a range of blood serine proteases: thrombin, factors IXa, Xa, XIIa, kallikrein, plasmin, and urokinase. Antithrombin III does not inhibit factor VIIa nor does it affect factors incorporated into membrane complexes; instead, it targets free enzymes in blood plasma, preventing the propagation of thrombosis throughout the Circulation.

The interaction between antithrombin and coagulation enzymes is accelerated in the presence of heparin. Heparin is a heteropolysaccharide synthesized by mast cells. Upon binding to heparin, antithrombin III undergoes a conformational change that increases its affinity for blood serine proteases. Following the formation of the antithrombin III-heparin-enzyme complex, heparin dissociates from it and can bind to other antithrombin molecules.

Hereditary antithrombin III deficiency manifests in early life with life-threatening thrombosis and vascular embolism.

α₂-Macroglobulin forms complexes with blood serine proteases. In such complexes, their active centers are not completely blocked, allowing them to interact with small-sized substrates. However, high-molecular-weight substrates, such as fibrinogen, become inaccessible to the action of proteases bound in the α₂-macroglobulin-thrombin complex.

Anticonvertin (tissue inhibitor of the extrinsic coagulation pathway) is synthesized in the vascular endothelium. It specifically binds to the Tf-VIIa-Ca²⁺ enzyme complex, after which it is taken up by the liver and degraded.

α₁-Antitrypsin inhibits thrombin, factor XIa, and kallikrein, although it is not considered a major inhibitor of blood coagulation factors. α₁-Antitrypsin primarily inhibits pancreatic and leukocyte proteases, collagenase, renin, and urokinase at THE TISSUE LEVEL.

Peptides generated during the proteolytic activation of zymogens and pro-factors also exhibit marked anticoagulant properties, though their MECHANISM OF ACTION remains to be fully elucidated.

D. The Role of Platelets in Hemostasis

The ability of platelets to adhere to the damaged surface of the vessel wall (adhesion) and to each other (aggregation), bind to fibrin to form a platelet plug, and secrete hemostatic factors at the site of vascular injury defines their crucial role in hemostasis.

Circulating Blood Platelets are disk-shaped and do not adhere to uninjured vascular endothelium. Adhesion and aggregation are prevented by mutual electrostatic repulsion between platelets and the intact endothelium, as well as by prostacyclin (PG I₂). The mechanism of Action of Certain platelet aggregation Inducers and repressors is illustrated in Fig. 14-17.

Prostacyclin is synthesized from arachidonic acid in the vascular endothelium and released into the bloodstream (see Section 8). The Synthesis and Secretion of prostacyclin by endothelial cells are stimulated by thrombin, histamine, angiotensin II, and kallikrein. It exerts its effects via the adenylate cyclase signal Transduction system (see Section 5). The binding of prostacyclin to its receptor activates protein kinase A. Active protein kinase A phosphorylates and thereby activates

Ca²⁺-ATPase and Ca²⁺-translocase. This leads to a decrease in cytoplasmic Ca²⁺ levels in platelets, helping them maintain their discoid shape and reducing their aggregation tendency.

Platelet activation is accompanied by the appearance of negatively charged patches formed by phosphatidylserine on the surface of the plasma membrane.

The main inducers of platelet activation and aggregation are von Willebrand factor, collagen, thrombin, and ADP.

Von Willebrand factor is a glycoprotein present in blood plasma, vascular endothelium, and platelet α-granules. Upon vessel wall damage, collagen, the basement membrane, and subendothelial myocytes interact with platelets via von Willebrand factor. The platelet plasma membrane contains several types of receptors for this factor. By interacting with these receptors, von Willebrand factor affects platelets through the Inositol phosphate signal transduction system (see Section 5). Ultimately, this leads to an increase in cytoplasmic Ca²⁺ levels in platelets and the formation of the calmodulin–4Ca²⁺–Myosin kinase complex. The enzyme myosin kinase within this complex phosphorylates the contractile protein myosin, which interacts with Actin to form actomyosin (thrombostenin). As a result, platelets acquire a spiky, spherical shape that facilitates their interaction with one another and with the surface of the damaged endothelium.

A decrease in von Willebrand factor concentration, a reduction in the number of its receptors, or alterations in their structure lead to impaired platelet adhesion and aggregation, which is accompanied by a bleeding tendency. This is observed in Bernard–Soulier syndrome, caused by a deficiency of the von Willebrand factor receptor glycoprotein Ia in platelets, and in von Willebrand disease, resulting from a deficiency of von Willebrand factor.

The most important primary inducers of platelet activation are thrombin and collagen. The interaction of these proteins with specific receptors on the platelet plasma membrane leads to the mobilization of Ca²⁺ from the dense tubular system into the Cytoplasm, which ultimately triggers their adhesion and aggregation.

Collagen induces the activation of phospholipase A2 in platelets, which releases arachidonic acid from their membrane phospholipids. Arachidonic acid serves as a substrate for the enzyme cyclooxygenase (COX). As a result of the reaction catalyzed by cyclooxygenase, cyclic endoperoxides—prostaglandin G₂ (PG G₂) and prostaglandin H₂ (PG H₂)—are formed. Under the action of thromboxane synthetase, these Prostaglandins are converted into thromboxane (see Section 8). Thromboxane A₂ decreases cAMP levels and, by activating phospholipase C, accelerates the release of Ca²⁺ from the dense tubular system (Fig. 14-17).

Fig. 14-17. Mechanism of action of certain inducers and an inhibitor of platelet aggregation. The interaction of prostacyclin with the R₁ receptor causes the activation of adenylate cyclase, an increase in cAMP concentration, and consequently a decrease in cytosolic Ca²⁺ concentration in platelets. The interaction of collagen with the H₄ receptor leads to the activation of phospholipase A₂, which hydrolyzes cell membrane phospholipids to yield arachidonic acid. Arachidonic acid is converted by the enzyme cyclooxygenase into prostaglandins PG G₂ and PG H₂, from which thromboxane A₂ is formed under the action of thromboxane synthetase. Thromboxane A₂ is secreted by activated platelets. Thromboxane and thrombin, by binding to their respective R₃ and R₂ receptors, activate phospholipase C. Phospholipase C hydrolyzes the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to form 1,4,5-inositol trisphosphate and 1,2-diacylglycerol. Inositol trisphosphate accelerates the influx of Ca²⁺ from the dense tubular system into the Cell Cytoplasm, where it binds to calmodulin. In the calmodulin–4Ca²⁺–myosin kinase complex, active myosin kinase phosphorylates myosin. Phosphorylated myosin interacts with actin to form the contractile protein actomyosin, which causes shape change, adhesion, and aggregation of platelets. The protein kinase C–phosphatidylserine–DAG–Ca²⁺ complex phosphorylates the protein pleckstrin. Phosphorylated pleckstrin triggers the release of the contents of platelet dense and α-granules: ADP, GDP, Ca²⁺, serotonin, von Willebrand factor, and β-thrombomodulin. R₁, R₂, R₃ are specific Membrane Receptors; AC is adenylate cyclase; G₁, G₂, G₃, G₄ are G proteins; PLC is phospholipase C; PLA₂ is phospholipase A₂; PIP₂ is phosphatidylinositol 4,5-bisphosphate; IP₃ is 1,4,5-inositol trisphosphate; DAG is 1,2-diacylglycerol; PKC is protein kinase C; COX is cyclooxygenase; TxA₂ is thromboxane A₂; TxA₂ synthetase is thromboxane synthetase.

Thrombin interacts with a specific receptor—an integral protein possessing 7 transmembrane domains. Thrombin activates the receptor through partial proteolysis by cleaving off its N-terminal peptide located on the outer plasma surface of the platelet. Therefore, unlike other activators, thrombin acts catalytically, and a single thrombin molecule can activate multiple receptors. Signal transmission is mediated via the inositol phosphate system, resulting in an increased Ca²⁺ concentration within the platelet and the activation of protein kinase C. The resulting calmodulin–4Ca²⁺–myosin kinase complex phosphorylates myosin, whose interaction with actin leads to platelet shape change, adhesion, and aggregation. Furthermore, protein kinase C phosphorylates the platelet protein pleckstrin. Phosphorylated pleckstrin triggers the "release reaction" of secondary inducers of platelet activation and aggregation contained within platelet granules. These substances include ADP, Ca²⁺, GDP, serotonin, and histamine found in dense granules, as well as the protein β-thromboglobulin, von Willebrand factor, fibronectin, thrombospondin, and HMWK present in α-granules. Thrombospondin participates in platelet-platelet interactions. β-Thromboglobulin decreases prostacyclin secretion and binds heparin. Fibronectin possesses binding sites for collagen, heparin, and platelets.

ADP is present in platelets and is also released into the blood upon erythrocyte destruction. ADP interacts with specific receptors and inhibits adenylate cyclase activity. This causes an increase in the mobilization of intracellular Ca²⁺ and ultimately leads to platelet aggregation.

Thus, platelet activation is accompanied by changes in their METABOLISM and the release of BIOLOGICALLY ACTIVE SUBSTANCES. These substances induce morphological changes, platelet adhesion and aggregation, and participate in thrombus formation.

Impairment of the functional activity of platelet receptors and secondary messenger systems leads to altered platelet function and may cause A number of diseases accompanied by thrombosis or Hemorrhage.

Drugs that inhibit platelet aggregation are used to prevent The Development of thrombosis. Aspirin (a cyclooxygenase inhibitor), nicotinic acid (a thromboxane synthetase inhibitor), and Ca²⁺ channel blockers suppress platelet aggregation by interfering with different stages of thrombogenic signal transduction.

E. Fibrinolysis

A thrombus is dissolved within a few days after its formation. Fibrinolysis is the Enzymatic Cleavage of fibrin fibers into soluble peptides that are cleared from the vascular bed. The breakdown of fibrin within a thrombus occurs under the action of the serine protease plasmin.

Plasmin is formed from plasminogen through the action of activators. The inactive plasmin proenzyme, plasminogen, is synthesized in the liver, Kidneys, and Bone Marrow.

Tissue plasminogen activator (tPA) is a proteolytic enzyme found in the vascular endothelium of all Tissues except the liver. The release of this activator into the bloodstream increases during emotional stress, pain, venous thromboembolism, and moderate physical exertion. Through partial proteolysis, tPA converts inactive plasminogen into active plasmin. Factor XIIa and kallikrein also serve as plasminogen activators.

Dissolution of the fibrin clot occurs via the interaction of fibrin, plasminogen, and tPA (Fig. 14-18).

Fig. 14-18. Scheme of fibrinolysis. 1 — plasminogen adsorbed onto the fibrin clot is converted into plasmin by partial proteolysis under the action of activators (factor XIIa, kallikrein, tPA); 2 — plasmin hydrolyzes fibrin to form soluble peptides X, Y, D, and E; 3 — in the bloodstream, tPA is inactivated by specific proteins PAI-1 and PAI-2; 4 — plasmin activity is reduced by nonspecific serine protease inhibitors (α₂-antiplasmin, α₂-macroglobulin, α₁-antitrypsin, antithrombin-heparin complex).

The formation of a fibrin fiber network during thrombus generation is accompanied by the sorption of plasminogen and its activators onto it. Plasmin and plasminogen molecules contain regions complementary to fibrin domains, with a single plasmin molecule capable of binding multiple fibrin molecules. tPA molecules also possess fibrin-binding sites. Plasmin, generated from plasminogen by the action of tPA, hydrolyzes fibrin to yield peptides X and Y (which promote fibrinolysis) and peptides D and E (which inhibit it). Soluble peptides X, Y, D, and E enter the bloodstream and undergo phagocytosis there. Thrombus destruction leads to the release of plasmin and tPA from it. Within the bloodstream, the latter are rapidly inactivated by specific inhibitors and cleared by the liver.

tPA is inhibited by tissue plasmin activator inhibitors type 1 (PAI-1) and type 2 (PAI-2), while plasmin is inhibited by α₂-antiplasmin or other serine protease inhibitors.

Urokinase, a proteolytic plasminogen activator, is synthesized in the kidneys; by converting plasminogen to plasmin, it helps clear fibrin fibers from the renal glomeruli.

Streptokinase, a protein isolated from β-hemolytic streptococci, forms a complex with plasminogen in which plasminogen is autocatalytically converted into plasmin.

Urokinase, streptokinase, and tPA are used in the thrombolytic therapy of myocardial infarction, venous and arterial thrombosis, and hemodialysis.

Such blood coagulation enzyme inhibitors as α₂-macroglobulin, α₁-antitrypsin, and the antithrombin III-heparin complex also exhibit modest fibrinolytic activity.

A decrease in blood fibrinolytic activity is associated with thrombosis. Impaired breakdown of the fibrin clot may result from a hereditary plasminogen deficiency or a genetic defect in its structure, reduced release of plasminogen activators into the bloodstream, or elevated levels of fibrinolysis inhibitors (PAI-1, PAI-2, α₂-antiplasmin).

Hereditary and acquired hemostatic disorders can lead to both hemorrhagic diseases, characterized by a tendency to bleed, and thrombotic disorders. However, it should be noted that an increased predisposition to thrombosis and intravascular coagulation (Thrombophilia) is far more common than hemophilia. For instance, the prevalence of Various Forms of hemophilia ranges across different countries from 6 to 18 per 100,000 males, whereas thrombophilia caused by antithrombin III deficiency occurs in 1 to 2 out of every 5,000 patients, and protein C deficiency affects approximately 1 in 15,000 individuals.