Extrinsic and intrinsic pathways of blood clotting

The extrinsic pathway of blood coagulation involves thromboplastin (tissue factor, factor III), proconvertin (factor VII), Stuart factor (Factor X), proaccelerin (factor V), as well as Ca²⁺ and phospholipids of membrane surfaces on which a thrombus is formed. Homogenates of many tissues accelerate blood clotting: this action is called thromboplastin activity. It is probably due to the presence in the tissues of some special protein. Factors VII and X are proenzymes. They are activated by partial proteolysis, turning to proteolytic enzymes – factors VIIa and Xa, respectively. Factor V is a protein that is under the influence of thrombin is converted to factor V', which is not an enzyme but activates the enzyme Xa according to the allosteric mechanism; activation is enhanced in the presence of phospholipids and Ca²⁺.

The blood plasma permanently contains trace amounts of factor VIIa. When there is tissue and the vessel walls damage factor III is released. It is a powerful activator of factor VIIa; activity the latter one is increased by more than 15,000 times. Factor VIIa cleaves the part of peptide chain of factor X, transforming it into an enzyme – factor Xa. Similarly Xa activates prothrombin; formed thrombin catalyzes the conversion of fibrinogen into fibrin, as well as the conversion of transglutaminase precursor into active enzyme (factor XIIIa). This cascade of reactions has positive feedbacks that enhance the end result. Factor Xa and thrombin catalyze the conversion of inactive factor VII in enzyme VIIa; thrombin converts factor V to factor V', which, together with phospholipids and Ca²⁺ in the 10⁴-10⁵ times increases the activity of factor Xa. Due to positive feedbacks the rate of formation of thrombin and, consequently, the transformation of fibrinogen into fibrin grow like an avalanche, and within 10-12 seconds blood is cloted.

Blood clotting according to the intrinsic mechanism is much slower and requires 10-15 minutes. This mechanism is called intrinsic because it does not require thromboplastin (tissue factor) and all relevant factors are contained in the blood. The intrinsic mechanism of coagulation is a cascade of successive activations of proenzymes. From the stage of conversion of factor X to Xa, the extrinsic and intrinsic pathways are identical. The intrinsic pathway of coagulation, as the extrinsic one, has positive feedbacks: thrombin catalyzes the conversion of precursor V and VIII into activators V 'and VIII', which ultimately increases the rate of formation of the thrombin.

External and internal mechanisms of blood coagulation interact to each other. Factor VII is specific to the extrinsic pathway of coagulation. It can be activated by factor XIIa, which is involved in the intrinsic pathway. This turns both pathways in a single system of blood clotting.

Hemophilias. Hereditary defects in proteins involved in blood coagulation, are manifested by increased bleeding. The most common disease is caused by a lack of Factor VIII. It is Hemophilia A. Factor VIII gene is localized in the X-chromosome; the gene damage is manifested as a recessive trait, so the women have not got hemophilia A. Men who have one X-chromosome inheritance of a defective gene causes hemophilia. Symptoms of the disease are usually detected at an early age: at the slightest cuts, or even spontaneously bleeding bleedings arise; it is characterized by intra-articular hemorrhage. Frequent blood loss leads to iron deficiency anemia. Fresh donor blood containing factor VIII, or preparations of factor VIII is injected to stop bleeding when there is hemophilia.

Hemophilia B. Hemophilia B is caused by mutations in the gene of factor IX, which, like the gene of factor VIII, is localized in the sex chromosome; mutations are recessive, therefore, hemophilia B is found only in men. Hemophilia B occurs in about 5 times less frequent than hemophilia A. Hemophilia B is treated with the introduction of drugs factor IX.

When there is increased blood clotting intravascular clots, plugging the intact vessels (thrombotic states, thrombophilia) may occur.

Fibrinolysis. Thrombus is dissipated within a few days after the formation. The main role of its dissolution belongs to proteolytic enzyme plasmin. Plasmin hydrolyzes peptide bonds in fibrin, formed by residues of arginine and tryptophan, with the formation of soluble peptides. The precursor of plasmin – plasminogen – is in the circulating blood. It is activated by the enzyme urokinase, which is found in many tissues. Plasminogen can be activated by kallikrein, which is also in the thrombus. Plasmin can be activated in the circulating blood without damaging the blood vessels. There plasmin is rapidly inactivated by protein inhibitor α₂-antiplasmin, while inside the clot it is protected from the action of the inhibitor. Urokinase is an effective agent for dissolving blood clots, or preventing their formation during thrombophlebitis, pulmonary vascular thrombosis, myocardial infarction, surgical intervention.

Anticoagulant system. During the development of the blood coagulation system in the course of evolution two opposite problems were solved: prevention the leakage of blood in damaged vessels and keeping the blood in the liquid state when in intact vessels. The second problem is solved by anticoagulation system, which is represented by a set of plasma proteins that inhibit proteolytic enzymes.

Plasma protein antithrombin III inhibits all proteases involved in blood clotting, except factor VIIa. It does not act on factors that are in the complexes with phospholipids, but only to those which are in the plasma in a dissolved state. Therefore, it is necessary not for the regulation of clot formation, but to eliminate the enzymes which enter the blood stream from the site of clot formation, thus it prevents the spread of blood clotting in uninjured areas of the bloodstream.

As a drug that prevents blood clotting, heparin is used. Heparin enhances the inhibitory effect of antithrombin III: joining heparin induces conformational changes that increase the affinity of the inhibitor to thrombin and other factors. After connecting this complex with thrombin heparin is released and can be attached to other molecules of antithrombin III. Thus, each molecule of heparin may activate a large number of molecules of antithrombin III; in this respect the action of heparin is similar to the action of catalysts. Heparin is used as an anticoagulant in the treatment of thrombotic conditions. Genetic defect is known when the antithrombin III concentration in the blood is half the normal; such people often have thrombosis. Antithrombin III is the main component of anticoagulation system.

In blood plasma there are other proteins – protease inhibitors, which can also reduce the probability of intravascular coagulation. Such a protein is α₂-macroglobulin, which inhibits many proteases, and not only those involved in blood clotting. α₂-macroglobulin contains sites of the peptide chain that are substrates of many proteinases; proteinases attach to these sites and hydrolyze in them some peptide bonds, as a result the conformation of α₂-macroglobulin is changed, and it captures the enzyme, like a trap. The enzyme is not damaged: in the complex with an inhibitor it is able to hydrolyze low molecular weight peptides, but for large molecules the active site of the enzyme is not available. α₂-macroglobulin complex with the enzyme is rapidly removed from the blood: time of its half-life in blood is about 10 minutes. During a massive entry into the bloodstream of activated clotting factors the power of anticoagulation system can be inadequate, and there is a risk of thrombosis.

Vitamin K. The peptide chains of factors II, VII, IX, and X contain an unusual amino acid – γ-carboxyglutamic. This amino acid is formed from glutamic acid as a result of the posttranslational modification of proteins:

Reactions, which involve factors II, VII, IX, and X, are activated by Са²⁺ ions and phospholipids: radicals of γ-carboxyglutamic acids form binding sites of Са²⁺ in these proteins. These factors, and factors V 'and VIII' are attached to the bilayer phospholipid membranes and to each other, with the participation of Са²⁺ ions; and there is the activation of factors II, VII, IX, and X in such complexes. Са²⁺ ion activates also some other reactions of coagulation. The decalcified blood does not clot.

The conversion of the glutamic residue into the residue of γ-carboxyglutamic acid is catalyzed by an enzyme, a coenzyme of which is vitamin K. Vitamin K deficiency appears to bleeding disorders, subcutaneous and internal hemorrhages. In the absence of vitamin K factors II, VII, IX, and X are formed; they do not contain γ-carboxyglutamic residues. These proenzymes cannot be converted into active enzymes.