How is blood coagulation carried out? Blood clotting. Blood coagulation pattern Temporary vasospasm

There are three main stages of hemocoagulation:

1. formation of blood thromboplastin and tissue thromboplastin;

2. formation of thrombin;

3. formation of a fibrin clot.

There are 2 mechanisms of hemocoagulation: internal clotting mechanism(it involves factors that are inside the vascular bed) and extrinsic clotting mechanism(in addition to intravascular factors, external factors also participate in it).

Internal mechanism of blood coagulation (contact)

The internal mechanism of hemocoagulation is triggered by damage to the vascular endothelium (for example, with atherosclerosis, under the action of high doses of catecholamines) in which collagen and phospholipids are present. Factor XII (trigger factor) joins the altered area of ​​the endothelium. Interacting with the altered endothelium, it undergoes conformational structural changes and becomes a very powerful active proteolytic enzyme. XIIa factor simultaneously participates in the coagulation system, anti-coagulation system, kinin system:

1. activates the blood coagulation system;
2. activates the anticoagulant system;
3. activates platelet aggregation;
4. activates the kinin system;

1 stage internal mechanism of blood clotting formation of complete blood thromboplastin.

XII factor, in contact with the damaged endothelium, passes into active XII. XIIa activates prekallikrein (XIY), which activates kininogen (XY). Kinins, in turn, increase the activity of factor XII.

Factor XII activates factor XI, which then activates factor IX (f. Christmas). Factor IXa interacts with factor YIII and calcium ions. As a result, a complex is formed, including the enzyme, coenzyme, calcium ions (f.IXa, f.YIII, Ca 2+). This complex activates factor X with the participation of platelet factor P 3 . As a result, a active blood thromboplastin, including f.Xa, f.Y, Ca 2+ and R 3 .

P 3 - is a fragment of platelet membranes, contains lipoproteins, rich in phospholipids.

Stage 2 - the formation of thrombin.

Active blood thromboplastin triggers the 2nd stage of blood coagulation, activating the transition of prothrombin to thrombin (f. II → f. II a). Thrombin activates the external and internal mechanisms of hemocoagulation, as well as the anticoagulant system, platelet aggregation and the release of platelet factors.

Active thrombin starts the 3rd stage of blood coagulation.

3 stage lies in formation of insoluble fibrin(I factor). Under the influence of thrombin, soluble fibrinogen sequentially passes into fibrin monomer, and then into insoluble fibrin polymer.

Fibrinogen is a water-soluble protein consisting of 6 polypeptide chains, including 3 domains. Under the action of thrombin, peptides A and B are cleaved from fibrinogen, and aggregation sites are formed in it. Fibrin strands are connected first into linear chains, and then covalent interchain crosslinks are formed. Factor XIIIa (fibrin-stabilizing) is involved in their formation, which is activated by thrombin. Under the action of factor XIIIa, which is a transamidinase enzyme, bonds between glutamine and lysine appear in fibrin during its polymerization.

Blood clotting should be normal, so hemostasis is based on equilibrium processes. It is impossible for our valuable biological fluid to coagulate - this threatens with serious, deadly complications (). On the contrary, it can result in uncontrolled massive bleeding, which can also lead to the death of a person.

The most complex mechanisms and reactions, involving a number of substances at one stage or another, maintain this balance and thus enable the body to quickly cope on its own (without the involvement of any outside help) and recover.

The rate of blood clotting cannot be determined by any one parameter, because many components are involved in this process, activating each other. In this regard, blood coagulation tests are different, where the intervals of their normal values ​​mainly depend on the method of conducting the study, and in other cases, on the sex of the person and the days, months, and years he has lived. And the reader is unlikely to be satisfied with the answer: Blood clotting time is 5-10 minutes". A lot of questions remain...

Everyone is important and everyone is needed

Stopping bleeding is based on an extremely complex mechanism, which includes many biochemical reactions, which involves a huge number of different components, where each of them plays a specific role.

blood coagulation pattern

Meanwhile, the absence or inconsistency of at least one coagulation or anticoagulation factor can upset the whole process. Here are just a few examples:

• An inadequate reaction from the side of the walls of the vessels violates the platelets - which “feels” the primary hemostasis;
• The low ability of the endothelium to synthesize and secrete inhibitors of platelet aggregation (the main one is prostacyclin) and natural anticoagulants () thickens the blood moving through the vessels, which leads to the formation of clots in the bloodstream that are absolutely unnecessary for the body, which for the time being can calmly “sit” attached to the wall of which or a vessel. These become very dangerous when they break off and begin to circulate in the bloodstream - thereby creating the risk of a vascular accident;
• The absence of such a plasma factor as FVIII is due to a sex-linked disease - A;
• Hemophilia B is detected in a person if, for the same reasons (a recessive mutation on the X chromosome, which, as is known, there is only one in men), Christman factor deficiency (FIX) occurs.

In general, it all starts at the level of the damaged vascular wall, which, by secreting the substances necessary to ensure blood clotting, attracts platelets circulating in the bloodstream - platelets. For example, “inviting” platelets to the accident site and promoting their adhesion to collagen, a powerful stimulator of hemostasis, must start its activity in a timely manner and work well so that in the future one can count on the formation of a full-fledged plug.

If platelets use their functionality at the proper level (adhesive-aggregation function), other components of primary (vascular-platelet) hemostasis quickly come into play and form a platelet plug in a short time, then in order to stop the blood flowing from the vessel of the microvasculature , you can do without the special influence of other participants in the blood coagulation process. However, for the formation of a full-fledged plug capable of closing an injured vessel, which has a wider lumen, the body cannot cope without plasma factors.

Thus, at the first stage (immediately after the injury of the vascular wall), successive reactions begin to take place, where the activation of one factor gives impetus to bringing the rest into an active state. And if something is missing somewhere or the factor turns out to be untenable, the process of blood coagulation slows down or breaks off altogether.

In general, the clotting mechanism consists of 3 phases, which should provide:

• The formation of a complex complex of activated factors (prothrombinase) and the conversion of a protein synthesized by the liver - into thrombin ( activation phase);
• The transformation of the protein dissolved in the blood - factor I ( , FI) into insoluble fibrin is carried out in coagulation phase;
• Completion of the coagulation process by the formation of a dense fibrin clot ( retraction phase).

Blood clotting tests

A multi-stage cascade enzymatic process, the ultimate goal of which is the formation of a clot that can close the “gap” in the vessel, will surely seem confusing and incomprehensible to the reader, so it will be sufficient to remind that this mechanism is provided by various coagulation factors, enzymes, Ca 2+ (ions calcium) and a variety of other components. However, in this regard, patients are often interested in the question: how to detect if something is wrong with hemostasis or to calm down, knowing that the systems are working normally? Of course, for such purposes, there are tests for blood clotting.

The most common specific (local) analysis of the state of hemostasis is considered to be widely known, often prescribed by therapists, cardiologists, as well as obstetrician-gynecologists, the most informative.

Meanwhile, it should be noted that carrying out such a number of tests is not always justified. It depends on many circumstances: what the doctor is looking for, at what stage of the cascade of reactions he focuses his attention, how much time is available to medical workers, etc.

Simulation of the external pathway of blood clotting

For example, an extrinsic clotting activation pathway in the laboratory can mimic what the medical profession calls Quick Prothrombin, Quick Test, Prothrombin Time (PTT), or Thromboplastin Time (all different names for the same test). This test, which depends on factors II, V, VII, X, is based on the participation of tissue thromboplastin (it joins citrate recalcified plasma during work on a blood sample).

The limits of normal values ​​for men and women of the same age do not differ and are limited to the range of 78 - 142%, however, in women who are expecting a child, this figure is slightly increased (but slightly!). In children, on the contrary, the norms are within the limits of smaller values ​​and increase as they approach adulthood and beyond:

Reflection of the internal mechanism in the laboratory

Meanwhile, in order to determine a violation of blood clotting due to a malfunction of the internal mechanism, tissue thromboplastin is not used during the analysis - this allows the plasma to use only its own reserves. In the laboratory, the internal mechanism is traced, waiting for the blood taken from the vessels of the bloodstream to clot itself. The onset of this complex cascade reaction coincides with the activation of the Hageman factor (factor XII). The launch of this activation is provided by various conditions (contact of blood with a damaged vessel wall, cell membranes that have undergone certain changes), therefore it is called contact activation.

Contact activation also occurs outside the body, for example, when blood enters an alien environment and comes into contact with it (contact with glass in a test tube, instruments). The removal of calcium ions from the blood does not affect the launch of this mechanism in any way, however, the process cannot end with the formation of a clot - it breaks off at the stage of factor IX activation, where ionized calcium is no longer enough.

The time of blood clotting or the time during which it, while in a liquid state, pours into the form of an elastic clot, depends on the rate of conversion of the fibrinogen protein dissolved in plasma into insoluble fibrin. It (fibrin) forms threads that hold red blood cells (erythrocytes), causing them to form a bundle that closes the hole in the damaged blood vessel. Blood clotting time (1 ml taken from a vein - Lee-White method) in such cases is limited on average to 4-6 minutes. However, the blood coagulation rate, of course, has a wider range of digital (temporary) values:

1. Blood taken from a vein goes into the form of a clot from 5 to 10 minutes;
2. The Lee-White clotting time in a glass tube is 5-7 minutes, in a silicone tube it is extended to 12-25 minutes;
3. For blood taken from a finger, indicators are considered normal: the beginning - 30 seconds, the end of bleeding - 2 minutes.

An analysis that reflects the internal mechanism is turned to at the first suspicion of gross violations of blood coagulability. The test is very convenient: it is carried out quickly (until the blood flows or forms a clot in the test tube), it does without special reagents and sophisticated equipment, and the patient does not need special preparation. Of course, blood clotting disorders detected in this way give reason to assume a number of significant changes in the systems that ensure the normal state of hemostasis, and force further research to identify the true causes of the pathology.

With an increase (lengthening) of the blood clotting time, one can suspect:

• Deficiency of plasma factors designed to ensure coagulation, or their congenital inferiority, despite the fact that they are at a sufficient level in the blood;
• Serious pathology of the liver, resulting in functional failure of the parenchyma of the organ;
• (in the phase when the ability of blood to clot is on the wane);

Blood clotting time is lengthened in cases of using heparin therapy, so patients receiving this drug have to take tests that indicate the state of hemostasis quite often.

The considered indicator of blood clotting reduces its values ​​(shortened):

• In the phase of high coagulation () DIC;
• In other diseases that caused a pathological state of hemostasis, that is, when the patient already has blood clotting disorders and is assigned to a group of increased risk of blood clots (thrombosis, etc.);
• In women who use for contraception or for the purpose of treatment for a long time, oral agents containing hormones;
• In women and men taking corticosteroids (when prescribing corticosteroid drugs, age is very important - many of them in children and the elderly can cause significant changes in hemostasis, therefore they are prohibited for use in this group).

In general, the norms differ little

Indicators of blood coagulation (norm) in women, men and children (meaning one age for each category), in principle, differ little, although individual indicators in women change physiologically (before, during and after menstruation, during pregnancy), therefore, the sex of an adult is still taken into account in laboratory studies. In addition, in women during the period of bearing a child, individual parameters should even shift somewhat, because the body has to stop bleeding after childbirth, so the coagulation system begins to prepare ahead of time. An exception for some indicators of blood coagulation is the category of children in the first days of life, for example, in newborns, PTT is a couple of times higher than in adult males and females (the norm for adults is 11-15 seconds), and in premature babies, the prothrombin time increases for 3 - 5 seconds. True, already somewhere by the 4th day of life, PTV decreases and corresponds to the norm of blood clotting in adults.

To get acquainted with the norm of individual indicators of blood coagulation, and, possibly, to compare them with their own parameters (if the test was carried out relatively recently and there is a form with a record of the results of the study on hand), the table below will help the reader:

In conclusion, I would like to draw the attention of our regular (and new, of course) readers: perhaps reading the review article will not be able to fully satisfy the interest of patients affected by hemostasis pathology. People who first encountered a similar problem, as a rule, want to get as much information as possible about systems that provide both stopping bleeding at the right time and preventing the formation of dangerous clots, so they start looking for information on the Internet. Well, you should not rush - in other sections of our website, a detailed (and, most importantly, correct) description of each of the indicators of the state of hemostasis is given, the range of normal values ​​\u200b\u200bis indicated, and indications and preparation for analysis are also described.

Video: just about blood clotting

Video: reportage on blood clotting tests

One of the most important processes in our body is blood clotting. Its scheme will be described below (images are also provided for clarity). And since this is a complex process, it is worth considering it in detail.

How is it going?

So, the designated process is responsible for stopping the bleeding that occurred due to damage to one or another component of the vascular system of the body.

In simple terms, three phases can be distinguished. The first is activation. After damage to the vessel, successive reactions begin to occur, which ultimately lead to the formation of the so-called prothrombinase. It is a complex complex consisting of V and X. It is formed on the phospholipid surface of platelet membranes.

The second phase is coagulation. At this stage, fibrin is formed from fibrinogen - a high-molecular protein, which is the basis of blood clots, the occurrence of which implies blood clotting. The diagram below illustrates this phase.

And finally, the third stage. It implies the formation of a fibrin clot, which has a dense structure. By the way, it is by washing and drying it that it is possible to obtain a “material”, which is then used to prepare sterile films and sponges to stop bleeding caused by rupture of small vessels during surgical operations.

About reactions

The scheme was briefly described above, by the way, it was developed back in 1905 by a coagulologist named Paul Oskar Morawitz. And it has not lost its relevance to this day.

But since 1905, much has changed in understanding blood clotting as a complex process. With progress, of course. Scientists have been able to discover dozens of new reactions and proteins that are involved in this process. And now the cascade pattern of blood coagulation is more common. Thanks to her, the perception and understanding of such a complex process becomes a little more understandable.

As you can see in the image below, what is happening is literally “broken into bricks”. It takes into account the internal and external system - blood and tissue. Each is characterized by a certain deformation that occurs as a result of damage. In the blood system, damage is done to the vascular walls, collagen, proteases (splitting enzymes) and catecholamines (mediator molecules). In the tissue, cell damage is observed, as a result of which thromboplastin is released from them. Which is the most important stimulator of the coagulation process (otherwise called coagulation). It goes directly into the blood. This is his "way", but it has a protective character. After all, it is thromboplastin that starts the clotting process. After its release into the blood, the implementation of the above three phases begins.

Time

So, what exactly is blood coagulation, the scheme helped to understand. Now I would like to talk a little about time.

The whole process takes a maximum of 7 minutes. The first phase lasts from five to seven. During this time, prothrombin is formed. This substance is a complex type of protein structure responsible for the course of the coagulation process and the ability of blood to thicken. Which is used by our body in order to form a blood clot. It clogs the damaged area, so that the bleeding stops. All this takes 5-7 minutes. The second and third stages happen much faster. For 2-5 seconds. Because these phases of blood clotting (diagram provided above) affect processes that occur everywhere. And that means at the site of damage directly.

Prothrombin, in turn, is formed in the liver. And it takes time to synthesize it. How quickly a sufficient amount of prothrombin is produced depends on the amount of vitamin K contained in the body. If it is not enough, the bleeding will be difficult to stop. And this is a serious problem. Since the lack of vitamin K indicates a violation of the synthesis of prothrombin. And this is a disease that needs to be treated.

Synthesis stabilization

Well, the general scheme of blood clotting is clear - now we should pay a little attention to the topic of what needs to be done to restore the required amount of vitamin K in the body.

For starters, eat right. The largest amount of vitamin K is found in green tea - 959 mcg per 100 g! Three times more, by the way, than in black. That is why it is worth drinking it actively. Do not neglect vegetables - spinach, white cabbage, tomatoes, green peas, onions.

Vitamin K is also found in meat, but not in everything - only in veal, beef liver, lamb. But least of all it is in the composition of garlic, raisins, milk, apples and grapes.

However, if the situation is serious, then it will be difficult to help with just a variety of menus. Usually, doctors strongly recommend combining your diet with the drugs they have prescribed. Treatment should not be delayed. It is necessary to start it as soon as possible in order to normalize the mechanism of blood coagulation. The treatment regimen is prescribed directly by the doctor, and he is also obliged to warn what can happen if the recommendations are neglected. And the consequences can be liver dysfunction, thrombohemorrhagic syndrome, tumor diseases and damage to bone marrow stem cells.

Schmidt's scheme

At the end of the 19th century, there lived a famous physiologist and doctor of medical sciences. His name was Alexander Alexandrovich Schmidt. He lived for 63 years, and devoted most of his time to the study of problems of hematology. But especially carefully he studied the topic of blood coagulation. He managed to establish the enzymatic nature of this process, as a result of which the scientist proposed a theoretical explanation for it. Which clearly depicts the scheme of blood coagulation provided below.

First of all, the damaged vessel is reduced. Then, at the site of the defect, a loose, primary platelet plug is formed. Then it gets stronger. As a result, a red blood clot (otherwise referred to as a blood clot) is formed. After which it partially or completely dissolves.

During this process, certain blood clotting factors are manifested. The scheme, in its expanded version, also displays them. They are denoted by Arabic numerals. And there are 13 of them in total. And you need to tell about each.

Factors

A complete blood coagulation scheme is impossible without listing them. Well, it's worth starting from the first.

Factor I is a colorless protein called fibrinogen. Synthesized in the liver, dissolved in plasma. Factor II - prothrombin, which has already been mentioned above. Its unique ability lies in the binding of calcium ions. And it is precisely after the breakdown of this substance that the coagulation enzyme is formed.

Factor III is a lipoprotein, tissue thromboplastin. It is commonly called the transport of phospholipids, cholesterol, and also triacylglycerides.

The next factor, IV, are Ca2+ ions. The ones that bind under the influence of a colorless protein. They are involved in many complex processes, in addition to clotting, in the secretion of neurotransmitters, for example.

Factor V is a globulin. Which is also formed in the liver. It is necessary for the binding of corticosteroids (hormonal substances) and their transport. Factor VI existed for a certain time, but then it was decided to remove it from the classification. Since scientists have found out - it includes factor V.

But the classification did not change. Therefore, V is followed by factor VII. Includes proconvertin, with the participation of which tissue prothrombinase is formed (first phase).

Factor VIII is a protein expressed in a single chain. It is known as antihemophilic globulin A. It is because of its lack that such a rare hereditary disease as hemophilia develops. Factor IX is "related" to the previously mentioned. Since it is antihemophilic globulin B. Factor X is directly a globulin synthesized in the liver.

And finally, the last three points. These are the Rosenthal, Hageman factor and fibrin stabilization. Together, they affect the formation of intermolecular bonds and the normal functioning of such a process as blood coagulation.

Schmidt's scheme includes all these factors. And it is enough to get acquainted with them briefly in order to understand how the described process is complex and ambiguous.

Anti-clotting system

This concept also needs to be noted attention. The blood coagulation system was described above - the diagram also clearly demonstrates the course of this process. But the so-called "anti-coagulation" also has a place to be.

To begin with, I would like to note that in the course of evolution, scientists solved two completely opposite tasks. They tried to find out - how does the body manage to prevent blood from flowing out of damaged vessels, and at the same time keep it in a liquid state in its entirety? Well, the solution to the second problem was the discovery of an anticoagulant system.

It is a specific set of plasma proteins that can slow down the rate of chemical reactions. That is to inhibit.

And antithrombin III is involved in this process. Its main function is to control the work of some factors that include the scheme of the blood coagulation process. It is important to clarify: it does not regulate the formation of a blood clot, but eliminates unnecessary enzymes that have entered the bloodstream from the place where it is formed. What is it for? To prevent the spread of clotting to areas of the bloodstream that have been damaged.

obstructing element

Talking about what the blood coagulation system is (the scheme of which is presented above), one cannot but note such a substance as heparin. It is a sulfur-containing acidic glycosaminoglycan (one of the types of polysaccharides).

It is a direct anticoagulant. A substance that contributes to the inhibition of the activity of the coagulation system. It is heparin that prevents the formation of blood clots. How does this happen? Heparin simply reduces the activity of thrombin in the blood. However, it is a natural substance. And it is beneficial. If this anticoagulant is introduced into the body, then it is possible to contribute to the activation of antithrombin III and lipoprotein lipase (enzymes that break down triglycerides - the main sources of energy for cells).

Now, heparin is often used to treat thrombotic conditions. Only one of its molecules can activate a large amount of antithrombin III. Accordingly, heparin can be considered a catalyst - since the action in this case is really similar to the effect caused by them.

There are other substances with the same effect contained in Take, for example, α2-macroglobulin. It contributes to the splitting of the thrombus, affects the process of fibrinolysis, performs the function of transport for 2-valent ions and some proteins. It also inhibits substances involved in the clotting process.

Observed changes

There is one more nuance that the traditional blood coagulation scheme does not demonstrate. The physiology of our body is such that many processes involve not only chemical changes. But also physical. If we could observe clotting with the naked eye, we would see that the shape of the platelets changes in the process. They turn into rounded cells with characteristic spiny processes, which are necessary for the intensive implementation of aggregation - the combination of elements into a single whole.

But that's not all. During the clotting process, various substances are released from platelets - catecholamines, serotonin, etc. Because of this, the lumen of the vessels that have been damaged narrows. What causes functional ischemia. The blood supply to the injured area is reduced. And, accordingly, the outpouring is also gradually reduced to a minimum. This gives the platelets the opportunity to cover the damaged areas. They, due to their spiny processes, seem to be “attached” to the edges of the collagen fibers that are located at the edges of the wound. This ends the first, longest activation phase. It ends with the formation of thrombin. This is followed by a few more seconds of the phase of coagulation and retraction. And the last stage is the restoration of normal blood circulation. And it matters a lot. Since the full healing of the wound is impossible without a good blood supply.

Good to know

Well, something like this in words and looks like a simplified scheme of blood coagulation. However, there are a few more nuances that I would like to note with attention.

Hemophilia. It has already been mentioned above. This is a very dangerous disease. Any hemorrhage by a person suffering from it is experienced hard. The disease is hereditary, develops due to defects in the proteins involved in the coagulation process. It can be detected quite simply - with the slightest cut, a person will lose a lot of blood. And it will take a lot of time to stop it. And in especially severe forms, hemorrhage can begin for no reason. People with hemophilia can become disabled early. Since frequent hemorrhages in muscle tissue (usual hematomas) and in joints are not uncommon. Is it curable? With difficulties. A person should literally treat his body as a fragile vessel, and always be careful. If bleeding occurs, donated fresh blood containing factor XVIII should be urgently administered.

Men usually suffer from this disease. And women act as carriers of the hemophilia gene. Interestingly, the British Queen Victoria was one. One of her sons contracted the disease. The other two are unknown. Since then, hemophilia, by the way, is often called the royal disease.

But there are also reverse cases. Meaning If it is observed, then a person also needs to be no less careful. Increased clotting indicates a high risk of intravascular thrombosis. Which clog entire vessels. Often the consequence can be thrombophlebitis, accompanied by inflammation of the venous walls. But this defect is easier to treat. Often, by the way, it is acquired.

It's amazing how much happens in the human body when he cuts himself with a piece of paper. You can talk for a long time about the features of blood, its coagulation and the processes that accompany it. But all the most interesting information, as well as diagrams that clearly demonstrate it, are provided above. The rest, if desired, can be viewed individually.

The process of blood coagulation begins with blood loss, but massive blood loss, accompanied by a drop in blood pressure, leads to drastic changes in the entire hemostasis system.

Blood coagulation system (hemostasis)

The blood coagulation system is a complex multicomponent complex of human homeostasis, which ensures the preservation of the integrity of the body due to the constant maintenance of the liquid state of the blood and the formation, if necessary, of various types of blood clots, as well as the activation of healing processes in places of vascular and tissue damage.

The functioning of the coagulation system is ensured by the continuous interaction of the vascular wall and circulating blood. Certain components are known that are responsible for the normal activity of the coagulation system:

• endothelial cells of the vascular wall,
• platelets,
• plasma adhesive molecules,
• plasma clotting factors,
• fibrinolysis systems,
• systems of physiological primary and secondary anticoagulants-antiproteases,
• plasma system of physiological primary reparants-healers.

Any damage to the vascular wall, “blood injury”, on the one hand, leads to varying severity of bleeding, and on the other hand, causes physiological, and subsequently pathological changes in the hemostasis system, which can themselves lead to the death of the body. The regular severe and frequent complications of massive blood loss include acute syndrome of disseminated intravascular coagulation (acute DIC).

In acute massive blood loss, and it cannot be imagined without vascular damage, there is almost always local (at the site of damage) thrombosis, which, in combination with a drop in blood pressure, can trigger acute DIC, which is the most important and pathogenetically most unfavorable mechanism for all the ills of acute massive blood loss. blood loss.

endothelial cells

Endothelial cells of the vascular wall maintain the liquid state of the blood, directly influencing many mechanisms and links of thrombus formation, completely blocking or effectively restraining them. Vessels provide laminar blood flow, which prevents the adhesion of cellular and protein components.

The endothelium carries a negative charge on its surface, as well as cells circulating in the blood, various glycoproteins and other compounds. Similarly charged endothelium and circulating blood elements repel each other, which prevents cells and protein structures from sticking together in the circulatory bed.

Keeping the blood fluid

The maintenance of a liquid state of the blood is facilitated by:

• prostacyclin (PGI 2),
• NO and ADPase,
• tissue thromboplastin inhibitor,
• glucosaminoglycans and, in particular, heparin, antithrombin III, heparin cofactor II, tissue plasminogen activator, etc.

Prostacyclin

The blockade of agglutination and aggregation of platelets in the bloodstream is carried out in several ways. The endothelium actively produces prostaglandin I 2 (PGI 2), or prostacyclin, which inhibits the formation of primary platelet aggregates. Prostacyclin is able to "break" early platelet agglutinates and aggregates, at the same time being a vasodilator.

Nitric oxide (NO) and ADPase

Platelet disaggregation and vasodilation are also carried out by the endothelial production of nitric oxide (NO) and the so-called ADPase (an enzyme that breaks down adenosine diphosphate - ADP) - a compound produced by various cells and which is an active agent that stimulates platelet aggregation.

Protein C system

The protein C system has a restraining and inhibitory effect on the blood coagulation system, mainly on its internal activation pathway. The complex of this system includes:

1. thrombomodulin,
2. protein C
3. protein S,
4. thrombin as an activator of protein C,
5. protein C inhibitor.

Endothelial cells produce thrombomodulin, which, with the participation of thrombin, activates protein C, converting it, respectively, into protein Ca. Activated protein Ca with the participation of protein S inactivates factors Va and VIIIa, suppressing and inhibiting the internal mechanism of the blood coagulation system. In addition, activated protein Ca stimulates the activity of the fibrinolysis system in two ways: by stimulating the production and release of tissue plasminogen activator from endothelial cells into the bloodstream, and also by blocking the tissue plasminogen activator inhibitor (PAI-1).

Pathology of the protein C system

Often observed hereditary or acquired pathology of the protein C system leads to the development of thrombotic conditions.

Fulminant purpura

Homozygous protein C deficiency (fulminant purpura) is an extremely severe pathology. Children with fulminant purpura are practically unviable and die at an early age from severe thrombosis, acute DIC, and sepsis.

Thrombosis

Heterozygous hereditary deficiency of protein C or protein S contributes to the occurrence of thrombosis in young people. Thrombosis of the main and peripheral veins, pulmonary embolism, early myocardial infarction, ischemic strokes are more common. In women with a deficiency of protein C or S, taking hormonal contraceptives, the risk of thrombosis (often cerebral thrombosis) increases by 10-25 times.

Since proteins C and S are vitamin K-dependent proteases produced in the liver, treatment of thrombosis with indirect anticoagulants such as syncumar or pelentan in patients with hereditary protein C or S deficiency may lead to an aggravation of the thrombotic process. In addition, a number of patients during treatment with indirect anticoagulants (warfarin) may develop peripheral skin necrosis (" warfarin necrosis"). Their appearance almost always means the presence of a heterozygous protein C deficiency, which leads to a decrease in blood fibrinolytic activity, local ischemia and skin necrosis.

V factor Leiden

Another pathology directly related to the functioning of the protein C system is called hereditary resistance to activated protein C, or V factor Leiden. Essentially V factor Leiden is a mutant V factor with a point replacement of arginine at position 506 of factor V with glutamine. Factor V Leiden has increased resistance to the direct action of activated protein C. If hereditary deficiency of protein C in patients predominantly with venous thrombosis occurs in 4-7% of cases, then V factor Leiden, according to different authors, in 10-25%.

tissue thromboplastin inhibitor

The vascular endothelium can also inhibit thrombosis when activated. Endothelial cells actively produce an inhibitor of tissue thromboplastin, which inactivates the tissue factor-factor VIIa complex (TF-VIIa), which leads to blockade of the external mechanism of blood coagulation, which is activated when tissue thromboplastin enters the bloodstream, thereby maintaining blood fluidity in the circulatory bed.

Glucosaminoglycans (heparin, antithrombin III, heparin cofactor II)

Another mechanism for maintaining the liquid state of the blood is associated with the production of various glycosaminoglycans by the endothelium, among which heparan and dermatan sulfate are known. These glycosaminoglycans are similar in structure and function to heparins. The heparin produced and released into the bloodstream binds to the antithrombin III (AT III) molecules circulating in the blood, activating them. In turn, activated AT III captures and inactivates factor Xa, thrombin, and a number of other factors of the blood coagulation system. In addition to the mechanism of inactivation of coagulation, which is carried out through AT III, heparins activate the so-called heparin cofactor II (CH II). Activated CG II, like AT III, inhibits the functions of factor Xa and thrombin.

In addition to affecting the activity of physiological anticoagulant-antiproteases (AT III and KG II), heparins are able to modify the functions of such adhesive plasma molecules as von Willebrand factor and fibronectin. Heparin reduces the functional properties of the von Willebrand factor, helping to reduce the thrombotic potential of the blood. Fibronectin, as a result of heparin activation, binds to various targets of phagocytosis - cell membranes, tissue detritus, immune complexes, fragments of collagen structures, staphylococci and streptococci. As a result of heparin-stimulated opsonic interactions of fibronectin, inactivation of phagocytosis targets in the organs of the macrophage system is activated. Purification of the circulatory bed from objects-targets of phagocytosis contributes to the preservation of the liquid state and fluidity of the blood.

In addition, heparins are able to stimulate the production and release into the circulatory bed of the tissue thromboplastin inhibitor, which significantly reduces the likelihood of thrombosis with external activation of the blood coagulation system.

The process of blood clotting

Along with the above, there are mechanisms that are also associated with the state of the vascular wall, but do not contribute to maintaining the liquid state of the blood, but are responsible for its coagulation.

The process of blood coagulation begins with damage to the integrity of the vascular wall. At the same time, the external mechanisms of the process of thrombus formation are also distinguished.

With an internal mechanism, damage to only the endothelial layer of the vascular wall leads to the fact that the blood flow comes into contact with the structures of the subendothelium - with the basement membrane, in which collagen and laminin are the main thrombogenic factors. They interact with the von Willebrand factor and fibronectin in the blood; a platelet thrombus is formed, and then a fibrin clot.

It should be noted that thrombi that form under conditions of fast blood flow (in the arterial system) can exist practically only with the participation of the von Willebrand factor. On the contrary, both the von Willebrand factor and fibrinogen, fibronectin, and thrombospondin are involved in the formation of thrombi at relatively low blood flow rates (in the microvasculature, venous system).

Another mechanism of thrombus formation is carried out with the direct participation of the von Willebrand factor, which, when the integrity of the vessels is damaged, increases significantly in quantitative terms due to the supply of endothelium from Weibol-Pallad bodies.

Coagulation systems and factors

thromboplastin

The most important role in the external mechanism of thrombosis is played by tissue thromboplastin, which enters the bloodstream from the interstitial space after a rupture of the integrity of the vascular wall. It induces thrombosis by activating the blood coagulation system with the participation of factor VII. Since tissue thromboplastin contains a phospholipid part, platelets participate little in this mechanism of thrombosis. It is the appearance of tissue thromboplastin in the bloodstream and its participation in pathological thrombosis that determine the development of acute DIC.

Cytokines

The next mechanism of thrombosis is realized with the participation of cytokines - interleukin-1 and interleukin-6. The tumor necrosis factor formed as a result of their interaction stimulates the production and release of tissue thromboplastin from the endothelium and monocytes, the significance of which has already been mentioned. This explains the development of local thrombi in various diseases that occur with pronounced inflammatory reactions.

platelets

Specialized blood cells involved in the process of its coagulation are platelets - non-nuclear blood cells, which are fragments of the cytoplasm of megakaryocytes. Platelet production is associated with a certain thrombopoietin that regulates thrombopoiesis.

The number of platelets in the blood is 160-385×10 9 /l. They are clearly visible in a light microscope, therefore, when conducting a differential diagnosis of thrombosis or bleeding, microscopy of peripheral blood smears is necessary. Normally, the size of a platelet does not exceed 2-3.5 microns (about ⅓-¼ of the diameter of an erythrocyte). Under light microscopy, unchanged platelets appear as rounded cells with smooth edges and red-violet granules (α-granules). The life span of platelets is on average 8-9 days. Normally, they are discoid in shape, but when activated, they take the form of a sphere with a large number of cytoplasmic protrusions.

There are 3 types of specific granules in platelets:

• lysosomes containing large amounts of acid hydrolases and other enzymes;
• α-granules containing many different proteins (fibrinogen, von Willebrand factor, fibronectin, thrombospondin, etc.) and stained according to Romanovsky-Giemsa in purple-red color;
• δ-granules are dense granules containing a large amount of serotonin, K + ions, Ca 2+ , Mg 2+ , etc.

α-granules contain strictly specific platelet proteins - such as platelet factor 4 and β-thromboglobulin, which are markers of platelet activation; their determination in blood plasma can help in the diagnosis of current thrombosis.

In addition, in the structure of platelets there is a system of dense tubules, which is, as it were, a depot for Ca 2+ ions, as well as a large number of mitochondria. When platelets are activated, a series of biochemical reactions occur, which, with the participation of cyclooxygenase and thromboxane synthetase, lead to the formation of thromboxane A 2 (TXA 2) from arachidonic acid, a powerful factor responsible for irreversible platelet aggregation.

The platelet is covered with a 3-layer membrane, on its outer surface there are various receptors, many of which are glycoproteins and interact with various proteins and compounds.

Platelet hemostasis

The glycoprotein Ia receptor binds to collagen, the glycoprotein Ib receptor interacts with von Willebrand factor, glycoproteins IIb-IIIa interact with fibrinogen molecules, although it can bind to both von Willebrand factor and fibronectin.

When platelets are activated by agonists - ADP, collagen, thrombin, adrenaline, etc. - the 3rd plate factor (membrane phospholipid) appears on their outer membrane, activating the rate of blood clotting, increasing it 500-700 thousand times.

Plasma clotting factors

Blood plasma contains several specific systems involved in the blood coagulation cascade. These are the systems:

• adhesive molecules,
• coagulation factors,
• fibrinolysis factors,
• factors of physiological primary and secondary anticoagulants-antiproteases,
• factors of physiological primary reparants-healers.

Plasma adhesive molecule system

The system of adhesive plasma molecules is a complex of glycoproteins responsible for intercellular, cell-substrate, and cell-protein interactions. It includes:

1. von Willebrand factor,
2. fibrinogen,
3. fibronectin,
4. thrombospondin,
5. vitronectin.

Willebrand factor

The von Willebrand factor is a high molecular weight glycoprotein with a molecular weight of 10 3 kD or more. The von Willebrand factor performs many functions, but the main ones are two:

• interaction with factor VIII, due to which antihemophilic globulin is protected from proteolysis, which increases its lifespan;
• ensuring the processes of adhesion and aggregation of platelets in the circulatory bed, especially at high blood flow rates in the vessels of the arterial system.

A decrease in the level of von Willebrand factor below 50%, observed in von Willebrand disease or syndrome, leads to severe petechial bleeding, usually of the microcirculatory type, manifested by bruising with minor injuries. However, in a severe form of von Willebrand's disease, a hematoma type of bleeding similar to hemophilia () can be observed.

On the contrary, a significant increase in the concentration of von Willebrand factor (more than 150%) can lead to a thrombophilic condition, which is often clinically manifested by various types of peripheral vein thrombosis, myocardial infarction, thrombosis of the pulmonary artery system or cerebral vessels.

Fibrinogen - factor I

Fibrinogen, or factor I, is involved in many intercellular interactions. Its main functions are participation in the formation of a fibrin thrombus (reinforcement of a thrombus) and the implementation of the process of platelet aggregation (attachment of some platelets to others) due to specific platelet receptors of glycoproteins IIb-IIIa.

Plasma fibronectin

Plasma fibronectin is an adhesive glycoprotein that interacts with various blood coagulation factors. Also, one of the functions of plasma fibronectin is the repair of vascular and tissue defects. It has been shown that the application of fibronectin to areas of tissue defects (trophic ulcers of the cornea of ​​the eye, erosions and ulcers of the skin) promotes the stimulation of reparative processes and faster healing.

The normal concentration of plasma fibronectin in the blood is about 300 mcg / ml. In severe injuries, massive blood loss, burns, prolonged abdominal operations, sepsis, acute DIC, the level of fibronectin decreases as a result of consumption, which reduces the phagocytic activity of the macrophage system. This can explain the high frequency of infectious complications in patients who have suffered massive blood loss, and the expediency of prescribing cryoprecipitate or fresh frozen plasma transfusions containing large amounts of fibronectin to patients.

Thrombospondin

The main functions of thrombospondin are to ensure the full aggregation of platelets and their binding to monocytes.

Vitronectin

Vitronectin, or glass-binding protein, is involved in several processes. In particular, it binds the AT III-thrombin complex and subsequently removes it from circulation through the macrophage system. In addition, vitronectin blocks the cellular-lytic activity of the final cascade of complement system factors (C 5 -C 9 complex), thereby preventing the implementation of the cytolytic effect of complement system activation.

clotting factors

The system of plasma coagulation factors is a complex multifactorial complex, the activation of which leads to the formation of a stable fibrin clot. It plays a major role in stopping bleeding in all cases of damage to the integrity of the vascular wall.

fibrinolysis system

The fibrinolysis system is the most important system that prevents uncontrolled blood clotting. Activation of the fibrinolysis system is realized by an internal or external mechanism.

Internal activation mechanism

The internal mechanism of activation of fibrinolysis begins with the activation of plasma XII factor (Hageman factor) with the participation of high molecular weight kininogen and the kallikrein-kinin system. As a result, plasminogen passes into plasmin, which splits fibrin molecules into small fragments (X, Y, D, E), which are opsonated by plasma fibronectoma.

External activation mechanism

The external pathway of activation of the fibrinolytic system can be carried out by streptokinase, urokinase, or tissue plasminogen activator. The external pathway of fibrinolysis activation is often used in clinical practice for the lysis of acute thrombosis of various localizations (with pulmonary embolism, acute myocardial infarction, etc.).

System of primary and secondary anticoagulants-antiproteases

A system of physiological primary and secondary anticoagulants-antiproteases exists in the human body to inactivate various proteases, plasma coagulation factors, and many components of the fibrinolytic system.

Primary anticoagulants include a system that includes heparin, AT III and KG II. This system predominantly inhibits thrombin, factor Xa, and a number of other factors of the blood coagulation system.

The protein C system, as already noted, inhibits plasma coagulation factors Va and VIIIa, which ultimately inhibits blood coagulation by an internal mechanism.

The tissue thromboplastin inhibitor system and heparin inhibit the extrinsic pathway of blood coagulation activation, namely the TF-VII complex. Heparin in this system plays the role of an activator of the production and release into the bloodstream of an inhibitor of tissue thromboplastin from the endothelium of the vascular wall.

PAI-1 (tissue plasminogen activator inhibitor) is the main antiprotease that inactivates tissue plasminogen activator activity.

Physiological secondary anticoagulants-antiproteases include components whose concentration increases during blood coagulation. One of the main secondary anticoagulants is fibrin (antithrombin I). It actively sorbs on its surface and inactivates free thrombin molecules circulating in the bloodstream. Derivatives of factors Va and VIIIa can also inactivate thrombin. In addition, thrombin in the blood is inactivated by circulating molecules of soluble glycocalycin, which are residues of the platelet glycoprotein Ib receptor. In the composition of glycocalycin there is a certain sequence - a "trap" for thrombin. The participation of soluble glycocalycin in the inactivation of circulating thrombin molecules makes it possible to achieve self-limitation of thrombus formation.

System of primary reparants-healers

In the blood plasma there are certain factors that contribute to the healing and repair of vascular and tissue defects - the so-called physiological system of primary repair-healers. This system includes:

• plasma fibronectin,
• fibrinogen and its derivative fibrin,
• transglutaminase or factor XIII of the blood coagulation system,
• thrombin,
• platelet growth factor - thrombopoietin.

The role and significance of each of these factors has already been discussed separately.

The mechanism of blood clotting

Allocate internal and external mechanism of blood coagulation.

Intrinsic pathway of blood clotting

In the internal mechanism of blood coagulation, factors that are in the blood under normal conditions participate.

In the internal pathway, the process of blood coagulation begins with contact or protease activation of factor XII (or Hageman factor) with the participation of high molecular weight kininogen and the kallikrein-kinin system.

Factor XII is converted to factor XIIa (activated) factor, which activates factor XI (the precursor of plasma thromboplastin), converting it to factor XIa.

The latter activates factor IX (antihemophilic factor B, or Christmas factor), converting it with the participation of factor VIIIa (antihemophilic factor A) into factor IXa. The activation of factor IX involves Ca 2+ ions and the 3rd platelet factor.

The complex of factors IXa and VIIIa with Ca 2+ ions and platelet factor 3 activates factor X (Stewart factor), converting it into factor Xa. The factor Va (proaccelerin) also takes part in the activation of factor X.

The complex of factors Xa, Va, Ca ions (IV factor) and the 3rd platelet factor is called prothrombinase; it activates prothrombin (or factor II), turning it into thrombin.

The latter splits fibrinogen molecules, converting it into fibrin.

Fibrin from a soluble form under the influence of factor XIIIa (fibrin-stabilizing factor) turns into insoluble fibrin, which directly reinforces (strengthens) the platelet thrombus.

extrinsic pathway of blood clotting

The external mechanism of blood coagulation is carried out when tissue thromboplastin (or III, tissue factor) enters the circulatory bed from tissues.

Tissue thromboplastin binds to factor VII (proconvertin), converting it to factor VIIa.

The latter activates the X factor, converting it into the X factor.

Further transformations of the coagulation cascade are the same as during the activation of plasma coagulation factors by an internal mechanism.

Mechanism of blood clotting briefly

In general, the mechanism of blood coagulation can be briefly represented as a series of successive stages:

1. as a result of a violation of normal blood flow and damage to the integrity of the vascular wall, an endothelial defect develops;
2. von Willebrand factor and plasma fibronectin adhere to the exposed basement membrane of the endothelium (collagen, laminin);
3. circulating platelets also adhere to collagen and basement membrane laminin, and then to von Willebrand factor and fibronectin;
4. adhesion of platelets and their aggregation lead to the appearance of the 3rd plate factor on their outer surface membrane;
5. with the direct participation of the 3rd plate factor, activation of plasma coagulation factors occurs, which leads to the formation of fibrin in the platelet thrombus - the reinforcement of the thrombus begins;
6. the fibrinolysis system is activated both by internal (through XII factor, high-molecular kininogen and kallikrein-kinin system) and external (under the influence of TAP) mechanisms, stopping further thrombosis; in this case, not only lysis of thrombi occurs, but also the formation of a large number of fibrin degradation products (FDP), which in turn block pathological thrombus formation, having fibrinolytic activity;
7. repair and healing of the vascular defect begins under the influence of physiological factors of the reparative-healing system (plasma fibronectin, transglutaminase, thrombopoietin, etc.).

In acute massive blood loss complicated by shock, the balance in the hemostasis system, namely between the mechanisms of thrombosis and fibrinolysis, is quickly disturbed, since consumption significantly exceeds production. The developing depletion of blood coagulation mechanisms is one of the links in the development of acute DIC.

Blood coagulation is an extremely complex and in many ways still mysterious biochemical process that starts when the circulatory system is damaged and leads to the transformation of blood plasma into a gelatinous clot that plugs the wound and stops bleeding. Violations of this system are extremely dangerous and can lead to bleeding, thrombosis or other pathologies, which together are responsible for the lion's share of death and disability in the modern world. Here we will consider the device of this system and talk about the most recent achievements in its study.

Anyone who has received a scratch or a wound at least once in his life, thereby acquired a wonderful opportunity to observe the transformation of blood from a liquid into a viscous non-fluid mass, leading to a stop of bleeding. This process is called blood clotting and is controlled by a complex system of biochemical reactions.

Having some kind of bleeding control system is absolutely essential for any multicellular organism that has a liquid internal environment. Blood clotting is also vital for us: mutations in the genes for the main clotting proteins are usually lethal. Alas, among the many systems of our body, violations of which pose a danger to health, blood clotting also occupies the absolute first place as the main immediate cause of death: people suffer from various diseases, but almost always die from blood clotting disorders. Cancer, sepsis, trauma, atherosclerosis, heart attack, stroke - for the widest range of diseases, the immediate cause of death is the inability of the coagulation system to maintain a balance between the liquid and solid states of the blood in the body.

If the cause is known, why not fight it? Of course, it is possible and necessary to fight: scientists are constantly creating new methods for diagnosing and treating clotting disorders. But the problem is that the clotting system is very complex. And the science of the regulation of complex systems teaches that such systems need to be managed in a special way. Their reaction to external influences is non-linear and unpredictable, and in order to achieve the desired result, you need to know where to apply the effort. The simplest analogy: to launch a paper airplane into the air, it is enough to throw it in the right direction; at the same time, for an airliner to take off, you need to press the right buttons in the cockpit at the right time and in the right sequence. And if you try to launch an airliner with a throw, like a paper airplane, then it will end badly. So it is with the coagulation system: in order to successfully treat, you need to know the “control points”.

Until very recently, blood clotting has successfully resisted attempts by researchers to understand its workings, and only in recent years has there been a quantum leap. In this article, we will talk about this wonderful system: how it works, why it is so difficult to study it, and - most importantly - we will talk about the latest discoveries in understanding how it works.

How is blood clotting

Stopping bleeding is based on the same idea that housewives use to prepare jelly - turning a liquid into a gel (a colloidal system where a network of molecules is formed that can hold in its cells a liquid that is a thousand times greater in weight due to hydrogen bonds with water molecules). By the way, the same idea is used in disposable baby diapers, in which material that swells when wetted is placed. From a physical point of view, there you need to solve the same problem as in curtailment - the fight against leaks with minimal effort.

Blood clotting is central hemostasis(stop bleeding). The second link of hemostasis are special cells - platelets, - able to attach to each other and to the site of injury to create a blood-stopping plug.

A general idea of ​​the biochemistry of coagulation can be obtained from Figure 1, below which shows the reaction of the conversion of soluble protein fibrinogen in fibrin, which then polymerizes into a network. This reaction is the only part of the cascade that has a direct physical meaning and solves a clear physical problem. The role of the remaining reactions is exclusively regulatory: to ensure the conversion of fibrinogen into fibrin only in the right place and at the right time.

Figure 1. The main reactions of blood coagulation. The coagulation system is a cascade - a sequence of reactions, where the product of each reaction acts as a catalyst for the next. The main "entrance" to this cascade is in its middle part, at the level of factors IX and X: protein tissue factor(denoted as TF in the diagram) binds factor VIIa, and the resulting enzymatic complex activates factors IX and X. The result of the cascade is a fibrin protein that can polymerize and form a clot (gel). The vast majority of activation reactions are proteolysis reactions, i.e. partial cleavage of the protein, increasing its activity. Almost every coagulation factor is necessarily inhibited in one way or another: feedback is necessary for the stable operation of the system.

Designations: Reactions for converting coagulation factors into active forms are shown one-sided thin black arrows. Wherein curly red arrows show which enzymes are activated. Activity loss responses due to inhibition are shown thin green arrows(for simplicity, the arrows are depicted as simply "leaving", i.e. it is not shown which inhibitors bind to). Reversible complex formation reactions are shown bilateral thin black arrows. Coagulation proteins are indicated by either names, Roman numerals, or abbreviations ( TF- tissue factor, PC- protein C, APC- activated protein C). To avoid congestion, the diagram does not show: binding of thrombin to thrombomodulin, activation and secretion of platelets, contact activation of coagulation.

Fibrinogen resembles a rod 50 nm long and 5 nm thick (Fig. 2 a). Activation allows its molecules to stick together into a fibrin thread (Fig. 2 b), and then into a fiber capable of branching and forming a three-dimensional network (Fig. 2 in).

Figure 2. Fibrin gel. a - Schematic arrangement of the fibrinogen molecule. Its base is composed of three pairs of mirror-image polypeptide chains α, β, γ. In the center of the molecule, one can see the binding regions that become accessible when thrombin cuts off fibrinopeptides A and B (FPA and FPB in the figure). b - Mechanism of fibrin fiber assembly: molecules are attached to each other "overlapped" according to the head-to-middle principle, forming a double-stranded fiber. in - Electron micrograph of the gel: fibrin fibers can stick together and split, forming a complex three-dimensional structure.

Figure 3. Three-dimensional structure of the thrombin molecule. The scheme shows the active site and the parts of the molecule responsible for the binding of thrombin to substrates and cofactors. (The active site is a part of the molecule that directly recognizes the cleavage site and carries out enzymatic catalysis.) The protruding parts of the molecule (exosites) allow the "switching" of the thrombin molecule, making it a multifunctional protein capable of working in different modes. For example, the binding of thrombomodulin to exosite I physically blocks access to thrombin for procoagulant substrates (fibrinogen, factor V) and allosterically stimulates activity towards protein C.

The fibrinogen activator thrombin (Fig. 3) belongs to the family of serine proteinases, enzymes capable of cleaving peptide bonds in proteins. It is related to the digestive enzymes trypsin and chymotrypsin. Proteinases are synthesized in an inactive form called zymogen. To activate them, it is necessary to cleave the peptide bond that holds the part of the protein that closes the active site. Thus, thrombin is synthesized as prothrombin, which can be activated. As can be seen from fig. 1 (where prothrombin is labeled factor II), this is catalyzed by factor Xa.

In general, clotting proteins are called factors and are numbered with Roman numerals in the order of their official discovery. The index "a" means the active form, and its absence - the inactive predecessor. For long-discovered proteins, such as fibrin and thrombin, proper names are also used. Some numbers (III, IV, VI) are not used for historical reasons.

The clotting activator is a protein called tissue factor present in the cell membranes of all tissues, with the exception of the endothelium and blood. Thus, the blood remains liquid only due to the fact that normally it is protected by a thin protective membrane of the endothelium. In case of any violation of the integrity of the vessel, the tissue factor binds factor VIIa from the plasma, and their complex is called external tenase(tenase, or Xase, from the word ten- ten, i.e. number of activated factor) - activates factor X.

Thrombin also activates factors V, VIII, XI, which leads to an acceleration of its own production: factor XIa activates factor IX, and factors VIIIa and Va bind factors IXa and Xa, respectively, increasing their activity by orders of magnitude (the complex of factors IXa and VIIIa is called internal tenase). Deficiency of these proteins leads to severe disorders: for example, the absence of factors VIII, IX or XI causes severe disease. hemophilia(the famous "royal disease", which was ill with Tsarevich Alexei Romanov); and deficiency of factors X, VII, V or prothrombin is incompatible with life.

Such a device is called positive feedback: Thrombin activates proteins that speed up its own production. And here an interesting question arises, why are they needed? Why is it impossible to immediately make the reaction fast, why does nature make it initially slow, and then come up with a way to further accelerate it? Why is there duplication in the clotting system? For example, factor X can be activated by both complex VIIa-TF (external tenase) and complex IXa-VIIIa (intrinsic tenase); it looks completely pointless.

There are also clotting proteinase inhibitors in the blood. The main ones are antithrombin III and an inhibitor of the tissue factor pathway. In addition, thrombin is able to activate serine proteinase. protein C, which cleaves coagulation factors Va and VIIIa, causing them to completely lose their activity.

Protein C is a precursor of serine proteinase, very similar to factors IX, X, VII and prothrombin. It is activated by thrombin, as is factor XI. However, when activated, the resulting serine proteinase uses its enzymatic activity not to activate other proteins, but to inactivate them. Activated protein C produces several proteolytic cleavages in clotting factors Va and VIIIa, causing them to completely lose their cofactor activity. Thus, thrombin - a product of the coagulation cascade - inhibits its own production: this is called negative feedback. And again we have a regulatory question: why does thrombin simultaneously accelerate and slow down its own activation?

Evolutionary origins of folding

The formation of protective blood systems began in multicellular organisms over a billion years ago - in fact, just in connection with the appearance of blood. The coagulation system itself is the result of overcoming another historical milestone - the emergence of vertebrates about five hundred million years ago. Most likely, this system arose from immunity. The emergence of another system of immune responses that fought bacteria by enveloping them in fibrin gel led to an accidental side effect: bleeding began to stop faster. This made it possible to increase the pressure and strength of the flows in the circulatory system, and the improvement of the vascular system, that is, the improvement of the transport of all substances, opened up new horizons for development. Who knows if the appearance of folds was not the advantage that allowed vertebrates to take their current place in the Earth's biosphere?

In a number of arthropods (such as horseshoe crabs), coagulation also exists, but it arose independently and remained in immunological roles. Insects, like other invertebrates, usually get by with a weaker version of the hemorrhage control system based on the aggregation of platelets (more precisely, amoebocytes - distant relatives of platelets). This mechanism is quite functional, but imposes fundamental restrictions on the efficiency of the vascular system, just as the tracheal form of respiration limits the maximum possible size of an insect.

Unfortunately, creatures with intermediate forms of the clotting system are almost all extinct. Jawless fish are the only exception: a genomic analysis of the lamprey's coagulation system showed that it contains much fewer components (that is, it is much simpler). From jawed fish to mammals, coagulation systems are very similar. Cellular hemostasis systems also operate on similar principles, despite the fact that small, non-nucleated platelets are unique to mammals. In other vertebrates, platelets are large cells with a nucleus.

In summary, the coagulation system is very well understood. No new proteins or reactions have been discovered in it for fifteen years, which is an eternity for modern biochemistry. Of course, the possibility of such a discovery cannot be completely ruled out, but so far there is not a single phenomenon that we could not explain using the available information. Rather, on the contrary, the system looks much more complicated than necessary: ​​we recall that of all this (rather cumbersome!) cascade, only one reaction is actually involved in gelling, and all the rest are needed for some kind of incomprehensible regulation.

That is why now coagulologist researchers working in various fields - from clinical hemostasiology to mathematical biophysics - are actively moving from the question "How is folded?" to questions "Why is folded the way it is?", "How does it work?" and finally “How do we need to influence clotting to achieve the desired effect?”. The first thing to do in order to answer is to learn how to study the whole clotting, and not just individual reactions.

How to investigate coagulation?

To study coagulation, various models are created - experimental and mathematical. What exactly do they allow you to get?

On the one hand, it seems that the best approximation for studying an object is the object itself. In this case, a person or an animal. This allows you to take into account all factors, including blood flow through the vessels, interactions with the walls of blood vessels, and much more. However, in this case, the complexity of the problem exceeds reasonable limits. Convolution models make it possible to simplify the object of study without losing its essential features.

Let's try to get an idea of ​​what requirements these models should meet in order to correctly reflect the process of folding. in vivo.

The experimental model should contain the same biochemical reactions as in the body. Not only proteins of the coagulation system should be present, but also other participants in the coagulation process - blood cells, endothelial and subendothelium. The system must take into account the spatial heterogeneity of coagulation in vivo: activation from the damaged area of ​​the endothelium, the spread of active factors, the presence of blood flow.

Considering coagulation models, it is natural to start with methods for studying coagulation. in vivo. The basis of almost all approaches of this kind used is to inflict controlled injury on the experimental animal in order to cause a hemostatic or thrombotic reaction. This reaction is studied by various methods:

• monitoring bleeding time;
• analysis of plasma taken from an animal;
• autopsy of the slaughtered animal and histological examination;
• real-time monitoring of a thrombus using microscopy or nuclear magnetic resonance (Fig. 4).

Figure 4. Thrombus formation in vivo in a laser-induced thrombosis model. This picture is reproduced from a historical work, where scientists were able to observe the development of a blood clot "live" for the first time. To do this, a concentrate of fluorescently labeled antibodies to coagulation proteins and platelets was injected into the mouse blood, and, placing the animal under the lens of a confocal microscope (allowing three-dimensional scanning), an arteriole under the skin accessible for optical observation was selected and the endothelium was damaged with a laser. Antibodies began to attach to the growing clot, making it possible to observe it.

The classical setting of the clotting experiment in vitro consists in the fact that blood plasma (or whole blood) is mixed in a certain container with an activator, after which the coagulation process is monitored. According to the method of observation, experimental techniques can be divided into the following types:

• observation of the clotting process itself;
• observation of changes in the concentrations of coagulation factors over time.

The second approach provides incomparably more information. Theoretically, knowing the concentrations of all factors at an arbitrary point in time, one can obtain complete information about the system. In practice, the study of even two proteins at the same time is expensive and associated with great technical difficulties.

Finally, coagulation in the body proceeds inhomogeneously. The formation of a clot starts on the damaged wall, spreads with the participation of activated platelets in the plasma volume, and stops with the help of the vascular endothelium. It is impossible to adequately study these processes using classical methods. The second important factor is the presence of blood flow in the vessels.

Awareness of these problems has led to the emergence, since the 1970s, of various flow experimental systems. in vitro. Somewhat more time was required to realize the spatial aspects of the problem. Only in the 1990s did methods begin to appear that take into account spatial heterogeneity and diffusion of coagulation factors, and only in the last decade have they been actively used in scientific laboratories (Fig. 5).

Figure 5. Spatial growth of a fibrin clot in normal and pathological conditions. Coagulation in a thin layer of blood plasma was activated by tissue factor immobilized on the wall. In the photos, the activator is located left. Gray flared stripe- growing fibrin clot.

Along with experimental approaches, mathematical models are also used to study hemostasis and thrombosis (this research method is often called in silico). Mathematical modeling in biology makes it possible to establish deep and complex relationships between biological theory and experience. The experiment has certain limits and is associated with a number of difficulties. In addition, some theoretically possible experiments are not feasible or prohibitively expensive due to the limitations of the experimental technique. Simulation simplifies experiments, as you can pre-select the necessary conditions for experiments in vitro and in vivo, at which the effect of interest will be observed.

Regulation of the coagulation system

Figure 6. The contribution of external and internal tenase to the formation of a fibrin clot in space. We used a mathematical model to explore how far the influence of a clotting activator (tissue factor) can extend in space. To do this, we calculated the distribution of factor Xa (which determines the distribution of thrombin, which determines the distribution of fibrin). The animation shows the distributions of the factor Xa, produced by external tenase(complex VIIa–TF) or internal tenase(complex IXa–VIIIa), as well as the total amount of factor Xa (shaded area). (Inset shows the same on a larger scale of concentrations.) It can be seen that factor Xa produced on the activator cannot penetrate far from the activator due to the high rate of inhibition in plasma. On the contrary, complex IXa–VIIIa works away from the activator (because factor IXa is inhibited more slowly and therefore has a greater distance of effective diffusion from the activator), and ensures the distribution of factor Xa in space.

Let's take the next logical step and try to answer the question - how does the system described above work?

Cascade Device Coagulation System

Let's start with a cascade - a chain of enzymes activating each other. One enzyme, running at a constant rate, gives a linear dependence of the concentration of the product on time. At the cascade of N enzymes, this dependence will have the form t N, where t- time. For the effective operation of the system, it is important that the response be of just such an “explosive” character, since this minimizes the period when the fibrin clot is still fragile.

Coagulation Triggering and the Role of Positive Feedbacks

As mentioned in the first part of the article, many clotting reactions are slow. For example, factors IXa and Xa are themselves very poor enzymes and require cofactors (factors VIIIa and Va, respectively) to function effectively. These cofactors are activated by thrombin: such a device, when the enzyme activates its own production, is called a positive feedback loop.

As we have shown experimentally and theoretically, a positive feedback of factor V activation by thrombin forms an activation threshold - the property of the system not to respond to a small activation, but to quickly work when a large one appears. This ability to switch seems to be very valuable for curtailment: it helps to prevent "false positives" of the system.

The Role of the Intrinsic Pathway in the Spatial Dynamics of Coagulation

One of the intriguing mysteries that haunted biochemists for many years after the discovery of the major coagulation proteins was the role of factor XII in hemostasis. Its deficiency was found in the simplest clotting tests, increasing the time required for clot formation, however, unlike factor XI deficiency, it was not accompanied by clotting disorders.

One of the most plausible options for unraveling the role of the internal path was proposed by us with the help of spatially inhomogeneous experimental systems. It was found that positive feedbacks are of great importance precisely for the propagation of coagulation. Effective activation of factor X by external tenase on the activator will not help to form a clot away from the activator, since factor Xa is rapidly inhibited in plasma and cannot move far from the activator. But factor IXa, which is inhibited an order of magnitude slower, is quite capable of this (and factor VIIIa, which is activated by thrombin, helps it). And where it is difficult for him to reach, factor XI, also activated by thrombin, begins to work. Thus, the presence of positive feedback loops helps to create a three-dimensional bunch structure.

Protein C pathway as a possible mechanism for the localization of thrombus formation

The activation of protein C by thrombin itself is slow, but it accelerates sharply when thrombin binds to the transmembrane protein thrombomodulin synthesized by endothelial cells. Activated protein C is able to destroy factors Va and VIIIa, slowing down the coagulation system by orders of magnitude. Spatially inhomogeneous experimental approaches became the key to understanding the role of this reaction. Our experiments suggested that it stops the spatial growth of the thrombus, limiting its size.

Summarizing

In recent years, the complexity of the coagulation system has gradually become less mysterious. The discovery of all essential components of the system, the development of mathematical models and the use of new experimental approaches made it possible to lift the veil of secrecy. The structure of the coagulation cascade is being deciphered, and now, as we saw above, for almost every essential part of the system, the role that it plays in the regulation of the entire process has been identified or proposed.

Figure 7 presents the most recent attempt to rethink the structure of the clotting system. This is the same circuit as in Fig. 1, where parts of the system responsible for different tasks are highlighted with multi-colored shading, as discussed above. Not everything in this circuit is securely installed. For example, our theoretical prediction that factor VII activation by factor Xa allows clotting to threshold-response to flow rate remains as yet untested experimentally.