How a blood clot grows

Forty-five microns per minuteGalina Kostina, special correspondent of the Expert magazine.

Fazli Ataullakhanov, professor of the Department of Biophysics of the Faculty of Physics of Moscow State University, fifteen years ago, while giving students a familiar lecture on blood clotting, suddenly realized that he did not fully understand how this process actually happens. But he was in charge of the Laboratory of Physical Biochemistry of the Hematology Research Center of the Russian Academy of Medical Sciences — the main place in Russia, where blood diseases are studied and treated. It seems that in textbooks it is written quite neatly that many factors in the blood plasma are involved in the process. It seems that all biochemical reactions are known. It's clear where it all starts. But no one has ever described in detail how the whole process of clotting and thrombus formation occurs in dynamics, and most importantly, why this process stops. Fazli was stung. He rummaged through the literature and realized that no one had thought to put quite simple experiments that would simulate the process of clotting in the body. And these experiments would be much more informative than existing clotting tests. One of the most common diagnostic methods is that an appropriate substance is added to a test tube with blood, which initiates clotting, shaken and waiting for the blood to curdle. If the blood coagulates within about 30 seconds, it is considered that everything is fine, if more than a minute — it may be hemophilia, a genetic disorder of clotting, widespread in the august surnames of Europe and Russia. The problem, however, was the diagnosis of the reverse condition — a tendency to increased clotting, or thrombophilia. Fazli Ataullakhanov thought that experimental clarification of the details of the mechanism of blood clotting might solve the problem of diagnosis. But at the first stage, he was seized by the purely scientific excitement of solving the mysterious question of what laws the folding process obeys.

The blood runs cold in the veinsEvery day, and maybe every hour, there are breakdowns in the blood vessels.

Even if it's a tiny hole in the capillary, all the blood could leak through it if we didn't have a special protective system. We can observe this protective process if, for example, we cut a finger. The blood thickens, forms first a film, then a thrombus, sealing a cut or a hole. After a few days, when a new tissue forms under the thrombus, it resolves. In such a case, the coagulation system acts as a protective one. Due to constant minor breakdowns, the system must be powerful. But this power has a downside. "Our blood can be called an explosive mixture," says Ataullakhanov. — It has everything to collapse it all, and it will look like a cast of our circulatory system. Therefore, the system quite often works at those moments when it is not required. This is because clotting is initiated due to inflammation in the tissues and cell death or due to infectious pathogens. As a rule, in youth, the body copes with this spontaneous formation of blood clots, anti-clotting agents are constantly working in the blood. However, it may happen that a blood clot forms in a “bad” place, for example, it will clog the vessel supplying the heart muscle or brain. And the defenses do not have time, a heart attack or stroke occurs. The older a person is, the higher the risk of spontaneous blood clots. Alas, the diagnosis of thrombophlebia often occurs after the fact of the formation of blood clots, the result is severe conditions and often death."

The study of the nature of clotting began at the end of the XIX century, but only by the middle of the XX century a certain general scheme was established. It became clear that blood cells — platelets — and about two dozen plasma proteins, the so-called blood clotting factors, are involved in this process. Initially, all of them are present in the blood. These factors are in an inactive state. In order for the process to be started, an appropriate signal is needed. The main one is the action of protein, which is found in all tissues of the body, with the exception of the endothelium lining the inner wall of blood vessels. It is called tissue factor. As soon as this insulating endothelium is disrupted as a result of a vessel breakdown, the blood comes into contact with the tissue factor, and the coagulation system is activated. A whole chain of biochemical reactions begins: a tissue factor activates one coagulation factor, one another, and so on. And interestingly, a process similar to an avalanche starts: clotting factors activate not only the following factors, but also indirectly themselves — and so the process of self-acceleration begins. As a result, many thrombin molecules are formed in the blood, which activate the main molecule for the formation of a thrombus — fibrin. Numerous fibrin molecules stick together, forming twisted threads and networks that entangle blood cells. This clot is a blood clot.

Ataullakhanov was occupied with the scenario of the process. If thrombin was formed only in the place where there is a tissue factor, then the thrombus would be too small. Imagine that the fibrin molecule forming a blood clot is measured in several tens of nanometers, and the hole it has to plug is in millimeters, a million times larger than the fibrin molecule. To overcome this gigantic chasm, the process must spread quite actively. "And I wanted to understand where the process is spreading and what its patterns are," says Fazli. "If it spreads due to the self—acceleration system, then why does a blood clot grow to a certain place, and then not, although according to the logic of this self-acceleration, blood should clot wherever there are relevant factors activated by thrombin — and they are in the entire circulatory system."

Surprised that no one had thought of putting quite simple experiments to look at the dynamics of coagulation, Fazli decided to evaluate it from the point of view of a physicist. He created a group that included some employees of his laboratory, in particular Vasily Sarbash and Rimma Volkova, as well as his graduate students and students. One of the students, Mikhail Panteleev, who took part in the development of some mathematical models necessary for research, later became an employee of the laboratory. Scientists have set up a fairly simple experiment. They imitated the process of coagulation in the body by growing fibroblasts on a special plate, on the surface of which there is a tissue factor. The plate was brought into contact with blood plasma and watched as a blood clot grew. The whole process was filmed on a digital camera. And we were surprised to find that the patterns of this process are not similar to the patterns of ordinary physical processes. At the beginning of the experiment, Ataullakhanov wanted to find out which physical method of perturbation propagation the folding process obeys — a conventional wave or an autowave. In the first case, it could be assumed that the tissue factor is the pebble that causes the usual wave, gradually fading. Or maybe this process will be like an autowave or gorenje process, when everything burns with maximum amplitude and stops only when the "fuel" runs out. During the experiments , it turned out: the method is neither one nor the other. In some cases (when the blood is healthy), the process begins as an autowave — thrombin is intensively formed in the blood, followed by fibrin, but it does not end as an autowave — the "fire" stops abruptly, despite the fact that there is plenty of "fuel" around. When, for example, the blood plasma of a hemophiliac was examined, the process was more like a spoiled gorenje — clotting started as usual, but then it went slowly, and the thrombus grew badly. "And when we discovered this new process, together with another employee of the laboratory, Georgy Guria, we published the first article in 1995 and then a bunch of articles in well—known physics journals and reported on it at many scientific conferences," says Fazli Ataullakhanov. — This came as a surprise to both biochemists and physicists. Now physicists are studying processes of this kind with might and main."

Thrombosis Fire ExtinguisherMost of all, Fazli was surprised by the unexpected stop of coagulation.

In fact, there is a self—accelerating process, it produces a lot of thrombin, and that produces a lot of fibrin, as if there is a powerful burning of an ignited gasoline film.gorenje. This self-acceleration is necessary in order to overcome the coagulation-inhibiting system in the blood. Normally, thrombin inhibitors, in particular antithrombin and alpha-2−macroglobulin, are constantly in the blood to prevent spontaneous formation of blood clots. But they are not able to stop the powerful wave. So, Fazli thought, this process must find something inside itself that was absent at the very beginning of the process (otherwise there will be no "gorenje"), but then it arose and served as a fire extinguisher that stops the whole fire. It was known that some proteins act to suppress thrombin, which are also activated during the clotting process, for example, the so-called protein C. It is curious what activates this protein With the same thrombin, which is the main "fire fan", that is, it acts not only on factors that trigger self-acceleration of clotting, but also on factors that will stop the process. But no one knew the details. In the experiment, it turned out that nature came up with a very original mechanism: despite the fact that thrombin starts both processes, the activation of the "fire extinguisher" is slower than the activation of the "fire" in order to make it possible to construct a thrombus of the right size.

Now Ataullakhanov's group has laid out the whole process on the shelves: what initiates, what ignites and accelerates, what stops — and how it happens in dynamics. A lot of experiments made it possible to track all these stages. The revealed characteristics of the process — the time of the onset of thrombus growth, the growth rate and the size of the thrombus — make it possible to differentiate not only hemophilia and thrombophilia, but also their various variations. And this means that they should be treated taking into account these characteristics.

Along the way, Ataullakhanov's group and the staff of the Hemophilia Center of the National Research Center of the Russian Academy of Medical Sciences, headed by Professor Lyudmila Plyushch, experimentally found out that the traditional treatment of hemophilia also needs correction. If there is a lack of the appropriate clotting factor, it is completely replaced. Previously, doctors intuitively noted that in such hemophiles, oddly enough, the risk of spontaneous blood clots increases. "The fact is that the body is trying to compensate for the lack of the necessary factor at least partially, so when it is added in full, it becomes more than necessary," says Fazli. — In experiments, we found that it is optimal to add about twenty percent of the desired clotting factor. And the effect is already visible on patients, they feel much better."

From their experiments, Fazli's group learned another thing: with the introduction of blood substitutes in the case of severe blood loss, the risk of thrombosis increases. The nature of this was unclear: it would seem that with blood dilution, clotting should be worse. But the doctors from the intensive care unit, with whom Elena Sinauridze, an employee of the laboratory, conducted research, knew that after such transfusions, blood clots often form. It turned out that both clotting factors and thrombin inhibitors — antithrombin, etc., are being eroded. But the latter are more important, because, unlike inactive blood factors, they are always active, and when their activation occurs, there is practically no one to fight them. Then scientists proposed to make a solution taking into account these features of the coagulation and anti-coagulation systems.

A device for the global marketFor their experiments, the scientists of the SSC created a laboratory device, which, as the head of the laboratory jokes, looked like a hut on chicken legs.

Only scientists could work with him. When it became clear that a unique diagnostic experience had matured, scientists thought about the practical side of the project. The company "Hematological Devices" was created, which at the first stage needed to create the first experimental industrial samples so that a laboratory assistant in any clinic could work with them almost automatically. "I took a graduate student in engineering from Baumanka — Sergey Karamzin, with him and my long—time employee and friend Vasily Sarbash, we began to improve the device," says Fazli. — And they transferred him from the laboratory to the diagnostic one." Igor Pivovarov, another former graduate student of Ataullakhanov, and now the CEO of a company producing laser medical equipment, took part in the grinding and manufacture of these devices. Special programs have been made for the computer connected to the device, and on the screen you can see online how the blood clot grows, as well as graphs from which all the main characteristics of the clotting process are visible — the start time, speed and size of the blood clot. The main indicator is the rate of thrombus formation. The norm fits into the indicator of 35-45 microns per minute. If the rate is lower, it shows a tendency to hemophilia, if higher — to thrombophilia.

We had to solve another important issue: it is impossible to imagine that fibroblasts with a tissue factor will be specially cultivated in the polyclinic, therefore, in the diagnostic device, the initiation of the process is performed by a special activator very similar to the tissue factor in the cell membrane. It was made using nanotechnology, for which Fazli attracted another of his graduate students, chemist-technologist Olga Fadeeva. "Fazli is a man of encyclopedic knowledge himself, so it's not surprising that he finds ideas at the intersection of sciences — biology, chemistry, physics," says Igor Pivovarov. But he always attracts young specialists from many universities to help, although, in fact, he helps them more by involving them in real, interesting science."

In general terms, it was clear that the young company should first create several devices and distribute them to leading clinics. One of the devices worked for some time at the Institute of the Red Cross. John Holand is in the USA and still works in one of the clinics of Lyon in France. The device was patented in Switzerland, but now you need to get national patents in different countries, which requires a lot of money. Funds are needed for the production of devices, which, most likely, will be made by Igor Pivovarov's company, and for their promotion to the world market. "The RFBR helped us a lot in the early stages," says Fazli. — But we have passed the R&D phase, and now we need a larger investor of a different competence. Since nanotechnology is used in the device, we submitted our project to Rusnano Corporation."

According to Igor Pivovarov, Rusnano was pleasantly surprised by its professionalism and efficiency: "Firstly, at the very beginning we were asked questions that allowed us to formulate our strategy more clearly. Secondly, the corporation has ordered a number of examinations, including international ones. Thirdly, it helped to assess the market prospects. And now we are modestly counting on at least ten percent of the European market with a capacity of 500 million dollars." In fact, it is modest, since no one is doing such tests yet. The new diagnostic method in the future will significantly expand the market, because for the first time it makes it possible to assess the tendency to blood clots, and quite quickly and efficiently.

Portal "Eternal youth" www.vechnayamolodost.ru22.12.2008