Thrombin
Text & References
H.C. Hemker
1
To the reader,
Here follows the text of my comic book on thrombin together with the appropriate references. In the booklet itself references have been omitted in order to maintain its unassuming character. However unassuming, it is meant to be a proper scientific text and thus the foundation of its statements should be asserted. Therefore they are published here for the interested reader. We cannot claim them to be complete however. In selecting the references we have – within the limits of our knowledge and possibilities - tried to do justice to the authors that did the original discovery.
One of the ways to become famous in science is to publish a review article in which the seminal articles are mentioned and then in later works refer to that review article. Another one is to contradict a seminal article and then, in a series of subsequent articles change one’s standpoint so as to finally arrive at that of the original article. With a bit of luck the original article is by that time forgotten. A third one is to bring about a minute modification to a novel technique and refer to that modification in further work. We have done our best to avoid such tricks. Nevertheless we may have made mistakes, so we invite the reader to extend, update and ameliorate the present references. A simple e-mail to hc.hemker@thrombin.com will do.
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Page 5.
Thrombin is the central enzyme in haemostasis1, 2. It is a potent proteolytic enzyme with numerous
different functions3, 4. It activates blood platelets and makes blood clot. By its action on platelets, on the
vessel wall and on blood proteins, thrombin repairs leaks in the circulation. If this function is triggered where it should not, normal blood flow is blocked (thrombosis) and tissue may die due to lack of oxygen (infarction). It is not surprising, therefore, that there is an extremely fine-tuned system for the regulation of thrombin formation.
Page 6.
Thrombosis and haemostasis always have a local and a systemic component. The local component can be a wound or internal vessel damage. When a vessel is wounded, an explosion of thrombin follows immediately. This results in the rapid formation of a plug consisting of platelets and fibrin. Once the wound is contained this process is switched off so that the plug remains limited to the damage area and does not grow into undamaged vessels. If it did, functional vessels would be occluded and the supply to or the draining of tissues would be impaired. The haemostatic reaction may also be triggered untowardly e.g. by a ruptured atherosclerotic plaque that then can cause a coronary or a cerebral infarction, or by the minimal venous damage that triggers the formation of a venous thrombus can grow. The flow conditions at the site of damage determine whether blood clotting will become evident or not, i.e. whether the thrombus will be white or red. If and where a thrombus or a haemorrhage will occur is determined by the damage of the vessel wall. How serious the event will be in relation to the damage depends on the blood. Apart from local measures such as closing an artery or removing a thrombus, management of haemostasis and thrombosis largely amounts to manipulation of the amount of thrombin that the blood can form.
Page 7.
The amount of thrombin that is formed must be large enough to ensure haemostasis and not so large as to cause thrombosis; hence: The first law of haemostasis and thrombosis:
“The more thrombin, the less bleeding but the more thrombosis; the less thrombin, the more bleeding but the less thrombosis.”
Thrombosis and bleeding together account for about half of all death and disease in the western hemisphere5. The importance of adequate thrombin formation therefore can hardly be overestimated.
In patients with a thrombotic tendency it may be necessary to decrease the amount of thrombin that is formed; a bleeding tendency may require increasing it. Luckily there are pharmacological tools to bring such changes about and diagnostic tools to measure their effect.
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As always in important biological systems, the delicate regulation of thrombin generation is ensured by a system of positive and negative feedbacks, in which thrombin influences its own formation and inactivation. This mechanism will be discussed hereafter. For daily medical practice it is not necessary to know the details of this beautiful but surprisingly complicated system. In fact the very simple scheme on the next page suffices.
Page 8.
The simplest clotting scheme
When the thrombin forming mechanism is triggered it takes some time before thrombin appears. As we will see later, a slow initiation mechanism is required to set thrombin generation going. The first traces of thrombin immediately make the plasma clot6 REF. They also, by positive feedback, set the production
mechanism of thrombin going at full speed7-9 REF. This makes that a large amount of thrombin activity
develops in the clot. The higher the thrombin concentration rises, the faster it will be inactivated by the antithrombins. After a few minutes prothrombin conversion gradually stops and the antithrombins get the better of the remaining thrombin. The situation resembles a sink into which a bucket of water is quickly emptied. The water level rises but the more the level builds up the faster the water drains away. When the inflow stops the level will gradually fall to zero. The course of the thrombin-in-time curve, the thrombogram, tells how much thrombin has been produced.
Page 9.
How readily blood will close a wound or form a thrombus is known as its “coagulability”.
This suggests that the clotting time might reflect the amount of thrombin formed. Unfortunately there is not necessarily a close connection between the two. Because the blood clots at the end of the initiation phase, the clotting time reflects the initiation mechanism only. We will see later that this mechanism is quite different from the mechanism that makes the bulk of thrombin. The clotting time therefore does not predict how much thrombin is going to appear. The length of the fuse may or may not predict the size of the bomb. Also a clotting time can hardly become shorter than normal so that it cannot indicate when too much thrombin is going to be formed. There is no escape from the second law of haemostasis and thrombosis: “If you want to know about thrombin - measure thrombin.”
Page 10.
The thrombogram
For medical purposes it thus is useful to quantify the amount of thrombin activity that develops in a sample of clotting plasma or blood. One can e.g. measure the peak level that is attained. However: not only the concentration of thrombin matters but also the time that it can act. During one minute, 100 nanomoles of thrombin can potentially convert as much product as 10 nanomoles thrombin can during
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ten minutes. That is why the time that thrombin is present, multiplied by its concentration, i.e. the area under the thrombin generation curve, also known as the Endogenous Thrombin Potential (ETP), in an important parameter10 REF. Further parameters that characterize the thrombogram are the lag-time, which for all practical purposes is equal to the clotting time, and the time to peak. Later we will see how we obtain the thrombogram in actual practice. First we discuss how thrombin is actually formed; how it is brought about that thrombin forms explosively and how that explosion remains limited in time and in space so as to ensure haemostasis and prevent thrombosis.
Page 11.
The mechanism of thrombin formation.
Thrombin is present in plasma in the form of an inactive precursor protein: Prothrombin (blood clotting factor II). This protein must be split in two places to become thrombin11 REF. This is an example of limited
proteolysis, a basic mechanism that can be recognized at every step of the coagulation process. The core reactions that lead to the formation of thrombin are very simple indeed. Tissue damage activates factor VII. Factor VIIa activates factor X, factor Xa activates prothrombin. This is the so called “clotting cascade” which has a strong amplifying effect12, 13 REF.
Page 12.
A second principle of blood coagulation is that the activation reactions do not, or hardly, proceed in free solution but rather at the surface of membranes10, 14 REF. By adsorbing to a membrane, prothrombin
becomes a better substrate for factor Xa and is converted some one thousand times faster than in free solution15 REF. Only membranes with negatively charged phospholipids (phosphatidyl serine,
phosphatidyl ethanolamine) can serve this purpose. In fact yet another protein is involved, factor Va. This protein itself is not a proteolytic enzyme but it is a cofactor that speeds up the activation of prothrombin by factor Xa again about a thousand times15, 16 REF. The process of thrombin formation
thus can be accelerated up to a million times as the three members of the prothrombinase complex combine.
Page 13.
The role of cell membranes in thrombin generation
Before explaining blood coagulation any further we must know more about cell membranes. They consist of a bilayer of phospholipids in and on which proteins are attached. These proteins, apart from tissue factor and thrombomodulin that we will meet later, do not concern us here. Phospholipid molecules consist of a hydrophilic head and two hydrophobic tails. The head can carry a neutral, a positive or a negative charge. The hydrophobic tails avoid contact with the surrounding solution and cluster together at the inside of the membrane, while the hydrophilic heads point outwards17 REF. It is
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very important to note that cell membranes are asymmetric 18REF. The outside hardly carries any
negative charge, the negatively charged heads are found almost exclusively at the inside of the cell. Only negatively charged membranes can carry clotting factors19, 20 REF. Intact cells therefore are not
procoagulant.
Page 14.
But how did it all start?
In the tissues around the vessels are cells that carry Tissue Factor (TF). This protein is fixed into the cell membrane and a big part shows at the outside of the cell 21REF. When a wound occurs, blood leaks into
the tissue and factor VII from plasma lands on the membrane next to TF 22REF. Factor VII is a proenzyme
of the same kind as factor X but a peculiar one that has a little bit of activity of its own 23REF. When it
interacts with TF, this inherent activity is strongly enhanced23 REF. This makes that the proenzyme factor
VII now can make some factor Xa out of factor X. Factor Xa in its turn acts on factor VII to make fully active factor VIIa 24REF. Such mutual activation leads to an explosion of activity that could become
dangerous. Below we will see how factors Xa and Va also will put a brake on this process.
Page 15.
The molecules of factor Xa thus generated can convert some molecules of prothrombin into thrombin, even without the help of factor Va. The first type of thrombin that is made here (meizothrombin) is enzymatically active but remains bound to the membrane25, 26 REF. It thus is in a perfect position to
activate factor V that itself also binds to the procoagulant membrane. Once factor V is activated it forms the prothrombinase complex with factor Xa and the production of thrombin can start in full force15.
Page 16.
When factor Xa is not bound to a membrane but is free in the plasma, it finds a protein there that is called TF-pathway inhibitor (TFPI), to which it binds27 REF. The resulting complex is a very efficient
inhibitor of the TF-VIIa complex28, 29 REF. In this way factor Xa blocks its own generation after having
stimulated it. Very recently we found that factor Va, when it is not bound to a membrane, also is a potent inhibitor of TF-VIIa 30REF. Both factors, Xa as well as Va, are therefore procoagulant when on a
membrane and anticoagulant when in solution. This is very important because it makes that thrombin is generated only there where procoagulant membranes are to be found, that is on damaged cells and activated platelets (as we will see below).
Page 17.
Factor Xa, by combining with TFPI and inhibiting the TF-Factor VII complex shuts off its own formation. The result is a short pulse of factor Xa activity. If there is an excess of TF around, as e.g. in a “prothrombin time“ measurement, this pulse will generate sufficient thrombin and one will measure the initiation
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mechanism only. When only small amounts of TF are available this direct pathway of factor Xa generation is not sufficient. For these cases nature has provided a side step, the Josso loop31, 32 REF. This
mechanism starts because factor VII and TF also activate factor IX (antihaemophilic factor B), in a similar way as it activates factor X. Factor IXa can activate factor X but for its activity it requires procoagulant phospholipid and activated factor VIII ((antihaemophilic factor A)33 REF. Together they form the tenase
complex that activates factor X in a manner similar to the way in which prothrombinase makes thrombin out of prothrombin34, 35 REF. In this way tissue factor and factor VII not only cause an immediate
generation of factor Xa but also give rise to the generation of a factor X activating enzyme complex. It now will be intuitively clear that haemophiliacs bleed more readily in organs with few TF, like joints and muscles than in organs that are rich in TF, like the brain or the lungs.
Page 18.
Unactivated factor VIII circulates in the plasma attached to Von Willebrand Factor (VWF)36-38 REF. This
is a very large protein consisting of a variable but large number of identical monomers39 REF. In Von
Willebrand Disease there is a lack of this protein and therefore also of factor VIII. Thrombin splits off a part of factor VIII, which causes it to lose its affinity for VWF and to bind to procoagulant membrane, where it readily combines with factor IXa35 REF. We recall that factor V was activated on the
procoagulant membrane by meizothrombin. Factor VIII cannot bind to a membrane because it is bound to VWF. Thrombin has to come and get factor VIII from free solution. On its way there it can be attacked by antithrombin especially when heparin is around (see later). That is why clotting times that are dependent upon the activation of factor VIII (such as the activated partial thromboplastin time) are sensitive to heparin whereas the thromboplastin time is not.
Page 19.
As soon as there is enough thrombin around, part of it will bind to thrombomodulin (TM). TM, like TF, is a protein that remains bound to the cell surface, but this time to the surface of endothelial cells40 REF.
Thrombin that is bound to TM loses all its procoagulant properties and acquires the capability to activate protein C41 REF. Activated protein C can land on procoagulant phospholipids and destroys the factors
Va and VIIIa that are adsorbed there42, 43 REF. In this process another plasma protein, protein S, also
plays a role 43-45REF. In this way both tenase and prothrombinase are destroyed and the formation of
thrombin stops. Protein S has other inhibitory actions, that we leave aside here46 REF. We notice that
thrombin, by activating factors V and VIII directly, enhances its own formation enormously but, by binding to TM and activating protein C it stops its own formation in a later stage. In this way a window in time is created during which thrombin can form. There is also a spatial effect here. Because TF is found on perivascular cells, i.e. in the wound, whereas TM is found on the endothelium, thrombin
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formation is fostered where a wound is and inhibited in the intact vessel. Thus thrombin formation, and hence the formation of a hemostatic plug, remains confined to the area of the wound.
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Primary and secondary haemostasis.
It is commonly taught that the first line defense in bleeding is by the blood platelets only. By adhesion to the wound surface and by aggregation between each other they would form a primary plug. Then, in a second stage, thrombin forms and fibrin will cement the aggregate together and form the secondary plug (e.g.47)REF. In the last ten years it has become clear that right from the beginning platelets and
thrombin generation cooperate. When a lesion is made in a vessel, fibrin develops some 10-15 seconds after the lesion is made 48, 49REF. As a clotting time is minimally 12 seconds, this means that there must
have been thrombin generation right from the very beginning. This leaves no room for the idea of primary and secondary haemostasis. We will now see how the interaction of the blood platelets with the connective tissue in the wound makes them contribute to thrombin generation by providing extra procoagulant membrane and factor V and how thrombin activates platelets. We present a schematic and simplified view of the blood platelet, only illustrating its close interaction with the blood coagulation system and omitting small molecular weight activators (e.g. serotonin, prostaglandins), the subtleties of shape change and passing by the intricate intracellular mechanisms that regulates platelet action.
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In the wound platelets come into contact with collagen to which they stick with a specific receptor. With another receptor they attach to VWF that is adsorbed onto the connective tissue50, 51 REF. In plasma
VWF is a long string of identical pieces that are rolled into a knot. (Only very few of these pieces carry factor VIII.) When the knot arrives in fast flowing fluid, as in a wound, and meets collagen then it sticks to the collagen surface and unrolls52 REF. The blood platelet is a cell surrounded by a membrane and
containing various types of organelles but no nucleus. One of the organelles is the α-granule, which, among other things, contains factor V53, 54 REF. By adhesion to collagen and by minute amounts of
thrombin platelets are activated. This again is an important positive feedback action of thrombin55, 56
REF. Platelets that are activated contract and form pseudopods. Others spread like a pan fried egg. Two essential reactions then follow almost immediately: The release reaction57 REF and the flip-flop reaction
55, 56REF. The release reaction is brought about by fusion of the α-granules with the cell membrane. This
causes these granules to shed their contents, such as factor V, in the surrounding medium.
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The flip-flop reaction concerns the membrane phospholipids. Platelets that stick to collagen and that are triggered by thrombin are able to rearrange their membrane in such a way that the procoagulant phospholipids that normally, as in any other cell, remain at the inside now appear at the outside 56REF.
In this way the negatively charged phospholipids appear at the side where the plasma proteins are and the platelet surface can serve as a support for clotting factors. The combination of thrombin and collagen is essential for provoking that reaction 56REF. Later we will see that fibrin together with Von
Willebrand Factor can replace collagen. We can now construct a diagram in which we integrate the reactions that lead to a thrombin explosion. To make it complete we have to add one reaction more: Thrombin can activate clotting factor XI (antihaemophilic factor C) that, in its turn activates factor IX
58REF. This is a lot more complicated than the scheme with which the whole process started because
the first molecules of thrombin engage in feedback reactions that set the full mechanism going. This stresses that the mechanism during the lag time, i.e. the mechanism that is responsible for the clotting time, is different from the mechanism that is responsible for bulk thrombin generation.
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The “extrinsic” and “intrinsic” system Up to here the reader will have missed the division between intrinsic system and extrinsic system that
is a standard item in all introductions to coagulation physiology (e.g.47) REF. The extrinsic system (left)
is operative when a tremendous excess of TF is added and sufficient phospholipid. In that case factors VII, X, V and II will directly produce enough thrombin to make a clot and factors VIII, IX, XI and platelets play no role. This happens in the measurement of the thromboplastin time (also called Quick time or prothrombin time, abbreviated as PT)6 REF. The intrinsic system (right) is the reaction mechanism of
thrombin formation when no TF is added at all but a foreign surface such as glass or added kaolin or ellagic acid6 REF, as is done for the measurement of the activated partial thromboplastin time (aPTT).
In that case Factor XII, the physiological role of which in the 60 years after its discovery still remains obscure, will interact with the foreign surface59. Together with prekallikrein and high molecular weight
kininogen it will activate factor XI 60, 61 REF. Activated factor XI then starts the coagulation reactions by
activating factor IX. In real life the amount of TF available will differ from situation to situation. The less TF is present the more will the reinforcement loop (Josso loop) via the antihaemophilic factors contribute to thrombin formation. The large excess of TF as in the prothrombin time or the total absence of TF as in the aPTT will be very rarely encountered in real life – if at all. Intrinsic and extrinsic system are useful concepts for the interpretation of clotting times. In everyday practice it suffices to know how much thrombin is formed.
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The most spectacular effect of the generation of thrombin is the clotting of fibrinogen. Thrombin splits of two fibrinopeptides (A and B) from fibrinogen and thus converts it into the fibrin monomer62, 63 REF.
Fibrin monomers then spontaneously polymerize to form long fibrin strands. Thrombin activates still another enzyme, factor XIII, which serves to crosslink the fibrin monomers so as to make a stable fibrin network 64REF. For a long time clotting has been regarded as the final act on the haemostatic scene. The
fibrin network is not an end point, however, because fibrin activates platelets and makes them procoagulant. As fibrin polymerizes, Von Willebrand Factor will adsorb onto it, assume its unrolled (activated) form and platelets can then bind to it and become activated. Fibrin thus takes the place of collagen as the wound is being closed65, 66 REF. Also thrombin adsorbs onto fibrin and is to some degree
protected from antithrombins there, whereas it maintains its capacity to activate clotting factors and platelets67, 68 REF.
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This explains how a thrombus can grow. The interaction of platelets with collagen can, by its very nature, do no more than coat the connective tissue in the wound with a monolayer of platelets. Also the tissue factor on the perivascular cells is no longer accessible then69 REF. Platelet to platelet aggregation70 REF
plays its role but does not explain the formation of thrombin and the appearance of tissue factor in the plug. The tissue factor (green) that is found in the platelet aggregate comes from the circulation REF. Monocytes contain tissue factor and carry it over to activated platelets REF. Tissue factor may also be present in circulating microparticles71, 72 REF. The fibrin becomes coated with VWF, to which more platelets bind and activate. Thus the plug can grow as long as blood streams past and brings more clotting factors and more platelets.The close interaction between platelets and the clotting system is illustrated by the observation that thrombin generation in platelet rich plasma is defective in congenital platelet defects like Glanzmann’s73, 74 REF and Bernard-Soulier’s diseases 74, 75REF. Thrombin generation
is inhibited by all antiplatelet drugs. It is also as well as by the effect of all “anti-aggregant” drugs such as abciximab 73REF. In Von Willebrands disease there are two reasons for thrombin generation to be
low (red line). Factor VIII is low and the platelets do not become procoagulant65 REF. If we add an excess
of factor VIII more thrombin is formed but it still is far from being normal (black) 65REF.
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The coagulation of all available fibrinogen in the plasma requires less than 5% of the total amount of thrombin acting for a few seconds only. After clotting, thrombin generation goes on in the clot and much more thrombin is produced than required for clotting alone. Why?76 REF. Hundreds of publications,
summarized in the “first law of thrombosis and haemostasis”, show that this “excess” thrombin is required for normal haemostasis and, when too much, promotes thrombosis. We have not a complete
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picture of what the functions of thrombin “beyond clotting” are. Several mechanisms may be responsible:
Thrombin that is produced locally in a clot or plug will diffuse out of that site and create a zone of high thrombin concentration in the fluid and tissues around the clot. In the blood it can activate platelets and clot fibrinogen; the higher the thrombin concentration in the clot, the broader the zone of high thrombin concentration around it, i.e. the faster the growth. Under high flow conditions the zone will be much smaller than in stagnant fluid, which explains the difference in aspect between arterial and venous thrombi, but it will always be there.
Thrombin that diffuses into the tissues has a large number of actions on a number of cells in connective tissue as well as in the vessel cells that we will leave aside here, but the more thrombin, the more reaction in the surrounding tissues.
Thrombin activates TAFI, the Thrombin Activatable Fibrinolysis Inhibitor77, 78 REF. If not enough
thrombin is available, not enough TAFI will be formed to protect the clot against fibrinolysis. This may explain the fibrinolytic character of the bleeding that is seen in hemophiliacs.
The velocity with which thrombin is formed influences the structure of the clot. Much thrombin gives a dense network of small fibres, little thrombin takes more time and makes a sparse network of thick fibres79, 80 REF.
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With all these procoagulant actions it is clear that the organism must get rid of thrombin once it fulfilled its hemostatic functions, otherwise any wound could result in massive thrombosis. This inactivation is caused by antithrombins. Through their action the half-life of thrombin in blood is only around one minute. They present themselves to thrombin as a substrate that is to be split. After engaging in an enzyme substrate complex, thrombin, to its surprise, will find that it cannot get lose from its prey anymore and is incapable of further enzymatic action81, 82 REF. The most important of the antithrombins
was formerly called antithrombin 3. It is responsible for the disappearance of around two thirds of all thrombin 83REF. The next important is α2-macroglobulin that takes care of about one quarter of the
thrombin84 REF. Finally there are miscellaneous inhibitors, like α1-antitrypsin and others that take care
of the 10% that remains. α2-macroglobulin is a particular one in that it surrounds thrombin so that it cannot interact with any protein anymore and loses its biological activity completely. Nevertheless the active center remains capable of converting small molecular weight substrates85, 86 REF.
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The efficacy of antithrombin (AT) can be enormously enhanced by the presence of heparin. Heparin is a chain of hundreds of different saccharide units and antithrombin binds strongly to a specific set of five
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sugars (“pentasaccharide”) that occurs occasionally in that chain87-89 REF. As soon as there are at least
12 saccharide units to one side of the pentasaccharide, thrombin can bind as well and thus meets antithrombin more easily90 REF. This can increase the velocity of thrombin inactivation up to a thousand
fold 91REF. In the “bucket end sink” model that we discussed earlier the drain is wider and the amount
of thrombin that is available in plasma becomes proportionally lower. Heparin molecules that do have the critical length of 5 plus 12 saccharides but are shorter than natural heparins have a favorable long in vivo half-life and less side effects than natural heparins have. It is a widespread delusion that they should work by inactivating factor Xa92-95 REF. Only molecules that contain the pentasaccharide but lack
12 extra units have no antithrombin action but still can still boost the inactivation of factor Xa. Factor Xa that is engaged in a prothrombinase complex is immune to this action. To inhibit thrombin generation to the same extend as larger heparin molecules do, ten to a hundred times higher concentrations are required96 REF.
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Vitamin K antagonists work both pro- and anticoagulant
Vitamin K antagonists make that the liver synthesizes less functional vitamin K dependent clotting factors: II, VII, IX and X97 REF. The factors that are produced, lack that part of the molecule that binds to
procoagulant phospholipid surfaces98, 99 REF. Because binding to phospholipids is essential for the
thrombin generation mechanism to proceed, for all practical purposes they are not there. Not only procoagulant factors are, also the anticoagulant factors protein C and protein S 44, 100REF. This makes
that thrombin generation diminishes, which has an antithrombotic effect but also it becomes to a certain extend resistant to the action of thrombomodulin, which has a prothrombotic effect 101REF. That
is probably why AVK treatment should be sufficiently deep and stable to be effective. There is a direct relation between the amount of thrombin formed and the degree of anticoagulation as indicated by the international normalized ratio (INR102, 103 REF). The picture shows thrombin generation curves at INR 1
(normal control) to INR 5 (blue). In our “bucket and sink” model there is less water and it is poured in more slowly.
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Direct inhibition of thrombin
Direct inhibition of thrombin itself, instead of impairing prothrombin activation or –synthesis, is a logical approach to anticoagulation. Hirudin, the anticoagulant from leeches, binds irreversibly to thrombin and kills it on a molecule for molecule basis. It is an effective antithrombotic but due to its all or none character the therapeutic range is narrow 104REF. Molecules that bind reversibly occupy the active
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therapeutic range. Moreover this type of molecules can be administrated orally 105REF. An important
consequence of inhibition of thrombin, with direct inhibitors as well as with heparin plus antithrombin, is that the feedback reactions are quenched. As a consequence inhibition of thrombin leads to inhibition of prothrombin conversion8, 106 REF. The degree of inhibition by this type of substances is dependent on
the concentration of the inhibitor (figure) but also on the individual properties of the plasma. In some people the same concentration of inhibitor may cause twice as much effect as in others107 REF.
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Direct inhibition of factor Xa
Like thrombin, also factor Xa, can be inactivated by antithrombin. The interaction is enhanced when the specific pentasaccharide from heparin binds to AT. Factor Xa that is engaged in a prothrombinase complex is immune to this action 108REF. For factor Xa, like for thrombin, small molecular weight
reversible inhibitors are available that can be taken orally. Unlike heparins they do inhibit factor Xa that partakes in a prothrombinase complex. Again the inhibition of thrombin generation that is observed is dependent upon the level of inhibitor in the plasma as well as upon the properties of the individual plasma. To obtain the same degree of inhibition, low responders may require twice higher concentrations than high responders do 107REF. All known antithrombotic drugs at their effective plasma
concentration inhibit thrombin generation. For all of them the degree of inhibition obtained is dependent upon the plasma concentration of the drug as well as on the individual properties of the plasma, i.e. for all of them there are low and high responders107, 109 REF. Ideally the dose of anticoagulant
should therefore be tailored to the needs of the patients.
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How about the possibility to measure thrombin generation in actual practice
Traditionally thrombin formation was assessed by subsampling from clotting blood or plasma110 REF.
This required much time and dexterity. Nowadays we calculate the course of thrombin in time from the conversion of a fluorogenic substrate added to the clotting blood (-plasma) and obtain the Thrombogram during the experiment on the screen of the computer 111REF. This technique can be used
to monitor thrombin generation in parallel in dozens of samples but also to measure thrombin generation in a drop of blood112 REF. A typical result with normal plasma, tested in triplo, is shown in
the figure. Parameters such as lag time (=clotting time), time to peak, peak value and ETP (the area under the curve) are rendered together with the curve. As we said earlier, managing haemostasis or thrombosis amounts largely to managing the amount of thrombin that a patient is making by pharmacological means. In fact that is what doctors have been doing all the time by administrating
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plasma or factor concentrates to bleeding patients and by giving anticoagulants to fight thrombosis. The clinical results of course are the ultimate criterion of success. A large amount of recent data indicate that the amount of thrombin formed is a good surrogate indicator of the clinical effect both in bleeding and in thrombosis. Efforts to make the method available beyond the research lab and for routine clinical practice are on their way.
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The thrombogram and risk of bleeding and thrombosis
Among normal people the capacity to form thrombin differs enormously111 REF; as much as body weight
in adults (but thrombin generation is not proportional to body weight). People with above average thrombin generation have a higher risk of venous thrombosis113 REF, people who are below average will
lose significantly more blood during an operation. All such conditions that bring a risk of thrombosis show an increased TG114 REF.
Application in the treatment of haemophilia
The picture shows what happens when you infuse a factor VIII concentrate into a patient with hemophilia. The black line is the thrombogram before infusion, the red one 1 hour after infusion, green after 24 and the blue after 60 hours. Different patients may react in a different way, in some TG increases twice as much as in others upon injection of the same dose. Also in some patients the effect lasts much longer than in others. Low thrombin generation is not restricted to hemophilia. All known blood based conditions, either congenital or acquired, that bring a risk of bleeding will also show low TG
114, 115REF.
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The thrombogram and risk of bleeding and thrombosis
Thrombin generation can be used find out more about the cause of a bleeding- or thrombotic tendency. By adding thrombomodulin e.g. one can test the function of the protein C system. To the left a normal thrombogram (black) is seen and also the inhibitory effect of added thrombomodulin (green). In the middle the same experiment in the plasma of a normal female person that uses oral contraceptives (black). If that person would not have used oral contraceptives but had a factor VLeiden trait, much the
same picture would have been obtained. In the rightmost picture is shown what would happen if a person with the factor VLeiden trait would use oral contraceptives (black). The effects on thrombin
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