Contents
It is essential for survival that a wound stops
bleeding and that childbirth be compatible with survival of the mother, i.e.
that the body possesses an adequate mechanism for haemostasis . If however
arrest of blood flow occurs in intact vessels, normal circulation is impaired,
which is deleterious to normal function. Tissues and organs can die when blood
supply is arrested for too long. Nature has provided an admiringly efficient and
extremely complicated mechanism, the haemostatic
system, to ensure the seemingly contradictory functions: adequate blood
flow in normal vessels and prompt arrest of bleeding in damaged ones.
Under primitive conditions, prompt arrest of bleeding
in individuals in the gestational age is of paramount importance for the
survival of the species. It therefore is no surprise that in vertebrates and
consequently in the human the haemostatic system is extremely effective. In
fact it is so forceful that, under conditions of modern life, where wounding
is much less common than in the wild and the age to which individuals live
surpasses all previous limits, it is slightly over-dimensioned. In fact half
of the people in the western world die of excessive haemostasis. Stroke and
coronary infarction are the best known diseases of this type. Like the skin,
the inside of the blood vessels looses its smoothness with age. In vessels
atherosclerotic changes are the main culprit. The blood may mistake the older
blood vessel for a wounded one and trigger the haemostatic mechanism. If this
happens in vital organs like the heart or the brain the damage to the area
downstream may be serious and lead to such serious disease as coronary
infarction and stroke. The phenomenon is not restricted to these two organs,
thrombosis is the general name for such obstructive disease. In order
to avoid thrombosis it is essential that the solidification of blood stays
confined to the wound area. So blood-clotting reactions should be triggered
promptly but also stop within reasonable limits. It is not surprising that the
haemostatic mechanism is a very complicated and extremely fine tuned one, replete
with checks and balances.
Haemostasis is brought about by the interplay between the minute blood cells, the blood platelets, and a set of proteins of the blood plasma, the coagulation system. Having past a damaged part of the vessel wall, platelets stick to the bare tissue in the wound and create a scenery in which blood can clot without the clot being washed away by the flowing blood. The interactions between the wound and the blood lead to the formation of thrombin and thrombin is responsible for a whole concert of reactions, of which the actual clotting, i.e. solidification, of the blood is only one: Thrombin activates the platelets and acts on the cell of the vessel wall. Activated platelets, in their turn, foster thrombin formation. Thrombin partakes in a whole set of positive and negative feedback reactions that first increase its own production enormously but inhibit it in a later stage. Therefore the way in which thrombin is formed and decreases again when blood coagulation is triggered, is the best indicator of the function of the haemostatic system. Strange enough there existed no good test for the function of the haemostatic system until recently. Specialists know all too well what the limitations are of measuring clotting- and bleeding times. One of the main activities of Synapse b.v. has been the development of easy ways to monitor the rise and fall of thrombin in clotting blood. To this end we developed the "Thrombogram" that estimates the course of thrombin in time. In the same way as we can get an impression of cardiac function by feeling the pulse but prefer to make an electrocardiogram, we also can obtain an impression of the function of the haemostatic -thrombotic system by measuring clotting times but it is much more informative to make a thrombogram. In contrast to clotting times, the thrombogram measures both low and high reactivity of the clotting system and is sensitive to the action of all types of antithrombotic drugs, so that it can be used as a universal monitor of clotting function.
Essential to the understanding is the concept of proenzyme-into-enzyme conversion. An enzyme is a protein capable to enhance a very specific chemical reaction. Proteolytic enzymes are proteins that can cut other proteins in pieces. They can therefore be extremely dangerous and usually are formed and transported in the body as proenzymes. Proenzymes are usually larger than enzymes and cannot, as such, do any harm. Only when they are cut at a specific place does their enzyme character appear. E.g. enzymes that are to digest the proteins of our food are formed as proenzymes in the pancreas and are only activated when secreted in the gut. The clotting mechanism is based on an ordered series of proenzyme - enzyme conversions. The first one of the series is transformed into an active enzyme that activates the second one, which activates the third one. In this way a few molecules in the beginning of the series create an explosion of the final active enzyme: thrombin, very much as a command that goes from the general to the officers, then to the sergeants and then to the men, takes little time to activate the whole army. This mechanism is often referred to as the coagulation cascade, even though this does not reflect the augmentation inherent to the system. Thrombin, in the blood, does not live long; otherwise the slightest wound could make all the blood clot. Its survival time in plasma is only a few minutes, due to the fact that it is bound by antithrombins, suicidal plasma proteins that inactivate thrombin by binding to it irreversibly. The haemostatic activity that develops in a wound or thrombus is essentially dependent upon the number of "man-hours" of thrombin that can develop in blood. That means that the amount of thrombin that generates as well as the length of time that it is active both count. The amount of work that can potentially be done by thrombin is reflected in the area under the curve that describes the concentration of thrombin in time during clotting, that is, the Thrombogram. We therefore baptized this area the "Endogenous Thrombin Potential"(the ETP). Another important property is the time it takes until thrombin formations starts in earnest, i.e. the lag time. This also happens to be the clotting time, because the clot appears when roughly 1% of thrombin is formed. You miss out on a lot of information if you just look at the clotting time as a determinant of coagulability: after the clot formation most of the thrombin action is still to come.
The former paragraph just explained that enzymes make more enzymes and so on and that therefore the clotting reaction is called a cascade. The cascade sequence only explains the explosive formation of thrombin, however, clotting is not as simple as that, the output is finely tuned by a web of positive and negative feedback reactions. Please look at the following picture:
|
|
Coagulation starts when a vessel is wounded. A vessel is wrapped in a layer that is full of cells containing Tissue Factor (TF). After injury this TF is exposed to blood, it binds with factor VIIa to form the first enzyme: TF:VIIa. This activates factor X to Xa, which in turn forms thrombin. |
This picture describes the Production Dimension; this certainly is a cascade, i.e. a series of proenzyme-enzyme conversions. All the plasma proteins involved in coagulation are clotting factors, indicated by Roman numerals. When a proenzyme is activated into an enzyme, an "a" is added to the number. Figure 1 shows that TF:VIIa activates X (TEN, not EX!), Xa activates prothrombin (factor II) into thrombin. Factor Xa is not a very efficient enzyme; it needs the help of factor V (a helper protein or cofactor) that helps to speed up prothrombin activation a thousand fold. Both factors bind to a phospholipid surface (to be discussed later in more detail) that also binds prothrombin and thus favors the meeting of substrate and enzyme. This offers the possibility to regulate the function of factor Xa, and hence thrombin production in space and time. Look at the next picture.
|
Factor V is activated by thrombin into factor Va. Xa and Va bind onto a phospholipid surface and form the prothrombinase complex which very efficiently converts more prothrombin into thrombin. Thrombin also binds to thrombomodulin. This complex converts protein C (again a proenzyme) into its activated (enzyme) form: APC. APC in turn attacks factor Va and inactivates it (factor Vi). |
Thrombin promotes its own formation by the activation of the helper protein factor V,
but it also inhibits its own formation. Thrombin on its own is able to
activate factor V into Va. The inactivation of this factor V takes extra steps
and therefore is slower. First thrombin has to bind to thrombomodulin (present
on the endothelial surface of the vessel and therefore hardly in a
wound) and then the thrombomodulin-thrombin complex activates a plasma
proenzyme, protein C, into its active form APC that degrades factor Va. So the
activation of V is direct but the inactivation goes via an intermediate step.
This creates a "window in time" in which prothrombinase can remain
active. We call this the Regulation Dimension.
Thrombin promotes its production by additional mechanisms, depicted in the next
picture.
|
VIIa:TF not only activates factor X but also factor IX (nine). Thrombin also activates factor VIII that, just like factor V, is a helper protein. Factor VIIIa and IXa form a complex called tenase, that produces more X. This reinforcement of X production is called the Josso loop. Like factor Va, factor VIIIa is degraded by activated protein C. |
The Josso loop makes extra Xa, so more prothrombinase is formed, which appears to be useful if a wound happens to bring insufficient tissue factor. Factor VIII is activated by thrombin and inactivated by protein C, just like factor V. Like the Xa-Va complex, the IXa-VIIIa complex only functions well when adsorbed onto a phospholipid surface (the importance of this will be shown later). Factors VIII and IX are known as the anti-haemophilic factors A and B, because if one of them is congenitally absent the bleeding disease haemophilia develops. The picture is not complete yet; please look at the next picture.
|
Factor Xa binds to TFPI, Tissue Factor Pathway Inhibitor. This complex binds so tightly to the VIIa-TF complex that X and IX cannot reach factor VIIa anymore and production of Xa and IXa stops. |
When factor Xa is not adsorbed onto phospholipid but swims free in plasma, it encounters the plasma protein TFPI to which it binds strongly. After this unit has formed, it binds to the VIIa:TF unit, and kills its activity. Note that again a window in time is created, we need enough Xa to get the TFPI:Xa complex formed. After this the Xa production will cease through binding of this complex to TF:VIIa. Actually, the pictures presented here in fact do not tell the whole story but they certainly give the main idea.
Not only regulation in time is important, also containment in space is necessary. Clotting should only occur at the site of injury.
|
|
Blood contains platelets (thrombocytes). As soon as they find an injury they bind to the exposed tissue (collagen) and, by this binding and the contact with thrombin, they activate. This results in a change of the surface of the platelets, that now becomes able to bind clotting factors such as the couples Xa-Va and IXa-VIIIa. This binding is essential for their proper functioning. |
Platelets bind to the site of injury and thrombin will activate them which will result in their surface becoming able to bind the enzyme complexes described above. This surface serves as a two-dimensional space where the clotting factors can find each other and more easily perform their reactions. This adsorption again increases thrombin formation by about a thousand fold. So without such a surface clotting will not occur. This provides the Localization Dimension: clotting only occurs where it is needed and remains contained to that area.
The result of this all is that a complicated web of positive and negative feedback loops governs thrombin production and confines it in time and space. Not only chemical reaction kinetics but also physical reactions like diffusion play a rate determining role. If one realizes that the clotting factor binding properties of the platelet surface changes in time with the degree of platelet activation and that the chemical kinetics of the activation complexes is dependent upon the properties of this surface, one realizes that this mechanism is so complicated as to make it (even theoretically) impossible to describe it in a mathematical model. Therefore it becomes extremely difficult to predict with any accuracy how the system will quantitatively answer to changes to one or more of its reactants.
Drugs that are designed to inhibit thrombosis by selectively attacking one single enzyme may in practice work out completely different than expected.
The consequence is that we are left with only one solution: measure the clottability of the blood rather than try to predict it.
Here the measurement of the rise and fall of thrombin in a medium that represents a sufficiently large portion of the physiological milieu, i.e. platelet rich plasma, presents itself as the logical choice. All clotting reactions have one final common path: it is all about thrombin production.
Clotting times only represent the lag phase before thrombin generation starts and therefore only tell a minor part of the story. The extent of the haemostatic- thrombotic reaction is also critically determined by the amount of thrombin that forms and by the time it remains active.
This made us formulate the first law of thrombosis and hemostasis:
More thrombin gives better hemostasis but more thrombosis. Less thrombin gives less thrombosis but a bleeding disorder.
If the argument developed in the previous section is valid, then the Thrombogram should reflect the large majority of disorders of the haemostatic mechanism and the effect of most antithrombotic drugs. We do not exclude that there are disorders or drugs that solely affect the adhesive- and/or aggregation function of platelets. In practice, however, we have been surprised not to encounter circumstances in which platelet inhibitors or – diseases did not influence thrombin generation.
This holds a fortiori for disorders of the coagulation mechanism and anticoagulant drugs: we have not found a single one yet that is not reflected in the thrombogram. The importance of thrombin generation for thrombus formation can be deduced from the fact that any drug that diminishes thrombin generation has an antithrombotic action, completely independent by the manner in which this is brought about. This will be explained in the next section.
In the normal situation prothrombin is converted by prothrombinase and thrombin disappears through binding to anti-thrombins. This is a non-equilibrium situation, comparable of emptying a bucket of water in a bathtub. When a large bucket of water is emptied in a bathtub that has no stopper, the level of water will increase and then decrease again. This change in level will depend on how large the opening in the sewer is and how fast water is poured in. Suppose the bucket size is the amount of prothrombin, prothrombinase activity determines how fast it is poured in, and the drain size is the action of antithrombin.
|
|
The dynamics of thrombin appearance and disappearance are like the filling of a bathtub without a stopper from a large bucket.. |
When we add heparin, the disappearance rate is increased, which results in less thrombin being present at any time.
|
|
Heparin
makes antithrombin a much more potent thrombin inhibitor, it works on the drain side. Thrombin comes in normally but goes out much
faster. The result is less thrombin. |
Now, what happens when we add a direct thrombin inhibitor? Either reversible (Melagatran e.g.) or irreversible (like Hirudin). Here production and disappearance are at its normal rates but the amount of thrombin is again decreased because of the direct inhibitory effect, for example the effect of Hirudin.
|
|
Hirudin binds directly to thrombin, so the IN- and OUT velocities remain unchanged but thrombin itself is inhibited. |
Oral anticoagulation diminishes prothrombin and factors VII, IX, X as well as proteins C and S. The net effect is a diminution of thrombin inflow at normal outflow.
|
A lack of vitamin K or drugs that prevent its functioning (vitamin K antagonists) make that several clotting factors including prothrombin, are not formed normally. However, thrombin decay remains normal, or: less in and normal out. |
These three antithrombotic drugs have only one thing in common: they diminish the amount of thrombin that appears in clotting plasma (Not necessarily the lag time, i.e. the clotting time!). This supports our formulation of the main law. At comparably effective clinically effective doses of these drugs the Thrombogram is similarly inhibited, which is additional evidence for its usefulness.
Next we will show you a few examples of the use of the measurement of the Thrombogram in different coagulation disorders, both hyperactivity (i.e. with a thrombosis risk) and hypoactivity (with a bleeding risk).
|
|
The Endogenous Thrombin Potential measured in men and women. The ETP is significantly higher in women using oral contaceptives (the pill). Also the clotting times are shown, it is seen that the pill or effect is not seen from the PT (prothrombin time) but is clear from the ETP. |
It is known that women using oral contraceptives have
a higher incidence of thrombosis, we now know that this is due to
hypercoagulability induced by the pill. The ETP is able to show this, in women
taking the pill the ETP is some 14% higher than normal. However, the clotting
time is not significantly different.
Another example:
Patients with Glanzmann Thrombasthenia do not have functional GPIIb-IIIa receptors on their platelets. This makes that their platelets do not stick to each other (aggregation) surprisingly it also has an effect on the amount of thrombin formed in platelet rich plasma. The Thrombogram clearly shows that thrombin generation is impaired. There are drugs that inhibit this receptor and cause a kind of Glanzmann Thrombasthenia, such as Reopro, that is used in acute coronary infarction. Such drugs also inhibit the thrombogram. Another drug family that influences coagulation are Coumarin congeners (such as Warfarin, ascenocoumarol (Sintrom) , phenprocoumon (Marcoumar)). These drugs are taken orally so patients are on Oral Anti Coagulation (OAC) mentioned in the previous section. Here is an example of a set of measurements of the Thrombogram in plasma of these patients compared to normal plasma.
The picture shows three Thrombograms of an average of 10 normal and 10 AVK-patients. The three blue curves, ranked by peak height, are: PPP with nothing added, PPP with thrombomodulin, PPP with APC added. The red, orange and yellow curves show the same set for the AVK patients. It can be seen that treatment with anti-vitamin K (AVK) clearly inhibits thrombin formation. Also when thrombomodulin or APC is added this formation is reduced. For the experts: note that the thrombomodulin-effect is stronger in normal subjects than in AVK patients. This is probably due to the fact that also the patient's protein C is affected by the treatment, therefore, thrombomodulin cannot work as effectively as in normal subjects. Addition of exogenous APC circumvents this effect; in AVK patients the APC addition completely kills thrombin formation.
A number of other drugs and diseases, formerly thought to affect platelet adhesion and aggregation only, also show aberrant Thrombograms, such as von Willebrands disease, Clopidogrel, Aspirin and more.
Blood coagulation is all about thrombin formation, more thrombin means more thrombosis and less bleeding, less thrombin means less thrombosis but more bleeding. The measurement of thrombin formation is the most sensitive and all-round tool to assess coagulability.
The Thrombogram is the only physiological function test of the thrombotic-hemostatic system that can measure hyper- as well as hypocoagulation. It can also measure the effect of treatment with a combination of drugs or a combination of pathological conditions.
|
Address Synapse BV University Maastricht P.O. Box 616 6200 MD, Maastricht The Netherlands |
Visiting/Delivery Address Synapse BV Oxfordlaan 70 6229 EV, Maastricht The Netherlands |
Phone:+31 (0)43 3885892 Fax: +31 (0)43 3884159 Email: info@thrombin.com |
