Blood Coagulation

Contents


Introduction


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.



How to assess the haemostatic function?


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.





The clotting mechanism


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.





Blood coagulation is not simply a cascade


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:

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.

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.

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.

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.

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.

Consulting Research and Development
in Thrombosis and Hemostasis

Address Synapse BV University Maastricht P.O. Box 616 6200 MD, Maastricht The Netherlands

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