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Sysmex EducationalEnhancement & Development
SEED-Arica Newsletter | No 1 | October 2010
Principles of haemostasis
The primary principle o haemostasis is to minimise blood
loss at sites o vessel injury by orming a thrombus (clot) and
at the same time maintaining blood low (igure ). In order
to achieve this, there is a highly regulated, ine-tuned inter-
action o multiple processes, involving the blood vessel wall,
principally the endothelium, platelets and non-cellular blood
constituents.
The core elements o haemostasis include blood vessel con-
striction, platelet activation, coagulation and ibrinolysis. All
o these processes are initiated at the same time in response
to blood vessel injury. Damage to the endothelial cell lining
o the blood vessel results in exposure o collagen which
under normal circumstances is not in contact with the blood.
When circulating platelets come into contact with collagen
they are activated and start to stick to the damaged surace.
At the same time a substance called tissue actor is alsoexposed which activates the coagulation cascade resulting in
the ormation o a ibrin clot. Platelet adhesion and activa-
tion is conventionally known as primary haemostasis and
the process o coagulation and ibrin ormation as secondary
haemostasis. In simplistic terms, the platelet plug can be con-
sidered to be the ‘bricks’ and the ibrin meshwork the ‘cement’,
both being required or the ormation o a stable thrombus.
Normal haemostasis depends on the interaction
of the following:
a) Blood vesselb) Platelets
c) Coagulation system
d) Fibrinolytic system
A deect in one or more o these systems will result in either
a bleeding disorder or a tendency to clot.
a) The blood vessel
The blood vessel wall which is lined with endothelial cells is
the irst line o deence or normal haemostasis. The endo-
thelium is a highly regulated ‘organ’ in that it is responsible
or ensuring that blood remains luid at all times so that
low continues without hindrance. The surace is thereore
regulated to be highly ‘anticoagulant’ most o the time.
Introduction to coagulation
The purpose o this newsletter is to provide laboratory sta with a simple outline o the principles o haemostasis as
the oundation or ensuring that only the best quality results are released and to acilitate logical troubleshooting when
problems do occur.
Figure 1 Schematic representation o the principles o haemostasis
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However, at the irst sign o injury where blood losscould be incurred, the endothelium switches to be highly
‘procoagulant’.
b) Platelets
Platelets are produced in the bone marrow. They circulate
in the blood and have a lie span o about days. Platelets
have a very complex structure which acilitates the critical
role that platelets play in the normal physiology o haemo-
stasis. The major components o the platelet include the
surace membrane composed mainly o phospholipids, in-
tracellular granules, surace receptors and the canalicular
system.
Ordinarily, platelets circulate as ‘inert’ particles, without
interacting with the blood vessel wall or any blood constit-
uent proteins. However, at sites o blood vessel damage,
platelets stick to a variety o structures via a multitude o
platelet surace receptors.
Von Willebrand actor (VWF) is a plasma protein the unc-
tion o which is to act as an anchor or platelets at sites o
injury. VWF and platelets ordinarily do not interact, but once
VWF has bound to subendothelial tissues, it changes its ownstructure which the platelet then recognizes and binds tightly.
The bond takes place via one o the major surace receptors,
which in turn triggers multiple reactions inside the platelet
which lead to ‘activation’ o the platelet.
Platelet activation includes several key end points:
n Shape change
Platelets undergo a shape change rom disc like to lattened
spread out structures with multiple inger-like extensions.
This physically assists in plugging the hole in the vessel wall.
The platelet is able to do this because it stores an abundanceo ‘excess membrane’ in the orm o the canalicular system.
This canalicular system is connected to the outside o the
platelet and when activated, this excess membrane is pushed
to the outside thereby greatly increasing the surace area.
n Granule release
Platelets contain granules that are secreted when platelets
are activated. These granules contain a number o key
elements, such as actor V, VWF and calcium which urther
ampliy the haemostatic process.
n Membrane flip-flop
Platelet activation results in an irreversible lip-lop o its
bilayer membrane resulting in the exposure o phospholipids
(especially phospatidylserine) to the plasma which arenormally housed primarily within the inner lealet o the
platelet membrane and hence separated rom plasma pro-
teins. This is a critical step in the haemostatic process as
phospholipid is an essential element in the support o the
coagulation cascade.
n Fibrinogen receptor
Another vital consequence o platelet activation is the in-
duction o a shape change in the main surace receptor or
ibrinogen. As the ibrinogen molecule has two identical
arms, one ibrinogen molecule is able to bind to ibrinogen
receptors on adjacent platelets thereby linking them together.
The overall eect o this is that there is now a physical seal
comprising o stretched out platelets which are tethered to
collagen in the vessel wall at the site o injury. The primary
platelet plug by itsel is however only a temporary seal, and
the ormation o a proper clot is needed in order to seal the
vessel wall securely whilst the damaged vessel repairs itsel.
The exposed phosphatidylserine on the activated platelets
surace is ready to support the coagulation cascade which
has its substrate, ibrinogen right there, holding the platelets
together.
c) Coagulation
Coagulation is the process o converting soluble ibrinogen
into insoluble ibrin in order to orm a stable blood clot
around the platelets that were initially trapped at the site
o injury. The plasma contains a large number o proteins
called coagulation actors that participate in a series o
enzymatic reactions that end in the ormation o insoluble
ibrin. A very small trigger results in the progressive ampli-
ication o each subsequent step which concludes in the
production o relatively large amounts o ibrin. This sequen-tial reaction is called the coagulation cascade. The clotting
actors involved are actors XI, X, IX, VIII, VII, V and II.
Factor II, also called prothrombin, becomes thrombin when
activated.
For normal haemostasis to occur, a suicient amount o
thrombin needs to be rapidly generated in order or ibrin-
ogen to be converted to ibrin. Fibrin is relatively ‘water-
tight’ and thereore urther blood loss is prevented. Fibrin is
also relatively resistant to ibrinolytic breakdown (see later).
When the surace o the endothelium is damaged, coagu-
lation is immediately activated by the exposure o tissue
actor (TF), which is present below the endothelial cells in the
vessel wall, to the coagulation actors present in plasma.
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n The coagulation cascadeThe coagulation cascade as we understand it to exist today
is shown in igure . The process o coagulation starts with
the presence o TF, which binds and activates FVII to FVIIa.
The activated clotting actors are enzymes that sequentially
cleave each other in a highly regulated ashion. This process
happens wherever TF has become exposed. Once the TF:FVIIa
complex is ormed, it has a potent positive eedback loop
on itsel thereby increasing the generation o more TF:FVIIa
several thousand old. TF:FVIIa in turn orms a complex with
FX and converts it to FXa. It is also able to bind and activate
FIX to FIXa although the intensity o this reaction is ar less
potent. FXa, together with the co-actor FVa, converts pro-
thrombin to thrombin (FIIa). This initial generation o throm-
bin is very short-lived as TF:FVIIa is very rapidly inactivated
by tissue actor pathway inhibitor (TFPI).
Furthermore thrombin activates FXIII to FXIIIa which
in turn is responsible or irreversibly cross-linking ibrin
creating insoluble polymers and a stable clot.
It can be said that the primary role o the initial small quan-
tity o thrombin generated is to activate platelets within
and close to the growing thrombus via thrombin plateletreceptors and the thrombin produced rom the secondary
positive eedback activation loop o FXI by thrombin is
responsible or the sustained burst o thrombin that gener-
ates the stable ibrin clot.
n The importance of vitamin K and calcium
An essential element in the success o coagulation cascade
is the assembly o the various proteases in an orderly ashion
on the surace o the platelet. The key to this process is
the presence o GLA (γ-carboxyglutamic acid) domains o
the vitamin K dependent clotting actors which are actors
II, VII, IX and X. These GLA domains o these clotting
actors are the key to the assembly o these complexes on
platelet suraces as they provide the critical link.
Calcium ions which are positively charged bind to various
GLA domains and to the phospholipid o the platelet,
both o which are negatively charged, thereby localizing
the clotting actors to the platelet surace. The calcium
ions essentially orm a bridge between the clotting actor
and the phospholipid on the activated platelet surace.
This is how the clotting process is kept localized at the
original site o endothelial injury. An absence o unctional
vitamin K will result in reduced ability or the coagulation
actors to assemble on the platelet surace and compromiseclot ormation. NB: Calcium is a critical element in the
laboratory tests o coagulation!
Figure 2 The revised coagulation cascade. PL reers to phospholipid
which is the phosphatidylserine provided by the activated platelet
surace.
Figure 3 The role o calcium in coagulation
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d) FibrinolysisIn the absence o adequate quantities o thrombin, a stable
clot cannot be ormed resulting in bleeding whereas un-
regulated production o thrombin will result in pathological
thrombosis. A variety o mechanisms that collectively inhibit
thrombin production have been well described. These
include:
n Removal o activated clotting actors by blood low
past the clot
n Inactivation o clotting actors by circulating natural
anticoagulants (protein C, protein S and antithrombin)
n Fibrinolysis
Fibrinolysis is the process o controlled clot breakdown.
As soon as clot ormation is triggered, so is the ibrinolytic
pathway activated. The primary enzyme involved here is
plasmin which degrades ibrin into ibrin breakdown prod-
ucts (FDPs) which includes D-Dimers.
The haemostatic balance
Despite the complexities o the control o haemostasis,
ultimately the outcome is quite simply a matter o balance
between clot ormation and clot breakdown, both o which
in turn are inluenced by blood low (or stasis), the vesselwall and the constituents o the blood (igure 4).
The next newsletter will ocus on the inluence o pre-
analytical actors on coagulation test results.
Figure 4 A schematic representation o the concept o haemostatic
balance. Normal haemostasis requires fne balance between clot orma-
tion and clot breakdown. Pathological thrombosis occurs when there is
excess clotting and/or diminished fbrinolysis and pathological bleeding
when clotting is reduced and/or fbrinolysis is enhanced. In this context
‘pathological thrombosis’ reers to thrombus ormation in excess o
what is required to seal any damaged blood vessel and ‘pathological
bleeding’ reers to any episode o bleeding which is more severe than
one would ordinarily expect based on the nature o the injury.
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