SEED No 1_COAG_Principles of Haemostasis

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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



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.

/4SEED-Africa Newsletter | No 1 | October 2010

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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

3/4SEED-Africa Newsletter | No 1 | October 2010




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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.

4/4SEED-Africa Newsletter | No 1 | October 2010


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SEED No 1_COAG_Principles of Haemostasis