Coagulation and the Clotting Cascade

almostadoctor.co.uk/encyclopedia/clotting-cascade

Dr Tom Leach

Introduction
Coagulation is the process by which blood changes from a liquid into a blood clot, to
cause the cessation of blood loss from a blood vessel.

The process involves the activation, adhesion and aggregation of platelets, and the
deposition of fibrin.

It can be divided into:

Primary haemostasis – the formation of a platelet plug

Secondary haemostasis – the activation of the clotting cascade which results in
despoliation of fibrin to strengthen the platelet plug

Haemostasis
Haemostasis – is the technical name for the cessation of bleeding, and has 3 separate
stages:

The vascular phase

The platelet phase
The coagulation phase

In reality, these are not distinctly separate, and all occur simultaneously as a result of
multiple cascades.

The vascular phase

Damage to the blood vessel wall will cause contraction in that particular area of the
blood vessel. This vasoconstriction can last from 30 minutes to a few hours and can
completely occlude the vessel. It occurs as a result of damage to the endothelial cells.
This damage causes them to release various factors, including:

ADP
Tissue Factor – aka factor III – required for the activation of thrombin from
prothrombin
Prostacyclin – kind of a feedback mechanism; thisprotein actually causes
vasodilation and prevents formation of the platelet plug
Endothelins – these are the primary hormones involved in the vascular phase.
They stimulate smooth muscle contraction and stimulate cell division of endothelial
cells, smooth muscle cells and fibroblasts, thus aiding repair of the damaged site.

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Endothelial cells also become ‘sticky’ and will express surface proteins that allow them to
‘stick’ to other endothelial cells, in an attempt to close of the damaged area.

The platelet phase

If the damage to a blood vessel is small enough, it can be ‘plugged’ by a platelet plug.
Platelets – aka thrombocytes:

Contain actin and myosin, and as a result are actually able to contract
They also contain another contractile protein known as thrmobosthenin
Have no nucleus, and are thus not able to reproduce
Have a large ER and golgi apparatus for storage of calcium ions
Have plenty of mitochondria for formation of ATP
Produce plenty of prostaglandins and fibrin-stabilising factor
Produce platelet derived growth factor (PDGF) – which helps vascular repair
Produce thromboxane A2 – which is a prominent vasoconstrictor
They are actually cell fragments rather than actual cells – as during their
development, their precursors break down to form them
Have a special glycoprotein membrane that prevents adhesion to normal
epithelium, but that promotes adhesion to damaged epithelium. they are
particularly adherent to collagen – which will only be present when deep areas
of the cell wall are exposed
About 1/3 of platelets are stored in the spleen and other vascular organs at
any one time – waiting to be mobilised
Low platelet count – thrombocytopaenia – this is a process either of high
platelet destruction, or low platelet production.
High platelet count – thrombocytosis – this is usually a result of increased
platelet formation, most commonly seen in response to infection, inflammation,
and cases of cancer.

Platelet formation is controlled by TPO (thrombopoietin – this is basically the platelet

version of EPO!). TPO is produced mainly by the liver – thus in diseases of the liver,
problems with clotting (mainly seen as manifestations of bruising) are commonly seen.
The platelet phase begins as soon as platelets begin to attach themselves to damaged
areas of endothelium – normally collagen. This process begins within 15 seconds of
injury.
The attachment of platelets to exposed surfaces is called platelet adhesion. As more and
more platelets arrive, we get platelet aggregation, and finally, once a certain mass of
platelets is reached, we have platelet plug formation.
This is a totally normally process, and will occur thousands of times a day. People
with problems with platelet formation may get thousands of tiny haemorrhages, and
problems bruising easily.

When a platelet becomes attached to a damaged endothelial surface, it will actually

changes it own size and shape:

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 It will swell, and become large and irregular
The contractile proteins contract causing the release of granules…
ADP, thromboxane and Ca2+ ions are all released – these can act on nearby
platelets, and attract them to the site, causing them to adhere to the platelets
already present. This creates a positive feedback loop, causing aggregation of
more and more platelets.

There are two types of granule released by platelets:

α – these contain growth factors, like fibrinogen and PDGF – a disease caused by
a lack of α granules is called grey platelet syndrome. This is a rare genetic
disorder (autosomal dominant) that causes. It will just basically cause reduced
clotting.
Dense – these contain non-protein things, like thromboxane, serotonin, adrenaline,
histamine, calcium, ATP and ADP

Negative feedback of the plug formation is controlled by prostacyclin released by the

endothelium. This reduces platelet aggregation. White cells in the area also release
proteins that prevent the clot getting out of control. Plasma enzymes will also break
down ATP that is found circulating near the plug, and thus reduce the amount of energy
available to the platelets.
Fibrin – is the activated form of fibrinogen – which is produced by the liver, and also by
platelets. It is activated in the clotting process, and forms lots of fibrin threads – which
help to stabilise a platelet plug, as well as isolating it from the normal circulation, thus
acting as a further feedback mechanism. Fibrin is possibly the single most important
protein involved in clotting!

Fibrinogen is soluble, but fibrin is insoluble, and thus once fibrin becomes activate
it begins to ‘precipitate’ out of the plasma.
Activated fibrin will basically entangle platelets, and passing red blood cells in a big
nasty ball – a blood clot!

The coagulation phase

This begins about 30 seconds after the initial injury. It involves a complex sequence of
events, that ultimately lead to the activation of fibrin from fibrinogen.

The Clotting Cascade

There are two separate clotting pathways, the intrinsic and extrinsic. These eventually
join together to form the common pathway.

The intrinsic pathway

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 Begins in the blood stream. It is basically activated when blood is exposed to
collagen (or other damaged surfaces, but collagen is the main thing involved).
Factor XII is activated to XIIa by exposed collagen
XIIa, with the help of HMW kininogen,activates XI to XIa. This can proceed more
quickly in the presence of prekallikrein.
XIa combines with calcium, and activates IX to IXa
Simultanesouly, platelets will release PF3, and also simultaneously, VIII will be
activated to VIIIa.
IXa, (with the help of PF3) will join together with VIIIa and form factor X activating
factor (‘tenase’)

The extrinsic pathway

Begins in the vessel wall. Damaged endothelial cells will release factor III (tissue factor),
and the greater the amount of damage, the more is released.
This combines with calcium, and activates factor VII and turns it into factor VIIa.
This VIIa-tissue factor complex is quickly inactivated by antithrombin III!

The Common Pathway

X is activated, either by VIIa or tenase , to form Xa – aka prothrombinase
Xa, with the help of calcium ions, and Va will turn prothrombin into thrombin!
Thrombin is factor IIa
Factor V is not activated until it has come into contact with thrombin itself.
Thus V is not required for this step, but when present will increase the rate.
Thrombin will then activate fibrinogen to fibrin. Fibrin strands will begin to join
together, and with the help of XIIIa this will cause the cross-linking of fibrin
strands.
XIII is also activated by thrombin. XIII is also known as fibrin stabilising
factor.

Note that – once activated, thrombin can act as a ‘catalyst’ in other areas of the
cascade to speed up the process:
It can activate factor VII directly
It activates factor V

The extrinsic pathway produces thrombin very quickly, but in small amounts, the
intrinsic pathway produces a large amount of thrombin, but takes a while to get
going.

Regulation of the pathways

TFPI – Tissue factor pathway inhibitor – this is a protein that directly inhibits Xa – even
when present at very low concentrations. It is important in regulating the clotting
cascade.
Antithrombin-III – this is am enzymes found circulating in the blood that binds to

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thrombin, thus preventing its action. It is crucial for the action of heparin!
Activated fibrin will remove and inactivate thrombin. About 85-90% of the thrombin
produced is inactivated in this way.

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 The Clotting Cascade

Drugs that affect coagulation

Heparin
This combines with antithrombin-III, and force a conformational change in the
compound, so that it acts at various stages of the clotting cascade to reduce clotting. It
increases the effectiveness of antithrombin-III by over 1000x.
Mechanism of action
Heparin does not act directly on the extrinsic pathway.
It will inactivate many of the factors produced in clotting:
2, 8, 9, 10, 11, 12 are the ones affected (thrombin and 8-12!)
It will also encourage the release of TFPI
Pharmacokinetics
Heparin is available as unfractioned heparin (‘normal’ heparin) or in various low
molecular weight preparations.
Note that heparin can be inactivated by nicotine!
Unfractioned Heparin
HL – approx 30 minutes – but this is unpredictable! It can increase to up to 2-3 hours
with larger doses.
The majority of heparin is metabolised by endothelial cells – hence its unpredictability.
The rest is mainly metabolised by the liver, and some is excreted by the kidney. The half-
life gets longer the greater the amount of drug you use because once you get past the
‘saturation point’ the endothelial cells can no longer cope with the drug, and thus slower
mechanisms, such as renal excretion are the primary method of metabolism, until the
level of drug drops below the saturation point again.
It can be given either subcutaneously or intravenously:

Subcutaneous – low doses are often given in this manner. Bio-availability is only
about 30% when you give it by this route though, so it is not good for large doses
or emergencies (i.e. PE). It also takes up to an hour before the action of the drug is
seen.
Intravenous – more rapid (immediate), and much greater bioavailability. Thus in
the emergency situation, it is often given as an initial IV dose, followed by
subcutaneous infusion.

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LMWH
Low molecular weight heparin – e.g. clexane (enoxaparin), Dolteparin, Nadroparin
These have approximately 2x the duration of action (HL), and are far more
predictable than unfractioned heparin.
They have a lower affinity for binding sites (particularly the binding sites on
endothelial cells) than unfractioned heparin, hence the greater duration of action
and greater predictability.
Note that this will also mean that more of the heparin is metabolised by the
liver than when you give unfractioned heparin.
They have a greater effect on factor Xa, than on IIa, which suggests that they
produce an equal anti-coagulant effect to heparin, but that they have a reduced
risk of causing bleeding – therefore good news all round!

In clinical practice, unfractioned heparin has to very closely monitored when it is

administered, by LMWH’s give you better freedom. They are also more effective when
give by subcutaneous injection than unfractioned heparin, and thus, for long-term
anti-coagulant therapy, patients may be given a couple of injections subcutaneously
each day, and this will provide sufficient anti-coagulation.
Side effects
Haemorrhage! – the risk is greatest in the elderly, and may be exacerbated by
alcohol intake. This is by far the most common side effect.
Protamine sulphate – This will prevent the action of unfractioned heparin – so
you can give it to reverse its effects. However, it DOES NOT work for LMWH’s.
Osteoporosis – can occur if the drug is used for more than a few weeks. This does
not occur with LMWH’s.
Thrombocytopaenia – can occur after 7-10 days of therapy. It is a result of
heparin induced antiplatelet antibodies.
Hyperkalaemia – due to inhibition of aldosterone secretion
Hypersensitivity

Nice to know
Heparin is found naturally in the body in small amounts and is produced by mast cells
and basophils.
Mast cells and basophils – basically the same cells, but mast cells are found in
connective tissue, and basophils are found in the blood stream.

Heparin is not actually one specific substance, the name actually encompasses a family
of glycosaminoglycans, commercially it is extracted from beef lung or hog intestines, and
due to variability of its potency, the drug is measured in standardised units, and not
mass or volume.

Vitamin K antagonsists– note that the ‘K’ stands for Koagulation in German!
e.g. warfarin
Vitamin K is a fat soluble vitamin, that occurs naturally in plants. It is essential for the
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formation of clotting factors 2, 7, 9 & 10. Proteins C and S are also dependent on
vitamin K.

Warfarin
It is the most important oral anticoagulant. Other examples with a similar mechanism of
action include pheninidione.
Patients on warfarin (and other vit K antagonists) need to have individualised doses, and
this means the treatment in both inconvenient and has a low margin of safety.
Mechanism
Inhibits enzymatic reduction of vitamin K to its active form – hydroquinone. Binding is
competitive.
The effect takes several days to develop, as it is dependent on the half life of the already
active factors, 2, 7, 9 and 10.
VII – has a half life of 6 hours
IX – has a half life of 24 hours
X – has a half life of 40 hours
II – has a half life of 60 hours

Pharmacokinetics
Absorbed rapidly and completely from the gut
Binds well to albumin
Peak time of action is about 48 hours after administration, but peak concentration
in the blood is about an hour after administration
The effect on prothrombin time is initially seen after 12-16 hours, and lasts
approximately 4-5 days.
Half life is very variable, but is on average about 40 hours
It crosses the placenta, and is teratogenic – thus it should not be given in
pregnancy at all! In the early stages it can causes defects, and in the later stages it
can cause haemorrhages in the foetus itself – usually intracranial haemorrhage.
It is metabolised by the cytochrome P450 system – thus it interacts with many
drugs – making administration and monitoring of dosage more difficult.
Warfarin is monitored by using the prothrombin time (PT), which is expressed as
the INR. The dose of warfarin is adjusted to give an INR of 2-4

Unwanted effects
Haemorrhage – this is especially common to the bowel and brain. This can be
counteracted by the administration of vitamin K, or giving fresh plasma containing
clotting factors.
Teratogenicity (causes birth defects)

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 Necrosis of soft tissues – this occurs mainly to tissues in the buttock and breast
and is a result of thrombosis in venules. It generally occurs shortly after
administration, and is a result of inhibition of synthesis of protein C – which is
another effect of warfarin, and which happens more quickly than the inhibition of
activation of vit K. thus for a short time after the initial administration, patients are
in a hypercoagulant state. This is rare, but serious.
To combat this issue, treatment is usually started with heparin, before treatment
with warfarin begins.

Interactions with warfarin

Things that potentiate the effects of warfarin
Disease
Liver disease – this reduces the number of clotting factors produced (2,7,9,10 are
affected)
High metabolis rate; e.g. thyrotoxicosis and fever – as these increase the rate at
which clotting factors are degraded

Drugs
Agents that inhibit hepatic metabolism – such as many antifungals, and other
specific drugs, including cimetidine, ciprofloxacin, chloramphenicol, co-
trimoxazole, imipramine, metronidazole and amiodarone.
Drugs that inhibit platelet function – Aspirin, and other NSAIDs – as these inhibit
platelet thromboxane synthesis. Also some antibiotics, including moxalactam and
carbenicillin
Drugs that displace warfarin from its binding site on albumin – e.g. NSAIDs and
chloral hydrate – as this will increase the concentration of warfarin in plasma
Drugs that inhibits synthesis of vitamin K – such as the cephalosporins

Things that decrease the effects of warfarin

Physiological state / disease
Pregnancy
Hypothyroidism – where there is reduced metabolis rate, and thus reduced
breakdown of coagulation factors

Drugs
Vitamin K – it is found in some vitamin preparations and some form of parenteral
feeding
Drugs that induce hepatic cytochrome P450 enzymes – this increases the
degredation of warfarin. Examples include rifampicin, carbamazepine,
barbiturates and griseofulvin

Nice to know
Named after the Wisconsin Alumni Research Foundation – after cows feed was
changed in the USA, to contain sweet clover, and loads of cows died from
haemorrhagic stroke.

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Warfarin acts on the extrinsic pathway, whilst heparin acts on the intrinsic
pathway. Thus, warfarin efficacy is monitored using the INR – which utilises the
prothrombin time. This is because warfarin acts on the extrinsic pathway – and the PT
is a measure of the extrinsic pathway. Remember that the extrinsic pathway produces
a bit of fibrin quickly whilst the intrinsic pathway produces large amounts but takes a
while to get going. Thus the PT, which is a measurement of how quickly a small clot
forms, relies on the extrinsic pathway.
Heparin is measured using the aPPT. This is the activated partial thromboplastin
time. This measures the intrinsic and common pathways.
Method – similar to the PT – a sample is taken, then mixed with some proteins,
and the time measured until a clot forms. The clot takes longer to form than in PT,
because the fluid that the blood is mixed with to stimulate clot formation has no
tissue factor (III) and thus the extrinsic pathway is not activated. The word ‘partial’
is used to indicate the lack of tissue factor.
The value obtained is normally between 25 and 39 seconds. A prolonged APPT can
be caused by:
Use of heparin
Haemophilia – coagulation factor deficiency

Signs of clotting deficiency

Easy bruising
Nosebleeds
Menorrhagia
Prolonged bleeding

The liver dependent clotting factors are 2 (thrombin), 7 9 & 10!

Thrombolysis and fibrinolysis
Fibrinolysis
When the clotting cascade is activated, so is the process of fibrinolysis. This is basically a
mechanism that prevents clotting from getting out of control, and prevents the excessive
deposition of fibrin.
The main mechanism involved is the release of tissue plasminogen activator
(tPA) by damaged endothelial cells.
Another activator; urokinase is produced and released by the kidneys as a means
of prevent small clots getting lodged in the kidney tissue.
Kallikrien and neutrophil elastase are further fibrinogenic factors.
These circulating activators will then convert plasminogen to plasmin, which
then breaks down fibrin. This actively breaks down the clot – as oppose to
anticoagulants, which just reduce the chance of further clot forming.
Fibrin fragments are broken down into D-Dimers.
tPA can itself be inhibited plasminogen aactivator inhibitor (PAI), and plasmin is
inactivated by α2 antiplasmin.
A lipoprotein, called lipoprotein a also inhibits tPA, and high levels of lipoprotein
a are associated with an increased risk of MI
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Plasminogen itself will deposit on fibrin strands, waiting to be activated, by the activated
factors circulating in the blood. It not only digests fibrin, but also fibrinogen, and factors
II (thrombin), V and VIII.
Only plasminogen that has been absorbed by fibrin will be activated by the
activators, (I guess that by joining to fibrin there is a structural change exposing the
active site), and plasminogen that escapes to the general circulation is broken down by
its inhibitors, particularly PAI.
Drugs affect this system by either increasing or reducing fibrinolysis
Fibrinolytic drugs e.g. streptokinase, alteplase, reteplase, tenecteplase
These are still relatively new and used with caution. It should not be used to treat DVT
(the risks outweigh the benefits, although it may still rarely be used) and is mainly only
used to treat MI. In some cases it may be used to treat a massive PE. See ‘clinical uses’
below.
Mehanism
They all activate plasminogen to enhance fibrinolysis.
Alteplase – this is basically synthetic tPA.
Reteplase – this is basically a modified synthetic tPA – it has reduced affinity for fibrin
and fibrinogen, and normal affinity for PAI, but has increased duration of action
Tenectplase – another synthetic tPA, it has increased affinity for fibrin, and reduced
affinity for PAI (which is good!), and also has a long duration of action.
Streptokinase – this is a bit different. it is inactive until it binds to plasminogen in the
blood, at which point it will form a streptokinase-plasminogen combination, which is able
to activate plasminogen. It is produced by streptococcal bacteria
Pharmacokinetics
They are all administered IV or intra-arterially.
Streptokinase – some is removed by streptococcal antibodies before it forms its
complex. Once it has formed its complex it is broken down enzymatically.
After streptococcal infection, or after the previous use of streptokinase, its
efficacy cn be seriously reduced to the presence of antibodies in the
circulation. These can persist for several years.
Streptokinase has quite a long half-life, similar to that of tenectplase (but longer
than that of alteplase and reteplase).
It is usually administered as a 1hr infusion for treatment of coronary artery
occlusion, although longer regimens may be used when treating PE or other
arterial blockages.

Alteplase and associated compounds are metabolised by the liver.

Altepplase is given as a long infusion of 3-24hr due to its short duration of action
patients will also be given 48hrs of heparin to prevent re-occulsion, again, a result
of the short half-life of the compound.
Reteplase is given as two bolus injections, and tenectplase as a single bolus.
Although there is little evidence to support it; heparin is also given with these
compounds (although a guess it would be given anyway in situations involving
clot! Just not necessarily for reasons related to the –plases.
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Unwanted effects
Haemorrhage – is usually minor, but can occasionally be serious, eg. Intracerebral
haemorrhage. Other haemorrhagic complications may involve GI bleeds and stroke.
This occurs with about 1% of those treated. It occurs slightly more frequenctly with
the –plases compared to streptokinase.
The bleeding can be stopped by antifibrinolytic drugs, or y giving fresh plasma
(which is full of nice clotting factors).

Hypotension – this is dose related and is most common with streptokinase. It is

possibly a result of release of bradykinin release which is a result of unbound
streptokinase circulation. The blood pressure will rapidly return to normal if the
treatment is stopped for a short while
Allergy – These are very rare, and only really occur with streptokinase as a result of its
bacterial origin.
Contraindications
Internal bleeding
Haemorrhagic cerebrovascular disease
Pregnancy
Uncontrolled hypertension
Recent trauma – including vigorous CPR

Which drug is best?

In MI the sooner you give the drugs, the greater their effects. You should try to ensure
they administered before 12 hours after the onset of symptoms.
Trials have shown that the drugs are all pretty much equal in their efficacy, but that
mechanical opening of the affected artery is more effective than using
thrombolysis. i.e. – angioplasty is more effective!
Clinical Use
MI – use only when there is ST segment elevation, and symptom duration is less than
12 hours
Acute thrombotic Stroke – within 3 hours of onset in selected patients
Clearing thrombosed shunts and cannulae
Acute arterial thromboembolism
Life-threatening PE (and rarely DVT)
Antifibrinolytic and haemostatic agents
Tranexamic acid – inhibits plasminogen activation, and thus prevents fibrinolysis.It
actually binds to plasminogen, and doesn’t actually de-activate it, as tPA is still allowed to
bind to it. However, the tPA binding action odes then not cause activation of
plasminogen, and thus plasminogen will not act on fibrin.
It can be given orally, or IV
It is used to treat conditions where there is serious risk of haemorrhage; commonly
including dental extraction, prostatectomy and menorrhagia, as well as for life-
threateneing bleeding following thrombolytic drug administration.

Can cause nausea, diarrhoea and vomiting

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Aprotinin – this is a broad spectrum protease inhibitor. It has many effects, including:
Inhibition of plasmin and therefore fibrinolysis
Inhibition of platelet activation
Anti-inflammatory effects
Inhibiton of kallikrien – therefore inhibition of the clotting cascade and thus
aprotonin also has anti-coagulant as well as antifibrinolytic effects.

It must be given intravenously due to poor absorption by the gut.

Can occasionally cause hypersensitivity

For both of these drugs, the theoretical risk of thrombotic tendency does not appear to
be a clinical issue

Related Articles
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Deep Vein Thrombosis – DVT
DVT Exam
Other Coagulation Factor Deficiencies
Polycythaemia Vera

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