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Homeostasis and drugs for maintaining homeostasis.MedicinePharmacy
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PHAY2003: Biological Systems and Therapeutics 2
Haemostasis and related drugs - Summary
Dr Rebecca Lever
Principles of Haemostasis
Haemostasis comprises the physiological response to injury and is a tightly regulated system.
Overactivity of haemostatic mechanisms predisposes the host to unwanted thrombosis (clotting of the
blood in vivo), whereas underactivity carries the risk of injury-related and spontaneous haemorrhage
(blood loss).
Physiologically, following loss of vascular integrity, local vasoconstriction occurs, which helps to limit
blood loss by limiting flow to the site of damage, and the following mechanisms are activated:-
• Platelets plug the damaged area
• The platelet plug is stabilised by fibrin (thrombus formation)
• Dissolution of the thrombus occurs
However, the process is dynamic and dissolution of the thrombus will start to occur at the same time as
it continues to develop. This point is important when considering, for example, how thrombosis can be
treated with anticoagulant drugs, whereby inhibiting the rate of thrombus formation allows the body’s
natural fibrinolytic mechanism to dominate and dissolve the thrombus. In pathophysiological
conditions, thrombosis can be treated or prevented by drugs that interfere with the various elements of
the above system. These classes of drugs are antiplatelet agents, which inhibit the activation and
aggregation of platelets, anticoagulant drugs, which inhibit the formation of fibrin by the plasma
coagulation cascade and fibrinolytic drugs (also referred to as thrombolytic), which act to dissolve a
thrombus that has already formed. Alternatively, anti-fibrinolytic drugs, which inhibit the natural
dissolution of fibrin, can be used in situations where excessive bleeding is a problem (e.g. menorrhagia,
certain types of surgery). Tranexamic acid is the main clinically-relevant example of an anti-fibrinolytic
drug.
Platelet activation
Following damage to the blood vessel, structures that are normally covered by the endothelial lining (a
single layer of endothelial cells that normally provides an anti-adhesive and anti-thrombotic surface to
the blood vessel wall) become exposed to the contents of the blood. Exposure of collagen, part of the
subendothelial matrix, allows the initial binding of platelets (thrombocytes) via formation of a ‘bridge’
formed between the platelet surface and the matrix by von Willebrand’s factor (vWF). vWF is stored
inside endothelial cells (in granules called Weibel-Palade bodies) and is released when they are
damaged. This mechanism of tethering platelets is thus localised to where it is required, i.e. the site of
damage. Other platelets adhere to the tethered platelets and in this way a platelet aggregate, or ‘plug’
is formed. However, this initial plug is not robust enough on its own to withstand the shear forces
presented by the blood flow and requires stabilisation by the formation of a fibrin meshwork, which
involves the plasma coagulation system. The activated platelet surface provides a ‘scaffold’ for the
generation of fibrin, again helping to localise the response to where it is required. Platelets are also a
source of vWF, where it is stored in alpha granules, along with other proteins involved in coagulation
(fibrinogen, factor V) and tissue repair (growth factors, fibronectin), and released upon activation. A
second type of granule within the platelet (dense granules) contains 5-HT, which promotes local
vasoconstriction, and adenosine diphosphate (ADP), which activates additional platelets. Platelets can
also be activated by thrombin (factor IIa; a key mediator of the coagulation cascade), thromboxane A2
(TXA2; produced by other activated platelets) and fibrinogen (the precursor of fibrin; found in the
plasma and released from activated platelets).
Antiplatelet drugs
Antiplatelet drugs work to prevent the activation of platelets in patients at risk of unwanted thrombosis
(usually as a result of atherosclerosis) by inhibiting the synthesis or actions of the mediators listed
above. Aspirin is an irreversible inhibitor of the enzyme cyclooxygenase (COX), which synthesises
prostanoids from the precursor arachidonic acid. TXA2 is the prostanoid generated by platelets (others
are produced by inflammatory cells, which is why aspirin is also an anti-inflammatory drug) and causes
vasoconstriction and aggregation of platelets. Because platelets lack a nucleus and aspirin is an
irreversible inhibitor of COX, once the enzyme is inhibited the platelet is prevented from generatingTXA2
for its entire lifespan (7-10 days) because it cannot synthesise more enzyme. For this reason, a low dose
(typically 75 mg per day) of aspirin provides an effective antiplatelet effect, whereas much higher doses
are required for anti-inflammatory and analgesic effects. Thienopyridine drugs such as clopidogrel are
irreversible P2Y12 receptor antagonists; the platelet receptor for ADP. In addition to activating platelets
directly ADP stimulates expression of the glycoprotein IIb/IIIa complex (GPIIb/IIIa) on the platelet
surface, the platelet receptor for fibrinogen. Other thienopyridine drugs that act in identical manner are
prasugrel and ticlopidine. Ticagrelor (not a thienopyridine chemically but often grouped with the other
drugs because of its similar mode of action) binds reversibly to the P2Y12 receptor. Binding of fibrinogen
to GPIIb/IIIa mediates the final common pathway of platelet activation and can be inhibited by
abciximab, a monoclonal antibody fragment (Fab region) that binds to the GPIIb/IIIa complex and
prevents fibrinogen from binding. Eptifibatide (peptide) and tirofiban (small molecule) are alternative
agents that act as competitive GPIIb/IIIa antagonists.
Coagulation cascade
The purpose of the coagulation cascade is ultimately the generation of fibrin. Although there is much
overlap between these pathways, the factors involved in the cascade can be grouped into an intrinsic
pathway, thus named because all of its components are present (intrinsic) within the plasma, an
extrinsic pathway, thus named because a key component (tissue factor) is found outside (extrinsic to)
the plasma and a common pathway into which the intrinsic and extrinsic pathways converge. It is
activation of the common pathway, by whichever route, that leads to the activation of thrombin and
generation of fibrin. The intrinsic pathway becomes activated by contact with a negatively charged
surface. In vivo, damage to the blood vessel wall exposes collagen and the subendothelial basement
membrane which can provide such a surface. In vitro, contact with glass for example would activate the
system (this is why blood clots in a test tube unless anticoagulant is added to it). The extrinsic pathway
becomes activated when the contents of the plasma come into contact with tissue factor, when blood
vessel integrity is lost (i.e. following injury).
The cascade involves the conversion of circulating inactive factors, known as zymogens, to active serine
protease enzymes. The zymogens are plasma proteins, produced by the liver, and are named by Roman
numeral. In their activated form, the letter ‘a’ is added to the Roman numeral (e.g. factor XII of the
intrinsic cascade becomes activated by a negative surface to form factor XIIa, which proteolytically
cleaves the next factor in the cascade, factor XI, to form factor XIa, and so on). Both pathways lead to
the activation of factor X, which in turn lead to the generation of thrombin (factor IIa). Thrombin is a
particularly important factor as it a) cleaves fibrinogen to form fibrin, the end product of the cascade, b)
activates factor XIII which stabilises the fibrin meshwork, c) is involved in positive amplification of the
cascade by activating cofactors (V and VIII) that promote the generation of further amounts of thrombin
(it also activates certain anticoagulant mechanisms so is also involved in negative regulation of the
system) and d) activates platelets, as mentioned above.
The importance of the coagulation cascade is illustrated by considering the condition haemophilia,
whereby the absence of a single factor of the system (factor VIII or factor IX in type A and type B
haemophilia, respectively) results in spontaneous and injury-related haemorrhage that is life-
threatening without treatment (replacement of the deficient factor). Interestingly, deficiency in factor
XI results in only a slight bleeding tendency by comparison, which goes some way to suggest that the
extrinsic pathway is the more critical in the initial response to injury. However, it is clear that, once
activated, the pathways act in concert.
Anticoagulant drugs
Warfarin is a synthetic coumarin derivative. Dicoumarol, the discovery of which led to the development
of warfarin, is found in mouldy hay containing the plant sweet clover. Around a century ago, it was
found that livestock fed silage made from sweet clover developed spontaneous haemorrhage that could
only be cured by blood transfusion (not practical). This was due to the presence of dicoumarol, a potent
anticoagulant substance, in the silage. Coumarins such as warfarin are classed as vitamin K antagonists.
Vitamin K is a fat-soluble vitamin that is required as a cofactor in liver cells for the synthesis of certain
factors of the coagulation cascade (namely factors II, VII, IX and X; ‘vitamin K-dependent factors’) that
require calcium and phospholipid binding for their biological activity. The natural anticoagulant factors
protein C and protein S are also vitamin K-dependent. These factors require post-translational
carboxylation of glutamic acid residues in order for phospholipid binding to occur and the factors are
non-functional without this. Vitamin K in a reduced form is required as part of a paired reaction that
leads to the carboxylation of the factors by the enzyme gamma glutamyl carboxylase. Vitamin K is
oxidised as part of this process and is regenerated in its reduced form by another enzyme, vitamin K
epoxide reductase (VKOR) and it is this enzyme that warfarin inhibits. As might be expected, the main
side effect of warfarin is haemorrhage. The drug is associated with a number of problems, mainly
associated with its narrow therapeutic window and propensity to interact with other drugs and dietary
factors. Warfarin is given orally as a racemic mixture. The isomers differ in that S-warfarin is
approximately five times as potent as R-warfarin as an anticoagulant, but it is also metabolised more
rapidly (different CYP enzymes involved). Drugs that increase the rate of metabolism of warfarin reduce
the anticoagulant (increased risk of thrombosis). Conversely, drugs that reduce the rate of metabolism
of warfarin increase the effective anticoagulant dose and thus increase the risk of bleeding. In addition
to this, circulating warfarin is approximately 99% albumin-bound. Therefore, other highly albumin-
bound drugs can act to displace warfarin and thus increase the anticoagulant effect. One such drug is
aspirin, which can further increase the overall risk of bleeding through its antiplatelet actions. Because
of these problems, patients taking warfarin need to undergo therapeutic monitoring to ensure that their
dose is both safe and effective. This is carried out by monitoring the international normalised ratio
(INR), which is the result of an in vitro clotting test (prothrombin time) of the patient’s plasma, divided
by that of normal reference plasma. The dose of warfarin in adjusted to ensure that the INR is kept
within an appropriate therapeutic range for the specific indication. It is particularly important that once
a patient is stabilised on warfarin therapy they do not significantly change their diet (due to different
types of food containing varying levels of vitamin K), or other medication (including over the counter
medicines) without advice. Warfarin cannot be given in pregnancy because it is teratogenic and its
effects take several days to become fully established. This is because the intact coagulation factors that
were synthesised before warfarin was started need to be cleared (all have different half lives) and
replaced with the non-carboxylated factors before the anticoagulant effect can stabilise. In overdose or
haemorrhage due to warfarin therapy, vitamin K1 (phytomenadione) can be given to replenish vitamin K
levels in the liver.
Heparin is a large, polyanionic, naturally-occurring molecule. It is a glycosaminoglycan, formed from
repeating disaccharide units of an amino sugar (glucosamine, galactosamine) linked to uronic acid
(glucuronic/iduronic acid). Heparin is the most negatively charged compound found in nature. Heparin
works indirectly as an anticoagulant by binding to and potentiating the effects of antithrombin, the
main natural inhibitor of coagulation. Heparin causes a conformational change in antithrombin that
makes its reactive arginine centre more accessible and the rate of inhibition of the serine proteases is
potentiated by up to 1000-fold. The most important inhibitory effects of heparin/antithrombin are on
factors Xa and IIa (thrombin), although factors VIIa, IXa, XIa and XIIa are also affected to varying extent.
Physiologically, a related substance, heparan sulphate, which is abundant on endothelial cells, is
thought to be responsible for the normal activity of antithrombin. Heparin has to be given parenterally
(intravenous or subcutaneous injection) because of its size and charge. Again, the main side-effect
associated with heparin is haemorrhage, although it can cause serious hypersensitivity reactions
(heparin-induced thrombocytopaenia) in some patients, especially following prolonged or repeated
therapy. Unlike warfarin, the anticoagulant effects of heparin are immediate because it works by
interacting with factors that are already present in the plasma (for this reason, heparin works in vitro
and in vivo, whereas warfarin would have no effect in vitro). Heparin is available as unfractionated
preparations or low-molecular weight preparations (e.g. enoxaparin, dalteparin, tinzaparin), the latter
having a longer half-life and more predictable pharmacokinetics. The positively charged molecule
protamine can be given to reverse the effects of heparin in overdose/haemorrhage due to heparin
therapy, although this approach is less effective against the low molecular weight heparins than the
unfractionated product. Low molecular weight heparins have greater effects on factor Xa than factor
IIa, whereas unfractionated heparin affects these factors equally. This is as a result of differences in the
way in which the antithrombin/heparin complex interacts with factor Xa versus factor IIa. Generally
speaking, the lower the mean molecular weight of the preparation, the greater the ratio of anti-Xa to
anti-IIa activity. An extreme example of this can be seen with fondaparinux, which is a pure factor Xa
inhibitor. Fondaparinux is a synthetic pentasaccharide which is identical to the high-affinity binding site
for antithrombin that occurs naturally in heparin.
Alternative and newer anticoagulants
Alternative anticoagulants that can be given by infusion when heparin is contra-indicated are bivalirudin
and argatroban. Both of these drugs are direct thrombin (IIa) inhibitors. Bivalirudin is a synthetic
peptide based on the naturally occurring peptide hirudin, which is secreted from the salivary glands of
leeches to facilitate extraction of blood from their host. Argatroban is a non-peptide, small molecule
inhibitor. Dabigatran is an orally active direct thrombin (IIa) inhibitor and rivaroxaban and apixaban
are orally active direct inhibitors of factor Xa. Dabigatran, rivaroxaban and apixaban have the
advantage over heparin of being orally active and the advantage over warfarin of predictable and stable
anticoagulant effects without the problems of interactions associated with warfarin. For this reason,
therapeutic monitoring is not required with these agents, which is a significant improvement. It is likely
that the therapeutic use of warfarin, and to an extent heparin, will gradually be replaced by these
agents, which currently share the vast majority of indications of the older drugs. Disadvantages of the
newer agents are cost, although this is offset to a large extent by the lack of requirement for monitoring,
and that there are currently no specific agents available that reverse their effects,
Fibrinolysis
Physiologically, once blood loss has been stopped and tissue repair is underway, there is a need for
removal of the thrombus. This is achieved by the natural process of fibrinolysis. Plasminogen is the
inactive precursor of another enzyme, plasmin, which digests the normally insoluble fibrin to soluble
degradation products that are readily cleared. Like the coagulation cascade, this process has to be very
tightly regulated in order that haemostasis can occur effectively. Plasminogen binds to both fibrin and
fibrinogen and, as a result, becomes incorporated into the thrombus as it is formed. This means that
plasminogen, and hence plasmin, is localised to where it is ultimately required, i.e. the site of the
thrombus. Plasminogen is activated by tissue plasminogen activator (tPA), which itself is released from
damaged endothelial cells and becomes active upon binding to fibrin (again localising it to the site
where it is required). Plasminogen activator inhibitors rapidly inactivate the system.
Fibrinolytic/Thrombolytic agents
These agents all work by activating plasminogen to form active plasmin, and thus act to promote
dissolution, or lysis, of a thrombus. These agents are often referred to colloquially as ‘clot-busting’
drugs. Streptokinase is a bacterial product and possesses the disadvantage that it is antigenic and can
cause severe hypersensitivity reactions in patients. In particular, streptokinase cannot be used more
than once in the same patient as antibodies will be produced against it on the first exposure. Alteplase
is human recombinant tPA, so is non-antigenic. Reteplase and tenecteplase are mutant forms of human
tPA that are longer-acting than alteplase. This feature is useful as it allows them to be administered by
bolus injection rather than infusion, which means that they can potentially be given before arrival at
hospital.
Major indications for drugs affecting haemostasis
Arterial thrombosis is usually as a consequence of atherosclerosis. The major problems associated with
arterial thrombosis are myocardial infarction (heart attack) and stroke. A key aim is to remove
modifiable risk factors, namely: smoking, hypertension, hypercholesterolaemia, uncontrolled diabetes,
lack of activity and obesity. Non-modifiable risk factors include advanced age, male gender and family
history. However, individuals with a non-modifiable risk should be particularly careful not to accumulate
additional risk factors.
Ischaemic stroke accounts for approximately 85% of all strokes, the remainder being due to
haemorrhage. In the majority of cases these are thrombotic in nature, whereby thrombosis occurs
within a cerebral artery and thus the brain tissue beyond the blockage becomes starved of oxygen and
glucose. Alternatively, the blockage may have originated elsewhere (usually the heart) and travelled to
the brain, in which case the stroke is considered to be embolic. In either case, if flow is not quickly
restored, the affected cells will die. Signs, symptoms and outcomes will depend upon the location and
size of the affected artery but typical signs and symptoms include sudden weakness on one side of the
body and/or drooping of one side of the face (usually the opposite side to that of the thrombus),
dizziness, confusion and speech problems. Stroke is a medical emergency and immediate treatment
should be sought. Antiplatelet drugs are useful in the prophylaxis of stroke, alongside management of
other risk factors such as smoking, high blood cholesterol and hypertension. The fibrinolytic agent
alteplase is indicated in selected patients, but is contra-indicated if the risk of haemorrhage is high.
However, alteplase is only useful within a limited timeframe (a few hours at most) and even then is
ineffective in restoring adequate blood flow in many patients. Anticoagulant drugs are useful in
selected patients at risk of thrombotic stroke, but are particularly useful and specifically indicated in
patients at risk of embolic stroke. Embolic stroke is strongly associated with atrial fibrillation (due to
stagnation of blood flow in the chambers of the heart, particularly the left atrium), but also with cardiac
valve dysfunction and recent heart attack. Emboli that form in the heart in this manner are more similar
in composition to a venous thrombus (i.e. fibrin-rich ‘red clot’) than an arterial thrombus (i.e. platelet-
rich ‘white clot’), hence why anticoagulant drugs are useful in preventing their development.
Myocardial infarction, comes under the umbrella term of Acute Coronary Syndrome. This
encompasses a range of acute myocardial ischaemic states and is a medical emergency. Again, the
problem is driven by atherosclerosis. Atherosclerotic plaques can be hard and stable, or soft and
unstable. It is the latter which are particularly problematic as they are prone to rupture, which leads to
thrombosis in the affected artery and potential embolism of the ruptured plaque material to an arterial
branch downstream of the site of origin. Occlusion of a coronary artery will lead to ischaemia of the
cardiac muscle supplied by that artery and, as with stroke, unless reperfusion is achieved promptly, the
tissue will start to die. Signs and symptoms may include chest pain, discomfort or a feeling of pressure
that may radiate to the left arm, jaw, neck, shoulder or back, breathlessness, autonomic features
(sweating, nausea, vomiting, pallor), palpitations, dizziness and collapse and intense fear. Immediate
treatment of acute coronary syndrome, following resuscitation (ideally defibrillation) if necessary,
follows the mnemonic ‘MONA’:-
Morphine (i.v.): For pain relief. Also provides a degree of venodilatation and non-specific anxiolytic
effect. Given with metoclopramide for relief of nausea (due to the condition as well as the morphine).
Oxygen: If required, as guided by pulse oximetry measurements .
Nitrate: Glyceryl trinitrate infusion. Coronary vasodilatation improves oxygenation and venodilatation
reduces the preload and hence oxygen demands of the myocardium.
Aspirin: 300 mg loading dose (usually chewed) is given for antiplatelet effects. This simple intervention
significantly improves survival outcomes.
In addition, an ECG would be performed as soon as possible and blood tests would be carried out, which
would include the measurement of cardiac enzyme levels in the plasma. The ECG would likely show
elevation of the ST segment (corresponding to the period of ventricular systole) in the case of
myocardial infarction (ST-elevation myocardial infarction, or STEMI), although this feature can
sometimes be absent (non-ST-elevation myocardial infarction, or NSTEMI). The presence of cardiac
enzymes in the plasma at levels above normal range would confirm myocardial injury; enzymes normally
found within cardiac myocytes become released when the cells die. The preferred marker is cardiac
troponin I or T. Troponins are contractile proteins that are normally absent in the plasma and the
cardiac forms are highly specific markers of myocardial damage.
In addition to drug therapy (a standard drug regimen would include aspirin, beta blocker, ACE inhibitor,
statin and insulin as appropriate), a patient with established myocardial infarction might undergo
thrombolysis or interventional cardiology. In both cases, time is of the essence in order to save as
much cardiac muscle as possible. Thrombolysis is contraindicated where a significantly increased risk of
haemorrhage exists, for example following recent major surgery or head injury. Interventional
Cardiology involves percutaneous coronary intervention (PCI), also referred to as coronary angioplasty,
whereby a guide-wire is inserted into the affected coronary artery via the aorta, following insertion
through an artery (usually the femoral artery). A balloon catheter is then threaded along the guide-wire
until it is in the correct position and the balloon is then repeatedly inflated. This restores flow through
the blocked artery by flattening the plaque against the vessel wall. The catheter and wire are
subsequently removed. A stent can be inserted to help keep the artery patent. This approach has the
advantages of mechanically improving myocardial perfusion and can be used when thrombolysis is
contraindicated. It is currently the preferred approach as outcomes are favourable compared with
thrombolysis, both immediately and longer term. Antiplatelet drugs are given before, during and after
and anticoagulants during the procedure.
Venous thrombosis tends to affect the deep veins, usually of the leg or hip. If deep vein thrombosis
(DVT) occurs a major concern is that the thrombus will partially or fully break off and embolise, i.e.
move with the venous return, resulting in pulmonary embolism (PE). In this situation, the thrombus is
carried to the right side of the heart and thus enters the pulmonary circulation, whereby it would lodge
in a branch of the pulmonary artery and thus prevent the flow of blood. DVT and PE comprise a single
clinical entity, venous thromboembolism (VTE). Risk factors for VTE include increasing age, immobility,
recent surgery (especially to the legs or hips, not least as this will likely result in a period of immobility),
pregnancy, use of the combined oral contraceptive pill or hormone replacement therapy, cancer and
chemotherapy, long-haul travel, personal or family history and genetic thrombophilia (increased
tendency for the blood to clot, often due to a deficiency in one of the natural anticoagulant
mechanisms). It is usually an accumulation of risk factors that results in VTE, which can be summarised
by Virchow’s triad. Stasis (i.e. poor flow/lack of movement of blood), hypercoagulability (i.e.
something promoting the tendency of the blood to clot) and vessel wall injury all individually promote
thrombosis, but a combination of these factors will greatly increase the chance of thrombosis occurring.
The main signs and symptoms of VTE would be pain, swelling and heat in the affected limb in the case of
DVT and acute chest pain with difficulty in breathing (dyspnoea) in the case of PE. Diagnosis would
involve considering the presence of relevant risk factors in conjunction with symptoms; in the case of PE
there may well be evidence of a prior DVT. In addition, Doppler ultrasound, venography or pulmonary
angiography and ventilation/perfusion scanning can be used to confirm the location of a thrombus.
Measurement of plasma D-dimers can also be useful; D-dimers are breakdown products of fibrin.
Because the body’s natural fibrinolytic mechanisms will be activated if a thrombus exists, levels of fibrin
breakdown products will be elevated. This test has fairly weak positive predictive value (i.e. false
positives can occur for a number of reasons, including inflammatory disease or general injury) but has
excellent negative predictive value (i.e. if D-dimer levels are not raised it is highly unlikely that a
thrombus is present).
VTE can almost always be treated with anticoagulant drugs (rarely, thrombolysis might be used to treat
massive PE). Inhibition of the further development of the thrombus will allow the body’s natural
fibrinolytic process to dominate and dissolve it. Heparin, dabigatran or rivaroxaban can be used to treat
active thrombosis. Prophylaxis, either for a limited period or indefinitely if an ongoing thrombosis risk is
present, can be achieved with warfarin or one of the newer oral anticoagulants.