Coagulation and the Clotting Cascade

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.

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:

• 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

The intrinsic pathway

• 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

• 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.

• It can activate factor VII directly
• It activates factor V

Regulation of the pathways

Drugs that affect coagulation

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.

LMWH

• 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!

• 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

• Mast cells and basophils – basically the same cells, but mast cells are found in connective tissue, and basophils are found in the blood stream.

Warfarin

• 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

• 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

• 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)
• 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.

• 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

• 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

• Pregnancy
• Hypothyroidism – where there is reduced metabolis rate, and thus reduced breakdown of coagulation factors

• 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

• 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.

• 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 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

• 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.

• The bleeding can be stopped by antifibrinolytic drugs, or y giving fresh plasma (which is full of nice clotting factors).

• Internal bleeding
• Haemorrhagic cerebrovascular disease
• Pregnancy
• Uncontrolled hypertension
• Recent trauma – including vigorous CPR

• 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.

• 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.

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