Haemostasis

Question 1

Why is balance important?

Answer 1

1 Allow stimulation of blood clotting processes following injury, in which case blood changes from its liquid stage - coagulation

2. Limit the extent of the response to the area of injury to prevent excessive or generalised blood clotting (thrombosis)
3. Start process that eventually leads to the breakdown of the clot as part of the process of healing (fibrinolysis)

Question 2

What is haemostasis and the processes?

Answer 2

Haemostatsis describes ‘halting of blood’ following trauma to blood vessels and results from three intertwined processes:
1. Contraction of blood vessels: vasoconstriction
2. Formation of unstable platelet plug at site of vessel wall damage (primary haemostasis)
3. Formation of stable fibrin clot (secondary haemostasis / coagulation)
Fibrin mesh binds and stabilises platelet plug and other cells

Question 3

Why do you need haemostasis mechanisms?

Answer 3

• Diagnose and treat bleeding sidoerders
• Identify risk factors for thrombosis
• Treat thrombotic disorders
• Monitor the drugs that are used to treat bleeding and thrombotic disorders
• Control bleeding in individuals who do not have an underlying bleeding disorder

Question 4

What is platelet adhesion?

Answer 4

• Platelets are discoid, non nucleated, granule containing cells that are derived from myeloid stem cells
• Platelets are formed in bone marrow by the fragmentation of megakaryocytic cytoplasm and have a circulating lifespan of around 10 days
• The plasma membrane contains glycoproteins (GPs) that are important for the platelets interactions
• Following injury to the vessel wall platelet stick to the damaged endothelium, either directly to collagen via the platelet GPIa receptor or indirectly via von Willebrand factor (VWF), which binds to the platelet GPIb receptor
• This adhesion of platelets causes them to become activated and changes their shape from a disc to a more rounded form with spicules to encourage platelet-platelet interaction

Question 5

What is platelet release reaction?

Answer 5

• The adhesion of platelets initiates their activation and the release of the contents of they storage granules
• Two main types of ultrastructurally-identifiable granules: alpha-granules and dense granules
• The platelet membrane is invaginated to form a surface connected canalicular system through which the contents of platelet granules are released
• Important components of these contents include: ADP, fibrinogen and von Willebrand factor

Question 6

What is thromboxane A2 synthesis?

Answer 6

• Platelets are stimulated to produce the prostaglandin thromboxane A2 from arachidonic acid that is derived from the cell membrane
• Role in platelet aggregation and known as a vasoconstrictor and important in tissue injury and inflammation

Question 7

What is platelet aggregation?

Answer 7

• The granular release of ADP and generation of thromboxane A2 have positive feedback effects resulting in further platelet recruitment activation and aggregation
• By binding to P2Y12 and thromboxane A2 receptor
• Platelet activation also causes conformational change in GPIIb/IIIa receptor which then provides binding site for fibrinogen
• Fibrinogen binding to GPIIb/IIIa causes ‘outside in’ signalling which further activates the platelets
• Fibrinogen key role in linking platelets together to form the platelet plug
• These effects usually counterbalanced by active flow of blood and the leases of prostacyclin (PGI2) from endothelial cells; prostacyclin is a powerful vasodilator and suppresses platelet activation, thus preventing inappropriate platelet aggregation

Question 8

What are anti-platelet dugs?

Answer 8

Antiplatlet drugs are widely used for the prevention and treatment of cardiovascular and cerebrovasuclar disease

Question 9

How does aspirin work?

Answer 9

• Aspirin inhibits the production of thromboxane A2 by irreversibly blocking the action of cyclo-oxygenase (COX), resulting in a reduction in platelet aggregation, although prostacyclin production is also inhibited by cyclo-oxygenase, endothelial cells can synthesise more COX whereas the non-nuclear platelet cannot
• The effect if a single dose of aspirin persists for around 7 days until most of platelets present at the time of aspirin ingestion have been replaced by new platelets

Question 10

How does clopidogrel work?

Answer 10

• Clopidogrel works by irreversibly blocking the ADP receptor (P2Y12) on the platelets cell membrane.
• Therefore the effect of clopidogrel ingestion also lasts for 7 days until new platelets have been produced

Question 11

Describe Von Willebrand Factor

Answer 11

• Von Willebrand factor (VWF) is a multimeric glycoprotein that is synethsisied by endothelial cells and megakaryocytic and circulates in plasma
• VWF mediates the adhesion of platelets to sites of injury and promotes platelet-platelet aggregation
• VWF also specific carrier for factor VIII (FVIII)

Question 12

Why is there secondary haemostasis?

Answer 12

• The primary platelet plug is sufficient for small vessel injury
• However in larger vessel it will fall apart
• Fibrin formation stabilises the platelet plug
• Blood coagulation pathways centre on the generation of thrombin which cleaves fibrinogen to generate a fibrin clot that stabilises the platelet plug at sites of vascular injury

Question 13

What are some clotting factors?

Answer 13

• Most clotting factors synthesised in liver (exceptions: factor VIII and VWF made by endothelial cells)
• VWF also made in megakaryocytic and incorporated into platelet granules
• Factors II (prothrombin) VII IX and X are dependent on Fit K for carboxylation fo their glutamic acid residues which is essential for the function fo these clotting actors

Question 14

What is each step in blood coagulation characterised by?

Answer 14

conversion of an inactive zymogen (proenzyme) into an active clotting factor by the splitting of one or more peptide bones and exposure of the active enzyme sire

Question 15

What are factors V and VIII?

Answer 15

Co-factors

Question 16

What do calcium ions do?

Answer 16

Calcium ions play an important role in the binding of activated clotting factors to the phospholipid surfaces of platelets

Question 17

Where do clotting factors work?

Answer 17

Many clotting factors are believed to work on the exposed phospholipid surface of platelets which helps to localise and accelerate these reactions

Question 18

What is the trigger to initiate coagulation at site of injury?

Answer 18

-tissue factor (TF) exposed on the surface of the endothelial cells and leukocytes and on most extravascular cells in an area of tissue damage
TF mainly located at sites that are not usually exposed to the blood under normal physiological conditions
As a result blood only encounter TF at sites of vascular injury

Question 19

What does binding of TF do?

Answer 19

• Binding of TF to factor VIIa leads to activation of factors IX to IXa and X to Xa
• This leads to the activation of prothrombin (factor II) to generate a small initial amount of thrombin (factor IIa) and this phase is known as the initiation phase
• This small amount of thrombin mediates the activation of the co factors V and VIII, the zymogen factor XI and platelets (Amplification phase)
• Factor XI convert more factor IX to IXa which inc enncret with factor VIIIa amplifies the conversion of factor X to Xa and there is consequently a rapid burst of thrombin generation (Propagation phase) which cleaves the circulating fibrinogen (soluble) to form the insoluble fibrin clot

Question 20

What are natural anticoagulant pathways?

Answer 20

• Number of inhibitory mechanisms prevent blood from clotting completely whenever clotting is initiated by vessel injury
• Action of these inhibitory mechanisms ensures that coagulation is confined to site of injury and prevent spontaneous activation of coagulation
• Most important of these are protein C, protein S and antithrombin

Question 21

What does thrombin do?

Answer 21

1. Thrombin binds to thrombomodulin on the endothelial cell surface leading to activation of protein C to activated protein C (APC) and AOC inactivates factors Va and VIIIa in the presence of co-factor protein S
2. Thrombin and factor Xa are inactivated by circulating inhibitor antithrombin and the action of it is markedly potentiated by heparin: this occurs physiologically by the binding of antithrombin to endothelial cell-associated heparins

Question 22

What does heparin do (anticoagulant drug)?

Answer 22

• Heparin works indirectly by potentiating the action of antithrombin leading to the inactivation of factors Xa and IIa (thrombin).
• Heparin is administered intravenously or by subcutaneous injection.

Question 23

What does warfarin do (anticoagulant drug)?

Answer 23

• Warfarin, derived from coumarin, is a vitamin K antagonist that works by interfering with protein carboxylation. It therefore reduces synthesis of functional factors II, VII, IX and X by the liver.
• Warfarin is given as an oral tablet and its anticoagulant effect needs to be monitored by regular blood testing (see ‘Tests of coagulation
• Because it reduces synthesis of coagulation factors rather than inhibiting existing factor molecules, it takes several days to take effect.

Question 24

What do direct oral anticoagulant (DOACs) do (anticoagulant drug)?

Answer 24

• Orally available drugs that directly inhibit either thrombin or factor Xa (i.e. without the involvement of antithrombin)
• These do not usually require monitoring

Question 25

What is the fibrinolytic system?

Answer 25

• After haemostasis has been achieved, the body has a mechanism to break down (lyse) clots
• The principal fibrinolytic enzyme isplasmin, which circulates in its inactive zymogen formplasminogen
• The activation of plasmin is mediated bytissue plasminogen activator (t-PA).
• However, t-PA does not activate plasminogen until these are both brought together by binding to lysine residues on fibrin. The breakdown of fibrin leads to the generation of fibrin-degradation produces (FDPs).
• Plasmin is not specific for fibrin and can also break down other protein components of plasma, including fibrinogen and the clotting factors Va and VIIIa.
• Plasmin is inhibited by antiplasmin which circulates in the blood

Question 26

What is thrombolytic therapy?

Answer 26

• Thrombolytic agents such as recombinant t-PA work by generating plasmin to lyse clots and are administered intravenously to selected patients presenting with ischaemic stroke
• The benefit is time-dependent and so t-PA needs to be given to eligible patients as quickly as possible, preferably within one hour of the onset of symptoms
• There is a high risk of bleeding associated with its use.
• Thrombolytic therapy can also be given to patients with life threatening pulmonary emboli and was previously used in patients with myocardial infarction, although this has largely been replaced with angioplasty and stenting of the diseased coronary vessels.

Question 27

What is antifibrinolytic drugs?

Answer 27

• Tranexamic acid is a synthetic derivative of the amino acid lysine that works by binding to plasminogen (see figure below).
• In doing so it prevents plasminogen from binding to the lysine residues of fibrin. This is known as competitive inhibition.
• This prevents the activation of plasminogen to plasmin, which would otherwise result in fibrinolysis
• Tranexamic acid is used widely to treat bleeding in trauma and surgical patients as well as in patients with inherited bleeding disorders.

Question 28

What are tests of coagulation?

Answer 28

• The initiation, amplification and propagation phases of coagulation described previously are an accurate physiological representation.
• This cellular-based model replaced the classical ‘intrinsic’ and ‘extrinsic’ coagulation cascade model (see figure below)
• ‘Intrinsic’ refers to a system in which all components are in the plasma (factors XII, XI, IX, X and co-factors VIII and V), while the ‘extrinsic’ system comprises TF and factors VII, X, and co-factor V.
• It was previously believed that the extrinsic and intrinsic pathways ran in parallel, with initiation of the intrinsic pathway resulting from contact activation of factor XII
• Through a greater understanding of factor XI and the recognition that people with inherited deficiencies of factor XII do not have bleeding problems, it became clear that the intrinsic-extrinsic model did not represent the physiological pathway of coagulation. However, the intrinsic-extrinsic model remains helpful in our understanding of the blood tests used to assess coagulation.

Question 29

What is prothrombin Time (PT)?

Answer 29

1. Measures the integrity of the ‘extrinsic’ pathway
2. Blood is collected into a bottle containing sodium citrate (usually blue-topped as in the picture), which chelates calcium thus preventing the blood from clotting in the bottle
3. The sample is spun to produce platelet-poor plasma
4. A source of TF and phospholipid is added to the citrated plasma sample, together with calcium to start the reaction; the length of time taken for the mixture to clot is recorded.
5. The PT may be prolonged if there is a reduction in the activity of factors VII, X, V, II (prothrombin) or fibrinogen i.e. (‘prothrombin’ is a misnomer)
6. Nowadays a recombinant thromboplastin is often used as the source of both TF and phospholipid
7. When the PT is used to monitor vitamin K antagonist anticoagulant therapy such as warfarin, the results are expressed as the international normalised ratio (INR). This involves a correction for the different thromboplastin reagents used by different laboratories and means that all laboratories would be expected to obtain the same INR result for a given sample irrespective of the source of thromboplastin.

Question 30

What are activated partial thromboplastin time (APTT)?

Answer 30

1. Measures the integrity of the ‘intrinsic’ pathway
2. Performed by the contact activation of factor XII by a surface such as glass, or using a contact activator such as silica or kaolin.
3. Contact activator, together with phospholipid, is added to the citrated plasmasample followed by calcium; the time taken for this mixture to clot is measured
4. Prolongation of the APTT is seen in a variety of situations where there is a reduction in a single or multiple clotting factors; in the the latter there may also be an associated prolonged PT
5. An isolated prolonged APTT (i.e. normal PT) is seen in patients with haemophilia A (factor VIII deficiency), haemophilia B (factor IX deficiency) and factor XI deficiency. However this may also be caused by factor XII deficiency which does not result in bleeding. (Note that FXII does not appear in the cell-based model described in ‘Coagulation (secondary haemostasis): formation of the stable fibrin clot’ and is not important for clottingin vivo).

Question 31

What is bleeding?

Answer 31

Loss of this balance may result inbleeding. This can be caused by:

1. Reduction in platelet number or function (primary haemostasis –platelet plug)
2. Reduction in coagulation factor(s) (secondary haemostasis – fibrin clot)
3. Increased fibrinolysis

Question 32

Thrombosis?

Answer 32

• Thrombosis is the term used to describe the formation of a blood clot within anintactblood vessel.
• This usually results in obstruction of the blood flow with serious and possibly fatal consequences.Over 150 years ago, the German physician, Rudolf Virchow recognised that there werethree contributory factors to pathological clotting orthrombosis. This is known as ‘Virchow’s triad’:
  1. Blood:dominant in venous thrombosis
  2. Vessel wall:dominant in arterial thrombosis
  3. Blood flow:complex, contributes to both arterial and venous thrombosis

Question 33

What increases risk of Venus thrombosis?

Answer 33

1. a) Reduced levels of anticoagulant proteins - usually genetic basis e..g inherited antithrombonin deficiency, an example of an inherited thrombophilia
   b) Reduced fibrinolytic activity e.g. pregnancy where inhibition of plasminogen activation through the production of. Specific inhibitor by placenta
2. Increases levels of clotting factors or platelets e.g.
   - Levels of factor VIII increase during pregnancy
   - Activity of factor V increased by asingle point mutation in the factor V gene, known as factor V Leiden. Factor V Leiden makes factor V more resistant to inactivation by protein C. Around 7% of the population are carriers (heterozygotes) for Factor V Leiden, making it the most common of the inherited thrombophilias.
   - Platelets are increased in some myeloproliferative disorders