Haemostasis

Question 1

What is the normal haemostatic balance?

Answer 1

State of equilibrium between fibrinolytic factors+anticoagulant proteins and coagulation factors+platelets

Question 2

Define haemostasis

Answer 2

Haemostais defines halting of blood following trauma to blood vessels

Question 3

Why is haemostatic balance important?

Answer 3

1. To allow stimulation of blood clotting processes following injury (coagulation)
2. Limit extent of response to area of injury to prevent excessive or generalised blood clotting (thrombosis)
3. Start process which leads to eventual breakdown of blood clot following healing (fibrinolysis)

Question 4

What are the 3 main processes of haemostasis?

Answer 4

1. Vasoconstriction
2. Primary haemostasis - formation of unstable platelet plug at site of damage
3. Secondary haemostasis - formation of stable fibrin clot

Question 5

What are the 5 stages of primary haemostasis?

Answer 5

1. Endothelial damage
2. Exposure
3. Adhesion
4. Activation
5. Aggregation

Question 6

What are platelets?

Answer 6

Platelets are discoid, non-nucleated, granule containing cells derived from myeloid stem cells, made in the bone marrow by fragmentation of megakaryocyte cytoplasm. Have a circulating lifespan of 10 days.

Question 7

Describe endothelial damage

Answer 7

Healthy endothelial cells produce nitric oxide and prostaglandins, however when damaged, these are reduced and endothelin is produced. When endothelial cell experiences damage, nerve cells detect this and cause a reflexive contraction, causing muscle cells to contract and lumen is narrowed to reduce blood loss. Endothelin also causes contraction.

Question 8

Describe exposure and adhesion

Answer 8

The damaged endothelial cells produce a protein called von Willebrand’s factor which binds to the exposed collagen and allows platelets to bind to them. The platelets can either stick directly to the collagen through binding of its GP1a receptor or indirectly through GP1b receptor binding with VWF. This binding activates the platelet causing conformational change from disc to a more rounded structure with spicules to encourage platelets to interact with one another.

Question 9

Describe activation

Answer 9

Contents of platelet store granules is released. There are two types of ultrastructurally identifiable granules: alpha granules and dense granules. Platelet membrane forms a surface connected cannalicular structure which allows this release and some of the important substances are: VWF, ADP, fibrinogen, serotonin to attract more platelets and thromboxane A2.

Question 10

What is the role of thromboxane A2?

Answer 10

It is a prostaglandin which is derived from arachidonic acid in cell membranes. Is also a vasoconstrictor and stimulates other platelets.

Question 11

Describe aggregation

Answer 11

Release of ADP and thromboxane A2 create positive feedback loops, by stimulating more platelets to bind to VWF and cause a conformational change to GP2b receptors, allowing binding with fibrinogen. Causes clustering around site of injury. However, healthy endothelial cells continue to produce prostacyclin which is a vasodilator to avoid excessive aggregation and make sure effects are only local.

Question 12

Why are antiplatelet drugs used and what is the most common?

Answer 12

Antiplatelet drugs are used to prevent and treat cardiovascular and cerebrovascular disease. Aspirin and clopidogrel are the most commonly used antiplatelet drugs.

Question 13

How does aspirin work?

Answer 13

Aspirin blocks action of cyclo-oxygenase irreversibly, hence preventing formation of thromboxane A2 reducing platelet aggregation. Although prostacylin production is also inhibited by blocking COX, endothelial cells can produce more COX and so prostacyclin made. Platelets however are unable to since they have no nucleus and hence single dose of aspirin lasts around 7 days till new platelets made.

Question 14

How does clopidogrel works?

Answer 14

Irreversibly blocks ADP receptor on platelet cell membrane (P2Y12) so effects are around for 7 days till new platelets made.

Question 15

What is Von Willebrand factor?

Answer 15

Glycoprotein made by endothelial cells and megakaryocytes that circulates in plasma. Mediates adhesion of platelets to sites of injury and promotes platelet aggregation. Also a specific carrier for factor VIII.

Question 16

What is secondary haemostasis and why is it necessary?

Answer 16

Primary platelet plug is sufficient for small vessel injury but falls apart in larger vessels and so fibrin formation stabilises the platelet plug. These blood coagulation pathways centre on generation of thrombin which cleaves fibrinogen to generate fibrin, stabilising platelet plug.

Question 17

Where are clotting factors synthesised?

Answer 17

Most clotting factors are synthesised in the liver. Exceptions are factor 5 and VWF, made by endothelial cells. Prothrombin, factor 7,9 and 10 are dependent on vitamin K for carboxylation of glutamic acid residue, essential for function of these factors.

Question 18

What is each step in blood coagulation characterised by?

Answer 18

Inactive zymogen converted into active clotting factor by splitting of one or more peptide bonds and exposure of active enzyme site.

Question 19

What are important co-factors and how do clotting factors work?

Answer 19

Ca2+ ions allow binding of activated clotting factor to phospholipid surfaces of platelet. Clotting factors work on exposed phospholipid surface of platelets, helping localise and accelerate these reactions.

Question 20

What is the significance of the tissue factor?

Answer 20

TF mainly located at sites not usually exposed to blood under normal physiological conditions and so blood only encounters TF at sites of vascular injury.

Question 21

What is the initiation phase?

Answer 21

Binding of TF to factor 7a leads to activation of factor 9 to 9a and then 10 to 10a. Leads to activation of prothrombin generating small amount of thrombin.

Question 22

What is the amplification phase?

Answer 22

Small amount of thrombin mediates activation of factor 5 and 8, zymogen factor 9 and platelets. Fcator 11 converts more factor 9 to 9a which along with factor 8a amplifies 10 to 10a and hence rapid burst of thrombin generation happens.

Question 23

What is the propagation phase?

Answer 23

Thrombin cleaves the circulating fibrinogen to form insoluble fibrin clot.

Question 24

What are important inhibitory mechanisms that prevent coagulation?

Answer 24

protein C, protein S and antithrombin

Question 25

How do protein C and protein S work?

Answer 25

Thrombin binds to thrombomodulin on edothelial cell surface, leading to activation of protein to activated protein C. APC inactivates factors 5a and 8a in presence of co-factor protein S.

Question 26

How does antithrombin work?

Answer 26

Thrombin and factor 10a inactivated by circulating antithrombin as antithrombin binds to heparins on edothelial cells.

Question 27

What are the main anti-coagulant drugs?

Answer 27

Main anti-coagulant drugs are heparin, warfarin and DOACs.

Question 28

What is heparin and how does it work?

Answer 28

Mixture of glycosaminylglycan chains extracted from porcine mucosa. Potentiates action of antithrombin, inactivating factor 5a and thrombin. Thrombin inactivation needs long chains of heparin which wraps around antithrombin and thrmobin. Administered intravenously or subcutaneously.

Question 29

What is warfarin and how does it work?

Answer 29

Is a vitamin K antagonist that interferes with protein carboxylation so reduces synthesis of factors 2, 7, 9 and 10 by liver. Takes several days to take effect as reduces synthesis of coagulation factors. Given as oral tablet and anticoagulant effect must be monitored via blood testing.

Question 30

What are DOACs and how does it work?

Answer 30

Orally available drugs that directly inhibit thrombin or factor 10a. Don’t require monitoring.

Question 31

What is the fibrinolytic system?

Answer 31

The mechanism of the body to lyse (break down) clots after haemostasis has been achieved.

Question 32

How is fibrinolysis achieved?

Answer 32

Principal fibrinolytic enzyme is plasmin, which circulates in inactive zymogen form plasminogen. Activation of plasmin mediated by tissue plasminogen activator (t-PA). t-PA does not activate plasminogen until both brought together by binding to lysine residues on fibrin.

Question 33

What is the nature of plasmin action?

Answer 33

Plasmin is not specific to fibrin and also breaks down other protein components of plasma like fibrinogen and cloting factors 5a and 8a. Plasmin inhibited by antiplasmin which circulates in blood.

Question 34

What is thrombolytic therapy?

Answer 34

Thrombolytic agents such as recombinant t-PA generate plasmin to lyse clots and administered IV to some patients with ischaemic stroke. Benefit is time-dependent so must be given within one hour of symptom onset. High risk of bleeding.

Question 35

Who is thrombolytic therapy used for?

Answer 35

Primarily, patients with ischaemic stroke. But also patients with life-threatening pulmonary emboli and previously in patients with myocardial infarction. Now largely replaced with angioplasty and insertion of stent into diseased CA.

Question 36

What is an example of an anti-fibrinolytic drug?

Answer 36

Transexamic acid, derived from lysine which works by binding to plasminogen. Prevents plasminogen binding to lysine residues of fibrin through competitive inhibition. Prevents activation of plasminogen to plasmin which would result in fibrinolysis.

Question 37

Who is transexamic acid used for?

Answer 37

Used to treat bleeding in trauma and surgical patients and in patients with inherited bleeding disorders.

Question 38

What is the cellular based model?

Answer 38

Replaced the classical intrinsic+extrinsic pathway moel as greater understanding of factor 11 and seeing that people with inherited deficiency disorders of factor 8 don’t have a bleeding problem, became clear the intrinsic-extrinsic model doesn’t represent the physiological mechanism.

Question 39

What is prothrombin time and how is it measured?

Answer 39

Measure integrity of extrinsic pathway. Blood collected into bottle containing sodium citrate which chelates calcium preventing blood from clotting. Sample spun to produce platelet poor plasma. Source of TF and phopholipid added to citrated plasma+calcium to start reaction. Length of time taken for clot to form is recorded.

Question 40

What does a prolonged PT mean? What is source of TF and phopholipid?

Answer 40

Reduction in activity of factors 7, 10, 5, 2 (prothrombin), or fibrinogen. Nowadays, thromboplastin.

Question 41

What is INR and when is it used?

Answer 41

INR = International Normalised Ratio. When PT used to monitor vitamin K antagonist anticoagulant therapy such as warfarin, results expressed in INR. All labs should obtain same INR for a given sample as each corrects for their own source of thromboplastin reagent.

Question 42

What is activated partial thromboplastin time and how is it measured?

Answer 42

Measures integrity of intrinsic pathway. Performed by contact activation of factor 12 by surface such as glass or activator such as silica or kaolin. This along with calcium added to citrated plasma sample and time for clot to form measured.

Question 43

What does prolonged APTT indicate?

Answer 43

Seen in variety of situations where there is reduction in a single or multiple clotting factors, in latter there may be associated prolonged PT.

Question 44

What does isolated prolonged APTT indicate?

Answer 44

Seen in patients with haemophilia A (factor 8 deficiency), haemophilia B (factor 9 deficiency) and factor 11 deficiency. Can also be caused by factor 12 deficiency but that doesn’t result in bleeding.

Question 45

What can loss of haemostatic balance be caused by?

Answer 45

1. Reduction in platelet number or function (primary haemostasis)
2. Reduction in coagulation factors (secondary haemostasis)
3. Increased fibrinolysis

Question 46

What are causes of reduction in platelet numbers?

Answer 46

This is known as thrombocytopenia. Can be due to failure of platelet production, shortened platelet survival or increased splenic pooling.

Question 47

What can cause failure in platelet production?

Answer 47

Drugs, viruses, bone marrow infiltration, megaloblastic anaemia resulting from B12 or folate deficiency, hereditary thrombocytopenia.

Question 48

What can cause shortened survival time of platelets?

Answer 48

Immune thrombocytopenia, disseminated intravascular coagulation.

Question 49

What causes reduction in platelet function?

Answer 49

Inherited causes and antiplatelet drugs like aspirin.

Question 50

What causes reduction in coagulation factors?

Answer 50

Can be congenital or acquired. Acquired more common. Congenital causes: Reduction in level+function of VWF (known as VW Disease and autosomal so affects males and females), Haemophilia A (factor 8 deficiency, X-linked so males affected and female carriers), Haemophilia B (factor 9 deficiency, X-linked). 1% of patients have deficiency of other factors which have autosomal recessive pattern usually and found in areas of high cosanguinity.

Question 51

How are congenital disorders causing reduction in coagulation factors treated?

Answer 51

With specific clotting factor concentrates, most of which are recombinants and synthesised in cell lines. Eliminates risk of pathogen transmission in case of plasma-derived concentrates.

Question 52

What are acquired causes of reduced coagulation factors?

Answer 52

Liver disease, Anticoagulant drugs, Disseminated intravascular coagulation

Question 53

What is Disseminated Intravascular Coagulation?

Answer 53

Process where there is generalised and uncontrolled activation of coagulation followed by marked activation of fibrinolytic system. Results from expression of TF within circulation leading to generation+dissemination of large amounts of thrombin, activation+consumption of platelets (leading to thrombocytopenia), widespread formation of thrombi in small blood vessels (microcirculation). Clotting factors+fibrinogen become depleted and high level of fibrin degradation products bec. fibrinolysis activation. Thrombi in small blood vessels can cause shearing of red blood cells leading to their fragmentation and red cell fragments schistocytes can be seen on blood film.

Question 54

What are the causes of Disseminated Intravascular Coagulation and how is it treated?

Answer 54

Bacterial Sepsis, advanced cancers and other obstetric emergencies. While replacement of missing clotting factors and platelets may help control bleeding symptoms, underlying cause needs to be addressed to switch off unregulated coagulation activation.

Question 55

What is thrombosis?

Answer 55

Describes formation of a blood clot within intact blood vessel. Usually results in obstruction of blood flow with serious and possibly fatal consequences.

Question 56

What is Virchow’s Triad?

Answer 56

The three contributory factors to pathological clotting: Blood (dominant in venous thrombosis), Vessel wall (dominant in arterial thrombosis) and Blood Flow (complex and contributes to both)

Question 57

How is risk of venous thrombosis increased?

Answer 57

Changes in blood constituents become more important in this case. Changes in blood that increase risk: reduced level of anticoagulant protein, reduced fibrinolytic activity (pregnancy-inhibition of plasminogen by specific inhibitor by placenta called PAI-2), increased level of clotting factors or platelets.

Question 58

When is level of clotting factors or platelets increased?

Answer 58

Factor 8 increases during pregnancy, activity of factor 5 increased by single point mutation in factor 5 gene (factor 5 Leiden). Factor 5 Leiden makes factor 5 more resistant to inactivation by protein C and 7% of population are carriers so it is most common of inherited thrombophilias, platelets increased in number in some myeloproliferative disorders where bone marrow output increased.