Haemostasis

Question 1

State of equilibrium in blood flow important to

Answer 1

Allow stimulation of blood clotting processes after injury, where blood changes from its liquid state (coagulation).
Limit the extent of the response to the area of injury to prevent excessive or generalised blood clotting (thrombosis).
Start the process that eventually leads to the breakdown of the clot as part of the process of healing (fibrinolysis).

Question 2

Haemostasis meaning

Answer 2

Halting of blood after trauma to blood vessels and results from 3 processes:

1. Contraction of blood vessels (vasoconstriction).
2. Formation of unstable platelet plug at site of vessel wall damage (primary haemostasis).
3. Formation of a stable fibrin clot (secondary haemostasis/coagulation)

Question 3

Understand haemostatic mechanisms important to

Answer 3

Diagnose and treat bleeding disorders,
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

Haemostasis overview

Answer 4

Response to injury:

1. Vessel constriction
2. PRIMAY HAEMOSTASIS Formation of unstable platelet plug - platelet adhesion and platelet aggregation
3. SECONDARY HAEMOSTASIS stabilisation of the plug with fibrin - blood coagulation
4. FIBRINOLYSIS - Dissolution of clot and vessel repair- fibrinolysis

Question 5

Platelet adhesion

Answer 5

Platelets are discoid, non-nucleated, granule-containing cells derived from myeloid stem cells.
Formed in bm by fragmentation of megakaryocyte cytoplasm, circulating lifespan of 10 days. The plasma membrane contains glycoproteins (GPs), important for the platelet’s interactions. Following injury to vessel wall platelets stick to 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. adhesion of platelets causes them to become activated and changes their shape from a disc to more rounded form with spicules to encourage platelet-platelet interaction.

Question 6

Platelet release reaction

Answer 6

Adhesion of platelets initiates activation and release of contents of their storage granules. 2 main types of ultrastructurally-identifiable granules: α-granules and dense granules. Platelet membrane is invaginated to form a surface-connected cannalicular system through which the contents of platelet granules are released. Important components of these contents include ADP, fibrinogen and von Willebrand factor.

Question 7

Thromboxane A2 synthesis

Answer 7

Platelets are stimulated to produce the prostaglandin thromboxane A2 from arachidonic acid that is derived from the cell membrane. thromboxane A2 is a known vasoconstrictor and is especially important during tissue injury and inflammation.

Question 8

Platelet aggregation

Answer 8

Granular release of ADP and generation of thromboxane A2 have + feedback effects resulting in platelet recruitment activation and aggregation by binding respectively to the P2Y12 and thromboxane A2 receptor.
Platelet activation also causes a conformational change in the GPIIb/IIIa receptor (known as ‘inside-out’ or ‘flip-flopping’) to provide binding sites for fibrinogen. Fibrinogen binding to GPIIb/IIIa causes ‘outside-in’ signalling which further activates the platelets. Fibrinogen has a key role in linking platelets together to form the platelet plug. These effects are normally counterbalanced by active flow of blood and the release of prostacyclin (PGI2) from endothelial cells; prostacyclin is a powerful vasodilator and suppresses platelet activation, thus preventing inappropriate platelet aggregation.

Question 9

Antiplatelet drugs

Answer 9

Prevention and treatment of cvd and cerebrovascular disease.
Aspirin and clopidogrel are the most commonly used antiplatelet drugs:

Aspirin inhibits production of thromboxane A2 by irreversibly blocking action of cyclo-oxygenase (COX) = 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 of a single dose of aspirin felt for week until most of platelets present at the time of ingestion have been replaced by new platelets.
Clopidogrel works by irreversibly blocking the ADP receptor (P2Y12) on the platelet cell membrane. Therefore the effect of clopidogrel ingestion also lasts for 7 days until new platelets have been produced.

Question 10

Von Willbrand factor

Answer 10

Glycoprotein, synthesised by endothelial cells & megakaryocytes, circulates in plasma as multimers of different sizes, mediates adhesion of platelets to sites of injury and promotes platelet-platelet aggregation. VWF is a specific carrier for factor VIII (FVIII).

Question 11

Coagulation (2ndary haemostasis)

Answer 11

The primary platelet plug is sufficient for small vessel injury.
However, in larger vessels 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 12

Site of Synthesis of clotting factors

Answer 12

Most clotting factors synthesised in the liver. Exceptions : factor VIII and VWF, made by endothelial cells. VWF also made in megakaryocytes and incorporated into platelet granules. Factors II (prothrombin), VII, IX and X are dependent on Vitamin K for carboxylation of their glutamic acid residues, which is essential for the function of these clotting factors.

Question 13

Process of blood coagulation

Answer 13

Each step: inactive zymogen (proenzyme) –> active clotting factor by splitting of 1 more peptide bones and exposure of the active enzyme site.
Factors V and VIII are co-factors.
Many clotting factors work on exposed phospholipid surface of platelets, localise and accelerate these reactions.
Ca ions role - binding of activated clotting factors to the phospholipid surfaces of platelets.
Trigger to initiate coagulation at site of injury - tissue factor (TF) exposed on the surface of endothelial cells and leukocytes and on most extravascular cells in area of tissue damage, at sites that are not usually exposed to the blood.
So blood only encounters TF at sites of vascular injury. Binding of TF to factor VIIa –> activation of factors IX to IXa and X to Xa –> activation of prothrombin (factor II) to generate a small initial amount of thrombin (factor IIa). {Initiation phase}

Small amount of thrombin –> activation of co-factors V and VIII, the zymogen factor XI and platelets (Amplification phase).

Factor XI converts more factor IX to IXa, which with factor VIIIa, amplifies the conversion of factor X to Xa, –> rapid burst in thrombin generation (Propagation phase), which cleaves the circulating fibrinogen (soluble) to form the insoluble fibrin clot.

Question 14

Natural anticoagulant pathways

Answer 14

Blood prevented from clotting completely when vessel injured. Inhibitory mechanisms ensures that coagulation is confined to site of injury, prevent the spontaneous activation of coagulation. most important of these are proteinC, protein S and antithrombin:
Thrombin binds to thrombomodulin on the endothelial cell surface leading to activation of protein C to activated protein C (APC). APC inactivates factors Va and VIIIa in the presence of a co-factor protein S.
Thrombin and factor Xa are inactivated by the circulating inhibitor antithrombin. The action of antithrombin is markedly potentiated by heparin: this occurs physiologically by the binding of antithrombin to endothelial cell-associated heparins.

Question 15

Main anticoagulant drugs

Answer 15

heparin, warfarin and the direct oral anticoagulants (DOACs). These drugs are widely used in the prevention and treatment of thrombosis.

Question 16

Heparin

Answer 16

Heparin is mix of glycosaminylglycan chains extracted from porcine mucosa.

Works indirectly by potentiating the action of antithrombin leading to the inactivation of factors Xa and IIa (thrombin).
Inactivation of thrombin requires longer chains of heparin chains, which are able to wrap around both the antithrombin and thrombin.

Administered intravenously or by subcutaneous injection.

Question 17

Warfarin

Answer 17

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


Question 18

Direct oral anticoagulants (DOACs)

Answer 18

Orally available drugs, directly inhibit either thrombin or factor Xa (i.e. without the involvement of antithrombin).
​Do not usually require monitoring

Question 19

Fibrinolytic system

Answer 19

After haemostasis, must lyse clots. Principal fibrinolytic enzyme is plasmin, circulates in its inactive zymogen form plasminogen. Activation of plasmin mediated by tissue 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 20

Thrombolytic therapy

Answer 20

Thrombolytic agents eg recombinant t-PA work by generating plasmin to lyse clots, administered intravenously to patients with ischaemic stroke. The benefit is time-dependent and so t-PA needs to be given to be given as quickly as possible, preferably within one hour of the onset of symptoms. 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 the insertion of stents to open the diseased coronary vessels.

Question 21

Antifibrinolytic drugs

Answer 21

Tranexamic acid is synthetic derivative of the amino acid lysin, binds to plasminogen, prevents plasminogen from binding to the lysine residues of fibrin.
Competitive inhibition.
Prevents the activation of plasminogen to plasmin, which would otherwise result in fibrinolysis.
Tranexamic acid used widely to treat bleeding in trauma and surgical patients as well as in patients with inherited bleeding disorders.

Question 22

Tests of coagulation

Answer 22

The initiation, amplification and propagation phases of coagulation are accurate physiological representation. This replaced the ‘intrinsic’ and ‘extrinsic’ coagulation cascade model. ‘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 believed that the extrinsic and intrinsic pathways ran in parallel, with initiation of the intrinsic pathway resulting from contact activation of factor XII. Greater understanding of factor XI and recognition that people with inherited deficiencies of factor XII do not have bleeding problems = 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 23

Prothrombin time (PT)

Answer 23

Measures integrity of ‘extrinsic’ pathway.
​1. Blood collected in bottle containing sodium citrate which chelates calcium, preventing the blood from clotting in the bottle.
2. Sample spun to produce platelet-poor plasma.
3. A source of TF & phospholipid is added to citrated plasma sample, with calcium to start the reaction; the length of time taken for the mixture to clot is recorded.
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)
Nowadays a recombinant thromboplastin is used as the source of both TF and phospholipid.
When PT is used to monitor vitamin K antagonist anticoagulant therapy eg warfarin, results expressed as the international normalised ratio (INR). This involves a correction for the different thromboplastin reagents used by different laboratories, all labs expected to obtain the same INR result for a given sample irrespective of the source of thromboplastin.

Question 24

Activated partial thrombinlastin time (APTT)

Answer 24

Measures integrity of the ‘intrinsic’ pathway.

1. Performed by the contact activation of factor XII by a surface eg glass, or using a contact activator such as silica or kaolin.
2. Contact activator, together with phospholipid, added to the citrated plasma sample followed by Ca; the time taken for this mixture to clot is measured.
3. Prolongation of the APTT is seen where there is a reduction in a single or multiple clotting factors; in the the latter there may also be an associated prolonged PT.

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. ( 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 clotting in vivo).

Question 25

Loss of balance may result in bleeding, caused by

Answer 25

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 26

Reduction in platelet number (thrombocytopenia)

Answer 26

Failure of platelet production: drugs, viruses, bone marrow infiltration, megaloblastic anaemia resulting from B12 or folate deficiency, hereditary thrombocytopenia
Shortened platelet survival – immune thrombocytopenia, disseminated intravascular coagulation (DIC) Increased splenic pooling

Question 27

Reduction in platelet function

Answer 27

Often due to antiplatelet drugs e.g. aspirin

Inherited causes

Question 28

Reduction in coagulation factors

Answer 28

Blood abnormalities in 2 categories: congenital and acquired. Acquired reductions in coagulation factors are much more common than congenital causes.

Question 29

Congenital causes of reduced coagulation factors

Answer 29

Reductions in the level or function of (VWF) are known as von Willebrand disease (VWD); VWD is most common inherited bleeding disorder and as it is autosomally inherited affects both males and females.
Smaller group of patients have congenital deficiencies of one of the clotting factors:
-​ 80-90% have haemophilia A (factor VIII deficiency, X linked – males affected, females carriers)

• 10-20% have haemophilia B (factor IX deficiency, X linked – males affected, females carriers)
• around 1% of patients have deficiencies of one of the other clotting factors. These usually follow an autosomal recessive inheritance pattern and are more common in areas with high levels of consanguinity.

Replacement of missing clotting factor with specific clotting factor concentrates forms the mainstay of treatment for patients with congenital forms of bleeding disorders: many of the factor concentrates now used iare recombinant and are synthesised in cell lines. This eliminates the risk of pathogen transmission in plasma-derived concentrates.

Question 30

Acquired causes of reduced coagulation factors

Answer 30

Liver disease,
Anticoagulant drugs,
Disseminated intravascular coagulation (DIC) – generalised and uncontrolled activation of coagulation followed by marked activation of the fibrinolytic system. Activation results from expression of TF within the circulation and leads to the generation and dissemination of large amounts of thrombin, activation and consumption of platelets (leading to thrombocytopenia) and the widespread formation of thrombi in the small blood vessels (microcirculation). The clotting factors and fibrinogen in the plasma become depleted, impairs haemostatic activity and may result in severe and life-threatening bleeding. There are high levels of fibrin degradation products (FDPs) as a result of fibrinolysis activation. The thrombi in the small blood vessels may cause shearing of the circulating red blood cells, leading to their fragmentation; red cell fragments (also known as schistocytes) may be seen on the blood film. There are many causes of DIC including bacterial sepsis, advanced cancer and a variety of obstetric emergencies. While replacement of missing clotting factors and platelets may help control the bleeding symptoms, the underlying cause needs to be addressed in order to switch off the unregulated coagulation activation.

Question 31

Thrombosis

Answer 31

Describes formation of a blood clot within an intact blood vessel. This usually results in obstruction of the blood flow with serious and possibly fatal consequences. Three contributory factors to pathological clotting or thrombosis. This is known as ‘Virchow’s triad’:

Blood: dominant in venous thrombosis
Vessel wall: dominant in arterial thrombosis
Blood flow: complex, contributes to both arterial and venous thrombosis

Question 32

Changes in blood that increase the risk of venous thrombosis include​: ​

Answer 32

1. a) Reduced levels of anticoagulant proteins, usually have a genetic basis, e.g. inherited antithrombin deficiency, an example of an inherited thrombophilia

b​) Reduced fibrinolytic activity, eg pregnancy where there is inhibition of plasminogen activation through the production of a specific inhibitor by the placenta (PAI-2)

2. Increased levels of clotting factors or platelets eg Levels of factor VIII increase during pregnancy.
   T​he activity of factor V is increased by a single 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 number in some myeloproliferative disorders, where the bone marrow output is increased.