Haematology - Haemostasis

Question 1

Under normal conditions

Answer 1

Blood flows within vascular system, transporting O2, nutrients and hormonal info around body + removing metabolic waste

Question 2

How is confinement of circulating blood and maintenance of fluid state achieved?

Answer 2

Balancing fibrinolytic factors, anticoagulant proteins, coagulation factors and platelets (allow stimulation of blood clotting processes (coagulation), limit extent of response to area of injury to prevent excessive/generalised blood clotting (thrombosis), start process that eventually leads to breakdown of clot as part of healing (fibrinolysis)

Question 3

Following trauma to blood vessels…

Answer 3

“Halting of blood”; vasoconstriction -> formation of unstable platelet plug at site of vessel wall damage (platelet adhesion and aggregation, primary haemostasis) -> formation of stable fibrin clot (blood coagulation, secondary haemostasis) -> dissolution of clot + vessel repair (fibrinolysis)

Question 4

Why is it important to understand mechanisms?

Answer 4

To diagnose and treat bleeding disorders, identify thrombosis risk factors, treat thrombotic disorders, monitor drugs used to treat bleeding and thrombotic disorders, control bleeding in individuals without underlying bleeding disorders

Question 5

Primary haemostasis

Answer 5

Platelet adhesion, discoid, non-uncleared, granule containing cells derived from myeloid stem cells, formed in bone marrow by fragmentation of megakaryocyte cytoplasm, circulating lifespan ~10 days ; plasma membrane with glycoproteins (GPs) important for interactions, following injury to vessel wall, platelets stick to damaged endothelium either directly to collagen via platelet GP1a receptor or indirectly via Von Willebrand fact or (VWF) which binds to GP1b; adhesion -> activation -> changing shape (disc to more rounded with spicules to encourage platelet-platelet interaction)

Question 6

Primary haemostasis - platelet release reaction

Answer 6

Activation = release of storage granules’ content (alpha and dense), platelet membrane invaginate to form surface-connected canalicular system for release of granule content (including ADP, fibrinogen, VWF)

Question 7

Primary haemostasis - thromboxane A2 synthesis

Answer 7

Prostaglandin, platelets stimulated to produce it by arachidonic acid derived from cell membrane, production blocked by aspirin

Question 8

Primary haemostasis - platelet aggregation

Answer 8

Granular release of ADP + generation of thromboxane A2 = positive feedback resulting in further platelet aggregation and activation; bind respectively to P2Y12 and thromboxane A2 receptor; activation = conformational change in GPIIb/IIIa receptor (“inside out”/“flip flopping”) to provide fibrinogen binding sites, fibrinogen binding to same receptor = “outside-in” signalling that further activates platelets; fibrinogen plays key role, effects usually counterbalanced by active blood flow and release of prostacyclin (PGI2, powerful vasodilator and suppresses platelet activation) from endothelial cells

Question 9

Primary haemostasis - antiplatelet drugs (general)

Answer 9

Prevention and treatment of cardiovascular and cerebrovascular disease

Question 10

Primary haemostasis - antiplatelet drugs (aspirin)

Answer 10

Inhibits production of thrombozane A2, irreversibly blocks action of cyclo-oxygenate (COX) = reduction in platelet aggregation; though prostacyclin production is also inhibited by COX, endothelial cells can synthesize more while nuclear platelets cannot -> effect of dose ~ 7 days (until most platelets present at ingestion have been replaced by new ones)

Question 11

Primary haemostasis - antiplatelet drugs (clopidogel)

Answer 11

Irreversibly blocks ADP receptor (P2Y12) on platelet cell membrane (same effect duration)

Question 12

Primary haemostasis - VWF

Answer 12

Glycoprotein, produced by endothelial cells and also found on megakaryocytes, in plasma as multiverse of different sizes, mediates adhesion of platelets to injury sites and promotes platelet-platelet aggregation, carrier for factor VIII (FVIII)

Question 13

Primary haemostasis - steps (platelet aggregation)

Answer 13

Arachidonic acid -cyclo-oxygenase-> cyclic endoperoxides -platelets + thromboxane synthetase-> thromboxane A2 -> platelet aggregation

Question 14

Primary haemostasis - steps (inhibited platelet aggregation)

Answer 14

Arachidonic acid -cyclo-oxygenase-> cyclic endoperoxides -endothelial cells + prostacyclin synthetase-> prostacyclin PGI_2 -> inhibits platelet aggregation

Question 15

Primary haemostasis - platelet adhesion and aggregation

Answer 15

• Adhesion - indirect = to VWF on endothelial cells through GIpIb receptor, direct = binding to collagen through GIpIa receptor
• Release of ADP and thromboxane
• Aggregation - thanks to fibrinogen and Ca2+, GIpIIb/IIIa receptors involved

Question 16

Secondary haemostasis - general

Answer 16

Formation of stable fibrin clot, blood coagulation pathways centre on generation of thrombin which cleaves fibrinogen to generate fibrin clot (stabilises platelet plug)

Question 17

Secondary haemostasis - clotting factor synthesis

Answer 17

• Most in liver (exceptions: FVIII and VWF made by endothelial cells, VWF also in megakaryocytes and incorporated into platelet granules); factors II (prothrombin), VII, IX, X dependent on vitamin K for carboxylation of glutamic acid residues (essential for function)
• Each step in blood coagulation characterised by conversion of an inactive zymogen (pro enzyme) into active clotting factor by splitting of one/more peptide bonds and exposure of enzyme active site, FV and FVIII = cofactors
• Believed to act on exposed phospholipid surface of platelets which helps localise and accelerate reactions, Ca2+ important role in this binding
• Trigger to initiate coagulation at site of injury is tissue factor (TF) exposed on surface of endothelial cells + leukocytes + most extra vascular cells in area of tissue damage

Question 18

Secondary haemostasis - clotting factor synthesis (TF)

Answer 18

Mainly located at sites that aren’t exposed to blood under normal physiological conditions so blood only encounters it at sites of vascular injury

• INITIATION PHASE: extra vascular cell, FVIIIa binds to TF -> activation of FIX to FIXa and FX to FXa -> activation of FII (prothrombin) -> small initial amount of thrombin (FIIa)
• AMPLIFICATION: activation of co-factors V (to Va) and VIII ((binds to VWF and becomes FVIIIa (not bound to VWF anymore)) (mediated by TF), zymogen (inactive substance converted into enzyme when activated by another enzyme) factor XI to XIa (via TF) and platelets (onto which FIIa, FV & then FVa, FXIa, FVIII and then FVIIIa are bound)
• PROPAGATION: FXI converts more FIX to FIXa with which FVIIIa amplifies conversion of FX to FXa which with FVa causes rapid burst in prothrombin (FII) to thrombin (FIIa) conversion (cleaves circulating soluble fibrinogen to form insoluble fibrin clot)

Question 19

Anticoagulant pathways - natural

Answer 19

• Ensure coagulation is confined to site of injury and prevent spontaneous coagulation activation; most important: act during propagation (secondary haemostasis), protein C & S and antithrombin
• thrombin binds to thrombomodulin on endothelial cell surface -> activation of protein C (APC) -> inactivates FVa and FVIIIa in presence of cofactor protein S
• thrombin and FXa inactivated by antithrombin (potentiated by heparin-binding of antithrombin to endothelial cell-associated heparins)

Question 20

Anticoagulant pathways - Drugs (general aim)

Answer 20

Prevent and treat thrombosis

Question 21

Anticoagulant pathways - Drugs (heparin)

Answer 21

Mixture of glycosaminoglycan chains from porcine mucosa, works indirectly by potentiating action of antithrombin (inactivation of FXa and FIIa), chains wrapping around both antithrombin and thrombin, intravenous/subcutaneous injection

Question 22

Anticoagulant pathways - Drugs (warfarin)

Answer 22

Derived from coumarin, vitamin K antagonist, interferes with protein carboxylation (reduces synthesis of FII, FVII, FIX and FX by liver), given as oral tablet and its effects need to be monitored with regular blood testing, several days for effect bc it reduces synthesis and not action of factors

Question 23

Anticoagulant pathways - Drugs (direct oral anticoagulants, DOACs)

Answer 23

Directly inhibit thrombin or FXa, no monitoring

Question 24

Fibrinolytic system - General

Answer 24

After haemostasis has been achieved, breaks down clots; principal enzyme = plasmin (circulates in its inactive zymogen form plasminogen, activation mediated by tissue plasminogen activator (t-PA), doesn’t activate it until they are brought together by binding to lysine residues on fibrin

Question 25

Fibrinolytic system - Fibrinolysis

Answer 25

Fibrin degradation products (FDPs), plasmin not specific to fibrin and can also break down other plasma components (ie fibrinogen, FVa and FVIIIa), inhibited by antiplasmin

Question 26

Fibrinolytic system - Thrombolytic therapy

Answer 26

Thrombolytic agents like recombinant t-PA work by generating plasmin to lyse clots; intravenous to patients presenting ischaemic stroke, benefit = time dependent, needs to be given ASAP (preferably within one hour of symptom onset); high bleeding risk, also given to patients with life threatening pulmonary emboli and previously for myocardial infarction (now largely replaced with angioplasty and stents)

Question 27

Fibrinolytic system - Antifibrinolytic drugs

Answer 27

Tranexamic acid = synthetic derivative of lysine, works by binding to plasminogen preventing its binding to fibrin’s lysine residues (competitive inhibition), treat bleeding in trauma + surgical patients and those with bleeding disorders

Question 28

Coagulation tests - general

Answer 28

Cellular-based “initiation amplification and propagation” model replaced classical intrinsic and extrinsic coagulation cascade model

Question 29

Coagulation tests - intrinsic pathway

Answer 29

All components in plasma (FXII, XI, IX, X, co-factors VIII and V)

Question 30

Coagulation tests - extrinsic pathway

Answer 30

Tissue factor (TF) and FVIII + X, co factor V

Question 31

Coagulation tests - intrinsic, extrinsic and common pathway (general)

Answer 31

Previously believed that pathways ran in parallel with initiation of intrinsic pathway resulting from contact activation of FXII, greater FXI understanding and recognition that ppl with inherited FXII deficiencies don’t have bleeding problems, doesn’t represent physiological coagulation pathway but helpful in understanding blood tests to assess coagulation

Question 32

Coagulation tests - intrinsic, extrinsic and common pathway (steps)

Answer 32

• INTRINSIC: contact activation (damaged surface/APTT (activated partial thromboplastin time)/contact system assembled on activation surface (FXII, prekallikrein (PK), High-molecular-weight kininogen)) -> activation of FXII which activates XI which activates IX which activates X
• EXTRINSIC: injury activates FVII which forms TF-VIIa complex which activates FX
• COMMON: prothrombinase complex activates prothrombin (FII) to thrombin (FIIa) which activates FI (fibrinogen) to FIa (fibrin) and FXIII leading to clot formation

Question 33

Coagulation tests - phrothrombin time (PT)

Answer 33

Measures integrity of extrinsic pathway; blood collected into bottle with Na citrate (usually blue-topped) that chelates (compound containing ligand, typically organic, bonded to central atom at 2+ pairs) Ca, preventing blood clotting in bottle -> sample spun to produce platelet-poor plasma -> source of TF and phospholipid along Ca to start reaction, length of time taken for mixture to clot is recorded, PT may be prolonged if there is reduction in activity FVII, X, V, II, or fibrinogen, recombinant thromboplastin often used as source of both TF and phospholipid

Question 34

Coagulation tests - activated partial thromboplastin time (APTT) (general)

Answer 34

Measures integrity of intrinsic pathway, contact activation of FVIII by surface (ie glass)/contact activator (ie silica/kaolin); contact activator together with phospholipid added to citrated plasma sample followed by Ca, time taken for mixture to clot is measured

Question 35

Coagulation tests - activated partial thromboplastin time (APTT) (prolongation)

Answer 35

Seen when there is a reduction in single/ multiple clotting factors, latter may also lead to prolonged PT

Question 36

Coagulation tests - activated partial thromboplastin time (APTT) (isolated prolonged APTT, normal PT)

Answer 36

Patients with haemophilia A (FVIII deficiency), haemophilia B (FIX deficiency) and FXI deficiency; may also be caused by FXII which does not cause bleeding (not important for in vivo clotting)

Question 37

Coagulation tests - bleeding (normal haemostasis)

Answer 37

Fibrinolytic factors and anticoagulant proteins + coagulation factors and platelets, loss of balance = bleeding caused by reduction in platelet number/function (primary haemostasis - platelet plug), reduction in coagulation factors (secondary haemostasis - fibrin clot), increased fibrinolysis; bleeding overall = more fibrinolytic factors and less coagulation factors

Question 38

Coagulation tests - bleeding (reduction in platelet numbers)

Answer 38

Thrombocytopenia, failure of production (drugs, viruses, bone marrow infiltration, megaloblastic anemia from B12/folate deficiency, hereditary), shortened platelet survival, disseminated intravascular coagulation (DIC), increased splenic pooling

Question 39

Coagulation tests - bleeding (reduction in platelet function)

Answer 39

Antiplatelet drugs/inherited

Question 40

Coagulation tests - bleeding (reduction in coagulation factors - congenital blood abnormalities)

Answer 40

• Reductions of VWF = VW disease (VWD), most common inherited bleeding disorder, autosomal, not sex related
• Deficiency of clotting factors: 80-90% haemophilia A (FVIII deficiency, X-linked); 10-20% haemophilia B (FIX deficiency, X-linked); ~1% deficiency of another clotting factor (autosomal recessive, more common in areas with high consanguinity levels; replacement with specific clotting factor concentrates (recombinant and synthesized in cell lines, eliminating risk of pathogen transmission in plasma-derived concentrates

Question 41

Coagulation tests - bleeding (reduction in coagulation factors - acquired blood abnormalities - general)

Answer 41

• Acquired reductions in coagulation factors = more common

- Liver disease, anticoagulant drugs, disseminated intravascular coagulation

Question 42

Coagulation tests - bleeding (reduction in coagulation factors - acquired blood abnormalities - disseminated intravascular coagulation)

Answer 42

• DIC, generalised and uncontrolled activation of coagulation followed by marked activation of fibrinolytic system resulting from expression of tissue factor (TF) within circulation
• Leads to generation and dissemination of large thrombin amounts, activation and consumption of platelets and widespread thrombi formation in small blood vessels (microcirculation)
• Clotting factors and fibrinogen become depleted, impairing haemostatic activity and possibly leading to severe bleeding
• High levels of fibrin degradation products (FDPs) as result of fibrinolysis activation
• Thrombi in microcirculation may cause RBC shearing leading to fragmentation (red cell fragments, schistocytes, seen on blood film (triangular, helmet shaped, or comma shaped with pointed edges, most often microcytic with no area of central pallor))
• CAUSES: bacterial sepsis, advanced cancer, obstetric emergencies
• Replacement of clotting factors and platelets may help control bleeding symptoms but underlying cause be addressed

Question 43

Coagulation tests - bleeding (increased fibrinolysis)

Answer 43

Disseminated intravascular coagulation (DIC) and thrombolytic therapy

Question 44

Coagulation tests - thrombosis

Answer 44

• Formation of blood clot within intact blood vessel
• Obstruction of blood flow
• Virchow’s triad (contributory factors): blood (venous), vessel wall (arterial), blood flow (both)
• More coagulation factors, less fibrinolytic factors
• Changes in blood that increase risk of venous thrombosis: reduced levels of anticoagulant proteins (usually genetic, ie inherited antithrombin deficiency (inherited thrombophilia)), reduced fibrinolytic activity (pregnancy = inhibition of plasminogen activation through production of specific placenta inhibitor (PAI-2)), increased levels of clotting factors/platelets (ie increased FVIII in pregnancy), single single point mutation in FV gene (FV Leiden, makes FV more resistant to inactivation by protein C, ~7% population are carriers (most common inherited trombophilia)), platelets increased in several myoproliferative disorders (increased bone marrow output)