haemostasis 1: haemostasis

Question 1

what is haemostasis?

Answer 1

the cellular and biochemical processes that enable the specific and regulated cessation of bleeding in resonse to vascular insult

Question 2

what is the purpose of haemostasis?

Answer 2

to prevent blood loss from intact & injured vessels and enable tissue repair

Question 3

how does a haemostatic plug form?

Answer 3

1) vessel constriction - vascular smooth muscle cells contract locally -> limits blood flow to injured vessel
2) formation of unstable platelet plug - platelets adhere + aggregate -> limits blood loss + provides surface for coagulation
3) stabilisation of plug with fibrin (associated with cascade) - blood coagulation -> stops blood loss
4) vessel repair and dissolution of clot - cells migrate + proliferate and fibrinolysis takes place -> restores vessel integrity

Question 4

what is the structure of a blood vessel (in terms of coagulation properties)?

Answer 4

endothelial layer = anticoagulant, subendothelium = procoagulant

Question 5

what is Von Willebrand factor?

Answer 5

glycoprotein that circulates in globular conformation with platelet binding sites ‘hidden’

Question 6

how does the structure of VWF change in response to vessel injury?

Answer 6

globular VWF recognises collagen -> binds -> structural change to linear conformation -> platelet binding sites exposed -> platelets bind to Gp1b

Question 7

what are some important surface proteins οf platelets?

Answer 7

GPVI (interact with collagen), αIIbβ3 integrin that interacts with fibrinogen), α2β1 (integrin that interacts with collagen) GP1b (essential for capture by VWF)

Question 8

how does a platelet plug form?

Answer 8

platelets bind to GP1b on VWF

• > recruited to site of vessel damage
• > interact with collagen
• > collagen acts as stimulator of platelets
• > platelet activated
• > change shape + release granules + membrane changes conformation
• > platelets stick together (αIIbβ3)

Question 9

when can platelets directly interact with collagen?

Answer 9

low shear stress (ie not in arteries & capillaries)

Question 10

how does the shape of a platelet change upon activation?

Answer 10

flowing disc-shape -> ball-shaped on rolling -> hemisphere-shaped (firm but reversible adhesion) -> spreading platelet (like fried egg) - irreversible adhesion

Question 11

what happens in Von Willebrand disease?

Answer 11

not enough / dysfunctional VWF -> initial platelent recruitment step inefficient

Question 12

what do platelet disorders cause?

Answer 12

dysfunctional / not enough platelets -> bleeding phenotype

Question 13

below what platelet count is spontaneous bleeding common?

Answer 13

<40x10^9 (below 10x10^9 is severe)

Question 14

what is the major function of thrombin?

Answer 14

cleaves fibrinogen (soluble) -> fibrin (insoluble)

Question 15

where are most circulating clotting factors synthesised?

Answer 15

liver (endothelial cells + megakaryocytes also produce clotting factors)

Question 16

what are the main groups of clotting factors?

Answer 16

zymogens eg prothrombin, FVII, FX (activated to become serine proteases eg thrombin, FVIIa, FXa), cofactors eg TF, FVa, FVIIIa, inhibitors eg TFPI, protein C

Question 17

what do serine proteases have in common?

Answer 17

homologous serine protease domains

Question 18

what is tissue factor?

Answer 18

integral membrane protein that is the primary initiator of coagulation & is cell receptor for FVII - located at extravascular sites + expressed more in certain organs eg lungs, brain, heart
-> provides further haemostatic protection in these organs

Question 19

what is factor VII?

Answer 19

serine protease zymogen produced by liver - has Gla domain, 2 EGF-like domains + serine protease domain (1% circulates in active FVIIa form)

Question 20

what features do FVII, FIX, FX & PC share?

Answer 20

homologous modular structure (4 domains), Gla domain, EGF domain, all circulate in zymogen form, all activated by proteolysis

Question 21

what does a Gla domain do?

Answer 21

allows factors to interact with negatively charged phospholipid layers

Question 22

which factors contain Gla domains?

Answer 22

FVII, FX, prothrombin, FIX, protein C, protein S

Question 23

what is warfarin and how does it achieve its function?

Answer 23

vitamin K antagonist diminishes response of clotting factors (in Gla domains:
glutamic acid residues post-translationally modified by vitamin K dependent carboxylases
-> additional carboxylic acid added to glutamic acid
-> gives residues affinity to Ca2+
-> domains fold up in such a way that allows binding of phospholipids)

Question 24

what is the first main step of the clotting cascade?

Answer 24

FVIIa binds TF (via Gla domain) -> activates FIX or FX (by cleaving activation peptides)

Question 25

what does FXa do in the clotting cascade?

Answer 25

FXa liberates thrombin (from ProT) - very inefficient as only small quantities of thrombin are generated

Question 26

what happens when initial thrombin is generated in the clotting cascade?

Answer 26

thrombin feedbacks to FVIII & FV -> FVIIIa (cofactor for FIXa) & FVa (cofactor for FXa)

Question 27

what effects do FVIIIa & FIXa then have in the clotting cascade?

Answer 27

lots more FXa produced

Question 28

what cofactors in the clotting cascade then result in further thrombin production?

Answer 28

FXa + FVa -> 300,000x more thrombin released (compared with FXa alone)

Question 29

what is the overall effect of initial thrombin release in the clotting cascade?

Answer 29

small amounts of thrombin positively feedbacks on coagulation cascade to rapidly produce much more thrombin

Question 30

what anticoagulant pathways regulate the clotting cascade?

Answer 30

TFPI (tissue factor pathway inhibitor - initiation), APC & PS (FVa + FVIIIa), antithrombin (FXa + FIXa)

Question 31

how does TFPI work?

Answer 31

2nd domain binds to FXa -> complex rebinds to TF-FVIIa complex -> 1st TFPI domain binds to FVIIa -> locks all 4 proteins in inactive form

Question 32

what is the TFPI response mainly used for?

Answer 32

dampening response to very small injuries (only small amount of circulating TFPI)

Question 33

what happens in the protein C pathway?

Answer 33

thrombin binds with high affinity to thrombomodulin on endothelial cell -> becomes anticoagulant -> complex activates protein C -> APC cleaves FVa + FVIIIa at different locations -> cofactors fall apart

Question 34

what overall effect does the protein C pathway have?

Answer 34

"fences" clot ie where endothelium is present clot will not form as anticoagulant thrombin-TM complex present

Question 35

what happens to the rest of the thrombin in the clot?

Answer 35

antithrombin (a serine protease inhibitor (SERPIN)) inactivates many activated coagulation serine proteases eg Fxa, thrombin, FIXa, FXIa - essentially mops up free serine proteases that escape site of vessel damage

Question 36

how does heparin work?

Answer 36

acts as a cofactor for antithrombin: binds to AT -> enhances efficiency with which antithrombin can inhibit thrombin + FXa

Question 37

what happens in fibrinolysis?

Answer 37

tPA (tissue plasminogen activator) binds to fibrin -> becomes activated -> plasminogen converted to plasmin -> degrades fibrin clot -> fibrin degradation products removed by liver

Question 38

what drugs can be used to reduce haemostasis?

Answer 38

anticoagulants (heparin, warfarin, DOACs) & antiplatelet drugs (aspiring, P2Y12 blockers) - often anticoagulants given for protection against venous thrombi and antiplatelets for arterial thrombi

Question 39

what tests can be used to diagnose abnormal haemostasis?

Answer 39

PT & APTT (blood test looking at coagulation of blood), platelet function tests (analysis of platelets), d-dimer (fibrin degradation product)

Question 40

how can tPA be used therapeutically?

Answer 40

to treat MI, ischaemic stroke etc