Thrombin Generation and regulation Flashcards
(80 cards)
1
Q
Stages of coagulation
A
Initiation and proagation
2
Q
Haemostasis in this context
A
fine balance between pro- and anti- coagulant activities
3
Q
Excessive procoagulant and/or inadequate anticoagulant function →
A
Thrombosis
e.g. atherosclerosis, genetic risk factors, etc.
4
Q
Inadequate procoagulant and/or excessive anticoagulant function →
A
Bleeding
E.g. genetic deficiency such as haemophilia, drugs
5
Q
Necessities of clotting system
A
Rapid response
Localised to site of damage
Once bleeding stopped, must shut down system
6
Q
Haemostatic plug formation. Step 1
A
- Formation of unstable platelet plug: adhesion and aggregation of platelets - limits blood loss and provides surface for coagulation
7
Q
Haemostatic plug formation. Step 2
A
- Stabilisation of plug with fibrin: Blood coagulation - Stops blood loss
Note: temporary like plaster
8
Q
Haemostatic plug formation. Step 3
A
- Vessel repair and dissolution of clot: Cell migration/proliferation and fibrinolysis - Restores vessel integrity
9
Q
Platelet activation in step 1 of haemostatic plug formation
A
Platlets also become activated and change their membrane composition - get negatively lipid surface and becomes a mean of attracting platelets
10
Q
Clot composition
A
Mainly platelet aggregates and all held together by fibrin protein meshwork
11
Q
Where are proteins for coagulation cascade produced
A
MOSTLY LIVER - produces and secretes into plasma
b. Endothelial cells – VWF, TM, TFPI
c. Megakaryocytes – VWF, FV
12
Q
What are the clotting factors called when inactive precursors in circulation and why are they inactive
A
Zymogen
- Need them ready to respond to injury but not active else too much clotting
13
Q
What are active forms of clotting factors calls
A
Serine protease
14
Q
What are the cofactors
A
TF, FVa, FVIIIa
15
Q
What are the zymogens
A
Prothrombin (FII) FVII FIX FX FXI FXII FXIII
16
Q
What are the serine proteases
A
thrombin (FIIa) FVIIa FIXa FXa FXIa FXIIa
17
Q
Haemophilia a
A
Definiciency in FVIII
18
Q
Haemophilia b
A
Deficient in FIX
19
Q
What are the inhibitors
A
TFPI
Protein C
Protein S
Antithrombin
20
Q
Haemostasis - sequence of events
A
- Coagulation is initiated upon vessel damage, which leads to the exposure of TF to plasma clotting factors
- TF-FVIIa can activate FX and FIX.
- FXa activates prothrombin (ProT) inefficiently leading to the generation of trace amounts of thrombin.
- Thrombin can then activate FVIII and FV, which function as non-enzymatic cofactors for FIXa and FXa, respectively.
- FIXa-FVIIIa catalyses the conversion of increased quantities of FXa
- FXa-FVa catalyses enhanced generation of thrombin.
- Thrombin at the site of vessel damage converts fibrinogen (Fbg) to fibrin (Fbn), which is the molecular scaffold of a clot.
21
Q
What do majority of reactions in cascade require?
A
↘ Most reactions here require Ca2+ ions and phospholipid membrane
↘ Hence most take place primarily upon activated platelet surfaces
22
Q
What is FVIIIa a cofactor for?
A
FIXa
23
Q
What is FVa a cofactor for?
A
FXa
24
Q
What are coagulation factors made up of
A
4 common discrete domains: 1. Gla domain 2/3. EGF (2 of these) 4. Serine protease FIX, FX, FVII, Protein C
25
What is prothrombin made up of
1. Gla domain
2/3. Kringle domain (2 of these)
4. Serine protease
26
Which factors share same domain organization
FVII, FIX, FX and PC
• A homologous modular structure (4 domains)
• All circulate in plasma in zymogen form
• Hence they require activation to become proteolytically competent → activated by proteolysis (removal of activation peptide)
27
What does EGF domain do?
involved in protein-protein interactions
28
What does Gla domain do
binding to phospholipid surfaces
29
What does Serine protease domain do
cleave substrates after specific Arg (and Lys residues)
30
What are the serine protease domains
Homologous family of proteases
| Serine protease contain a catalytic triad His/Asp/Ser
31
How are serine proteases present in plasma
precursor zymogens – activated specifically upon demand
32
Serine protease domain-containing proteins
- FVII
- FX
- Prothrombin
- FIX
- FXI
- Protein C
Note:
Similar module structure Diverse roles
33
Gla domain containing proteins:
FVII
- FX
- FIX
- Prothrombin
- Protein C
- Protein S
34
What does Gla domain do
binding to phospholipid surfaces
| - Defines vit K dependent protein
35
Effect of Gla on Vit K dependent proteins
1. At the N-terminus of the Gla domain containing protein there are glutamic acid residues
2. These get post-translationally modified by vitamin K dependant carboxylase
3. This causes the domain to greatly elevate the binding of Ca2+ to the Gla domains
36
Gla domain structure
9 Gla residues with Ca2+ ions sandwiched in
If Gla domain does not have Gla residues → it does not bind to Ca2+
If it does not bind Ca2+ then it cannot fold up into required confirmation (as above) and therefore cannot bind phospholipid surfaces
37
Warfarin action
Vit K antagonist: Prevents gama carboxylation - messes up the synthesis of Gla domain containing proteins. Coagulation proteins still produced (no effect on expression levels) but cannot bind to phospholipid surfaces
Hence are not recruited to site of blood vessel damage
38
Omega loop part of hydrophobic residue
sually insert into the phospholipid membrane (where water is excluded)
39
What are the requirements for the initiation of coagulation?
1. Fast (to rapidly prevent bleeding)
2. Specific (must only be activated upon requirement)
3. Regulated (must be controlled to prevent thrombosis)
40
Property of epithelial cell subface vs subendothelial environent
Epithelial cell surface is anticoagulant Blood can flow over with no problem
- Underneath the endothelium the environment is procoagulant (various matrix proteins, collagens, etc.)
Plus other cell types that do not normally come into contact with blood (e.g. VSMCs, fibroblasts)
→ They contain tissue factor (the main initiator of the coagulation cascade)
41
What is TF
Integral membrane protein
| Cellular receptor and cofactor for FVII and FVIIIa
42
Location of TF
located at extravascular sites (usually not exposed to blood)
43
Distinction of TF from other procoagulant factors
Only procoagulant factor that does not required an activation event – it is ‘ready to go’
↘ Tissue factor as the primary initiator of coagulation
44
Organs where TF expressed higher and why
lungs, brain, heart, testis, uterus, and placenta
| → TF in these locations provide extra haemostatic protection in these organs
45
Ligand for TF
Factor VII
46
FVIIa presence in blood at all time
1% in blood circulates in active form
This is essential for clotting system
Although we still don’t know where this comes form
47
What does TF-FVII complex do
a. Activate FIX to FIXa
| b. Activate Factor X to FXa
48
FXa action
can activate prothrombin to generate thrombin
Activation is inefficient
Very small quantities of thrombin are generated
49
What does small amount of thrombin do
This small amount is able to feedback and amplify its own production
• Via activation of cofactor FVIII and FV
50
Effect of FVIII → FVIIa
Provides a cofactor for FIXa
| The complex formed then converts more FX → FXa Leads to ↑FXa
51
Effect of FVIIIa vs TF-FVIIa complex
much more efficient
| Hence a new way of producing more FXa
52
Effect of FV → FVa
Makes FXa a super activator of prothrombin → thrombin
| Enhances FXa function by ~300,000 times
53
Key thrombin action apart from co-factors
convert fibrinogen into fibrin
| Spontaneously polymerises into a 3D network
54
Regulation of initiation phase of coagulation (before thrombin produces)
Regulation of this part by TFPI
| Tissue factor pathway inhibitor
55
Structure of TFPI
43kDa Kunitz-type inhibitor TFPI has 3x Kunitz domains
56
What does TFPI inhibit
TF-FVIIa in a FXa-dependent manner
• Kunitz domain 2 binds and inhibits FXa
• Kunitz domain 1 binds and inhibits TF-FVIIa
Formation of a complex between these four proteins
Inactive quaternary complex (TF-FVIIa/FXa-TFPI) Has no more procoagulant or enzymatic function
57
Primary influence of TFPI
TFPI primarily influences the initiation of coagulation
So if there is only a very small amount of TF exposed, probably don’t need the coagulation cascade and thrombin generation
Hence TFPI will stop the system triggering and the system initiating to any extent - TFPI threshold must be breached before coagulation can proceed efficiently
58
TFPI pathway sequence of events
1. TF (transmembrane) bound to FVIIIa
2. Complex binds FX (converts it to FXa)
3. FXa dissociates from complex and moves on
4. TFPI binds to inhibit FXa via 2nd Kunitz domain
5. This complex can then dock back to the TF-FVIIa complex
6. And inhibit the FVIIa via its 1st Kunitz domain
7. Shuts down the initiation phase
59
Breaching of TFPI threshold
Thrombin feedback to activate FVIII and FV cofactors
Once FVIII is activated and forms a complex with FIXa
FXa can be produced via thrombin generation
This is the huge amplification effect
At this point we no longer need TF-FVIIIa complex
Hence TFPI is now of no use
60
Regulation when site of injury 'plugged'
Protein C
61
How is protein C activated
activated by thrombin-thrombomodulin
| complex on the surface of endothelial cells
62
Action of activated protein C
inhibits thrombin generation by proteolytically inactivating procoagulant cofactors FVa and FVIIIa
63
Which phase does protein C act on
APC acts primarily upon the propagation phase of coagulation
| No effect on initiation phase
64
Factor 5 Leiden
Procoagulant genetic trait (5% of population)
| Impairs function of Proetein C pathway and get activated protein C resistance
65
Domain organisation of Protein C
Gla domain, 2 EGF domains and Serine protease
66
How is protein C pathway switched on
When site of injury is filled
| → Thrombin then encounters the endothelial surface and binds to TM
67
What happens when thrombin reaches the endothelium and binds to thrombomodulin
If encountering endothelium - means that gap has been plugged so no longer need coagulation.
→ Modulates the action of thrombin
→ Causes it to become an anti-coagulation enzyme
Thrombin can then activate protein C
68
Activation of protein C by...
Thrombin–thrombomodulin complex
69
What does activated protein C do
(with cofactor protein S) then cleaves FVa (at 3 different sites) and FVIIIa - so they can no longer function as cofactors
Does not inhibit thrombin but downregulates production
70
Cofactor of protein C
Protein S.
| helps APC assemble on phospholipid surfaces
71
Where does protein C activation take place
Perimeter of site of vessel damage
72
What is antithrombin?
a 58kDa serine protease inhibitor (SERPIN)
73
Concentration of antithrombin in plasma
AT circulates in plasma at high concentration (2.5μM)
74
Action of antithrombin
- inactivates many activated coagulation serine
proteases (FXa, thrombin, FIXa, FXIa and FXIIa)
- mops up any free serine proteases that escape the
site of vessel damage
75
Effect of antithrombin on factors when bound or in comples
Does not inhibit factors when bound on a phospholipid surface or in complex.
Only those that escape the site of vessel damage
It is only FREE enzymes
76
What enhances antithrombin?
AT is enhanced by GAGs/heparin
| Heparin is not in itself an anticoagulant
77
Action of heparin
* Makes anti-thrombin more effective
| * It is a cofactor for anti-thrombin
78
Mechanism of antithrombin
1. AT presents a bait loop to target proteases (like mousetrap)
2. Bait loop contains a ‘mock substrate’
3. Bait loop binds active site of target protease Thrombin binds in an attempt to cleave the bait loop
4. AT undergoes a massive conformational change that locks the target protease in a covalent, inactive complex that can no longer function
5. AT inhibits FREE proteases.
6. AT cannot inhibit proteases in complexes
79
What is TAT level
(Thrombin anti-thrombin levels) Measure of thrombin generation in an individual
80
Use of TAT levels
Used in the clinic to look at whether pts have increased amounts of basal clotting
If people have a thrombotic event, ↑clotting
The TAT levels will go up
