Haem 7 Flashcards
Define haemostasis
the cellular and biochemical processes that enables both the specific and regulated cessation of bleeding in response to vascular insult
Generally, when can thrombosis and when can bleeding occur
Bleeding: INCREASED Fibrinolytic factors
Anticoagulant proteins,
DECREASED Coagulant factors
Platelets
Thrombosis: DECREASED Fibrinolytic factors
Anticoagulant proteins, INREASED Coagulant factors
Platelets
State how haemostatic plug is formed
Vessel constriction (vsmc contracts)
Formation of unstable platelet plug (Platelet adhesion and aggregation)
Stabilisation of the plug with fibrin
(blood coagulation)
Vessel repair and dissolution of clot
(Cell migration/proliferation & fibrinolysis)
Function of platelet adhesion and aggregation
Physical barrier and a site for coagulations reactions to occur
Why is the endothelial layer anticoagulant? Give examples of anticoagulants on the endothelial wall
So that normal blood flowing does not clot…..
TM, TFPI, GAG, EPCR,
Why are contents of subendothelium procoagulant
Give examples of procoagulant moecules
So in damage to vessel, you get clotting
Basement membrane: Elastin, collagen
VSMC - TF
Fibroblasts - TF
Where is tissue factor present
VSMC and fibroblasts
Where does vessel constriction occur
- Mainly important in small blood vessels
2. Local contractile response to injury
Size of platelet and life span
Small (2-4µm)
Life span: ~10 days
Normal platelet count
150-350 x 109/L
Characteristics of megakaryoctes
nuclear lobes and granulated cystomplasm
Outline the differentiation of haematopoetic stem cells to create platelets
MK looses its ability to divide, however continue to replicate its DNA becomes polyploid, cytoplasm enlarge. MK matures, becomes granular and form platelets that will be released in the circulation.
They form pseudopodia-like extensions (proplatelets) that extend in the lumen plt are released from tip of these long extensions by shear forces.
Each MK produces 4000 platelets
T/f/ platelets have no nucleus
T
How are utltrastrucural properties of platelet relevant to function
What is the function of PAR, gb1b, gp2b/3a, a2b1?
What are the different types of granules in a platelet
Lots of receptors e.g. PAR (protease activated receptors, responsive to thrombin), gp1b (vWF), gb2b/3a (other platelets and fibrinogen) so can be activated by lots of agonists and a2b1 (=Glp1a) (binds collagen directly)
P2Y12 is a receptor for ADP
Lots of granules. Dense granules contain ADP/ATP released to activate other platelets. a-granules contain growth factors
Cytoskeleton- allow platelets to change shape upon activation (actin and myosin) and microtubules
How are clotting factors recruited to platelet surface
The plasma membrane inverts upon platelet activation making the outside negatively charged, which recruits the clotting factors to the platelet surface
How does platelet acivation look
Why is the cytoskeleton important
spreads out-
Conversion from active to passive cell
Cytoskeleton important for Important for platelet morphology, shape change, pseudopods,
contraction and clot retraction.
Roles of platelets
Haem/thromb, cancer, atherosclerosis, infection, inflammation
Why does vWF not bind to platelet receptors all the time then!?
Multimeric VWF circulates in plasma in a globular conformation. Binding sites are “hidden” from platelet GpIb
Outline platelet adhesion
Vascular injury damages endothelium & exposes sub-endothelial collagen
Exposed sub-endothelial collagen binds globular VWF
Tethered VWF unravelled by rheological shear forces of flowing blood
Now, the platelet receptors on VWF can bind platelets
VWF unravelling exposes platelet binding sites
Platelets are tethered to VWF via Gp1b (this glycoprotein is present on the platelet surface and is a receptor for VWF)
Binding of VWF to platelet GpIb recruits platelets to site of vessel damage
T/F only VWF can bind platelets
F: Platelets can also bind directly to collagen via GPVI & α2β1 …. at LOW SHEAR NOT ARTERIES/CAPILLARIES
What will bound platelets release
ADP and thromboxane – activate platelets
Activated platelets (αIIbβ3,= same as GPIIb/IIIa) recruit additional platelets
αIIbβ3 (=gp2b/3a) function
recruit additional platelet and bind fibrinogen
Platelets will bind to each other via fibrinogen on activated aIIbb3 integrin. Platelet aggregation
– platelet plug develops
Helps slow bleeding & provides surface for coagulation
SIMULALTANEOUSLY, the coagulation cascade is occurring to try to help with clot stabilisation by fibrin formation
Symptoms of immune thrombocytopnia, and why
purpura,
multiple bruises,
ecchymoses
Because it’s depleting platelets, to a point where spontaneous bleeding is common (below 40 X 10^9)
If it was brought below 10, then there is SEVERE spontaneous bleeding, which can occur during treatment for leukaemias.
Between 40-100 there is no spontaneous bleeding, only bleeding with trauma
What is thrombin
end product of the coagulation cascade…. cleaves fibrinogen to allow them to self assocaite to make fibrin meshworks to hold platelets together
Why do some plateletes all off the end of a clot
if they are not consolidated by fibrin (and not all are), they will fall off
Where are coagulation proteins produced
- The liver – most plasma haemostatic proteins
- Endothelial cells – VWF, and TM & TFPI (anticoag.)
- Megakaryocytes – VWF, FV
What type of molecules are the activated coagulation proteins and where do they cleave and what is their catalytic triand
serine proteases
These serine protease cleave substrates after specific Arg (and Lys residues)
triad: His/Asp/Ser
Which coagulation factor does not need proteolytic actviaton and why
Tissue factor (NOT FVII)
Explain how coagulation initiates
How does FVII interact with TF
- FVII/FVIIa bind cell surfaces via Gla domain
- All domains of FVII/FVIIa interact with TF
- TF makes FVIIa 2x106 times more active
What is the structure of FVII and what other proteins share this structure. How is factor VII activated
What is the use of each domain
Factor VII is initiated by proteolysis, as are the others with the same structure
Contains Gla domain, 2*EGF domains and serine protease domain
It is a serine protease zymogen
This structure is shared by FVII, FIX FX and PC.
Gla: binding to negatively charged phospholipid surfaces
EGF domain is involved in protein-protein interactions
Where is tissue factor expressed more, and why is tissue factor important
Cellular receptor and cofactor for FVII/VIIa
Only procoagulant factor that does not require proteolytic activation
It is the initiator of coagulation
In tissues where you really don’t want bleeding…. brain, lungs, heart, testis, uterus and placenta
How does factor VII circulate
circulates in plasma at ~10nM
~1% of plasma FVII circulates in its activated form (FVIIa)
Differentiate FVII and TF location
FVII= plasma glycoprotein 48kDa
TF= integral membrane 47kDa
TF is regarded as a cofactor whereas FVII is a serine protease zymogen
Via which receptor on the platelet does vWF bind
And where can collagen bind on the platelet
vWF:Gp1b
Collagen: GPVI &α2β1
Which proteins contain Gla domain, and what is gla domain required for
NB this is the same as the vit K dependent factors because vit K is needed to make these using gamma- VITAMIN K carboxylase
so…
FVII
FX
Prothrombin
FIX
Protein C
Protein S
Required for the binding to phospholipid surfaces (i.e. for FVII to bind to VSMC membrane before interaction with TF, or for thrombin to bind to platelet membrane or for any of the coagulation that requires PL to occur)
Outline how Gla domains are produced
All this is, is that protein is produced with Glutamic acid residue.
This residue has another negatively charged carboxylic acid groups added to it by: VITAMIN K carboxylase..
to produce gamma-carboxyglutamic acid (=GLA!!)
Because of the 2 negatively charged carboxylic acid groups, they can now bind 6 or 7 calcium ions which causes a structural transition so they are in a conformation that can bind phosphoplipid
this is so that they can bind cell surfaces such as on VSMC/fibroblast for TF interaction for factor VII,
and for the other factors to bind to platelet cell surfaces where all the coagulation cleavage reactions happen
Mechanism of warfarin action
It in inhibits the vitamin K dependent carboxylase, so the proteins can’t bind Ca2+ and make the right shape to bind to phosopholipid membranes…. it interferes with production of all the gla domain proteins….
therefore the clotting factors cannot bind phospholipids….
for FVII, it means it cannot bind to TF on VSMC and cannot initate coagulation
Now we have produced TF/FVIIa complex, what does this do
Proteolytically cleaves
FX–> FXa and
FIX–>FXIa and
(removes activation peptide)
What does FXa do in initiation of coagulation
FXa can activate prothrombin to generate thrombin
Activation is inefficient - only small quantities of thrombin are generated
What is the function of the gla domain for factor VIIa
helps it to bind to tissue factor on extravascular cell phospholipid
How does propogation of coagulation cascade occur
So the factor Xa generated by the TF-FVIIa complex can only convert a small amount of pro-thrombin to thrombin…..
So what happens is that the small amount of thrombin produced due to factor Xa then cleaves factor VIII –> factor VIIIa.
Factor VIIIa is a COFACTOR (not a serine protease), and it is a cofactor to factor IX.
This factor VIIIa/XIa complex can activate factor X –> Xa… MUCH more Xa produced
Factor V is also activated to factor Va by thrombin, which is a cofactor for factor Xa. The factor Va/factor Xa complex can convert pro-thrombin to thrombin very very rapidly!
So it is a positive feedback loop
What is the function of the thrombin produced through the clotting cascade
It can convert fibrinogen to fibrin and this can then consolidate the platelet plug
Examples of deficiency in procoagulant factors
FVIII deficiency –> haemophilia A
FIXa deficiency –> haemophilia B
These ones are X linked
- SPONTANEOUS joint and muscle bleeds
- Severe but compatible with life
Prothrombin deficiency is lethal
FXI you bleed after trauma but not spontaneously
FXII no bleeding at all
All other factor defiencies apart from FVIII/FIX are recessive
Type 2 VWF disease is AD, and type 1 and 3 are AR
How is the coagulation system regulated
TFPI
Protein C pathway (with protein S cofactor)
Antithrombin
Explain mechanism of TFPI
Inhibits the INITIATION phase of the coagulation cascade
TF-VIIa can cleave FX–>FXa, which can then dissociate and set off the cascade (through propogation).
When FXa dissociates, K2 domain of TFPI binds to active site of Xa and inhibits it. The TFPI/factor X then goes back to the TF/FVIIa complex and, binding to the active site of this complex via K1, shuts down the complex
If TFPI is always in the blood, how can clotting ever occur
It is present at very low levels so is easily overwhelmed when the TF/VIIa compex makes a larger amount of Xa during coagulation….. but it is an important mechanism
How does protein C pathway regulate coagulation cascade
Protein C is localised to the endothelium by endothelial protein C receptor (EPCR), which is an anticoagulant molecule on the endothelial surface.
Protein C activated when thrombin binds to thrombomodulin on EC.
Activated protein C (APC) inhibits thrombin generation by protealiytically inactivating cofactors FVa and FVIIIa (to FVai and FVIIIai)
Where is thrombomodulin present
On surface of enthothelial cells
What is the effect on thrombin of thrombin binding to thrombomodulin
It turns thrombin from an procoagulant to an anticoagulant molecule…
this happens when thrombin is produced outside of the site of vessel damage (i.e. when the endothelium damage has been plugged, and you don’t need more clotting), as the thrombomodulin is present on endothelial cells
Where does activated protein C inhibit clotting
On the edge of the clot (the clot in at the site of vessel damages spills onto the healthy endothelium, the thrombin binds to the thrombmodulin on the surface of the EC)… so it ring fences clot
T/f protein c inhibits thrombin
F! ACTIVATED PROTEIN C inhibits FV and FVIII, so it inhibits thrombin GENERATION
Why is antithrombin needed
Because TFPI and protein C interfere with generation of thrombin, but what about inhibiting the thrombin that has already been made and has been swept away by the blood flowing over the site of vessel damage!? It could go and cause clots elsewhere so needs to be directly inhibited
What type of molecule is antithrombin
Serine protease inhibitor (SERPIN)
T/F: antithrombin only inactivates thrombin
F! Inactivates FXa, thrombin, FIXa and FXIa
How does antithrombin work
Inactivates FXa, thrombin, FIXa and FXIa and mops up serine proteases that escape the site of vessel damage
How does heparin work
Not an anticoagulant molecule….
binds to antithombin and makes it more effective at inhibiting thrombin and the other factors
Those with deficiency in TFPI/protein C or S or antithombin are at risk of what
thrombosis… can’t control their haemostatic processs
How does dissolution of the clot occur
Plasminogen is converted to plasmin by tPA (tissue plasminogen activator)… plasmin can then break fibrin clot down into fibrin degradation products
Where does tPA bind
to fibrin (where it is converted to active protease), to convert plasminogen to plasmin
Therapeutic use of tPA
THROMBOLYSIS (different to fibryonolysis, which is physiological!)
clot busters in MI, or ischaemic stroke etc
(i.e. alteplase is a human recombinant tPA)
3 types of anticoagulant
and
2 antiplatelet agents
anticoag: heparin, warfarin and DOACs
antiplatelet: aspirin, P2Y12 blockers
What tests assess haemostasis
PTT, APTT
platelet function tests
d-dimer (a fibrin degradation product)
What is the d-dimer
a fibrin degradation product…. presence indicates possibility of a thrombosis.. absence practically rules it out
