Haemostasis Flashcards
What are the different components of haemostasis
– i.e. vessel wall, platelets and clotting factors
Clotting factors can be found in the blood, on the surface of the endothelium or expressed on the surfaces of some extravascular cells
How can clinical bleeding manifest
e.g. thrombocytopenia, haemophilia, VWD
How can thrombosis manifest
.g. thrombophilia, venous/arterial thrombosis, cancer
In many diseases (cancers, myocardial infarction) a haemostatic/thrombotic challenge is often the cause of death for these patients
Summarise the therapeutic intervention to manage the disorders of haemostasis
anticoagulant/antiplatelet drugs, replacement therapy (for haemophilia)
What is haemostasis
“the cellular and biochemical processes that enables both the specific and regulated cessation of bleeding in response to vascular insult”
Essentially the balance between bleeding and thromboisis
Don’t want to bleed excessively once we cut ourselves, so it acts rapidly to prevent blood loss.
What is haemostasis needed for
“to prevent blood loss from intact and injured vessels, enable tissue repair”
meshwork of fibrin which is a temporary structure- to allow subsequent repair mechanisms to kick in
Describe a delicate balance in normal haemostasis
Maintaining the integrity and patency of the vascular system is essential to life. Although there are many regulators that ensure this process operate within the normal range
Balance between pro-fibronylytic factors and pro-coagulant factors to ensure balance between bleeding and thrombosis.
Essentially, what is meant by normal haemostasis
Equilibrium: normal haemostasis is a balance between bleeding and thrombosis
Describe how the balance can be tipped towards bleeding
Increase in fibrinolytic factors or anticoagulant proteins
Decrease in coagulant factors and platelets
Describe how the balance can be tipped towards thrombosis
Decrease in fibronlytic factors or anticoagulant proteins
Increase in coagulant factors and platelets
What is a major complication of DVT and what will most people die from
Pulmonary embolism is a major risk factor of DVT- clots float away and get trapped in the lungs (DVT not a risk factor for strokes)
Most people die with a ‘haemostatic end-point’! And this is modifiable with therapeutic intervention.
What is the first step in the response to injury to the endothelial cell lining
Vessel constriction
Vascular smooth muscle cells contract locally
Limits blood flow to injured vessel
But vessel constriction (although it reduces the blood flow)- is strictly not a haemostatic response
Summarise primary haemostasis
Formation of an unstable platelet plug
platelet adhesion
platelet aggregation
Limits blood loss + provides surface for coagulation
Platelets act as a physical barrier to stop the flow of blood
Summarise secondary haemostasis
Stabilisation of the plug with fibrin
blood coagulation
Stops blood loss
Activation of coagulation cascade and deposition of fibrin
Only stop bleeding when you get fibrin mesh on top of the platelets (i.e once you get primary and secondary haemostasis).
Summarise vessel repair and dissolution of the clot
Vessel repair and dissolution of clot
Cell migration/proliferation & fibrinolysis
Restores vessel integrity
What is important to remember about vessel constriction
§ Vessel constriction:
o Mainly important in SMALL blood vessels.
o Local contractile responses to injury but the precise mechanisms are uncertain.
This is in itself sometimes sufficient to temporarily restrict blood loss from a wound in small blood vessels.
Describe a key property of the arterial endothelial cells
Anti-coagulant by nature- provide a surface for the blood to irrigate
EC - anticoagulant barrier
TM, EPCR, TFPI, GAG
Describe the key properties of the sub-endothelium
Sub-endothelial structures are pro-coagulant in nature
So the tunica intima, media and adventitia (which also has its own microvessels) have tissue factors and various ECM proteins which helps to recruit platelets to the site of injury.
Summarise the subendothelium
Subendothelium - procoagulant Basement membrane Elastin, collagen VSMC - TF Fibroblasts - TF
Internal elastic lamina seperates the tunica intima and media
External elastic lamina seperates the tunica media and tunica adventitia.
Describe the normal intact blood vessel wall
Intact blood vessel with endothelium expressing anti-coagulant factors such as EPCR and TM.
Various plasma proteins (clotting factors) whicha re present constituitively in the blood, but normally in a latent form.
VWF, PI
FVII, FX, Prothrombin (FII), FV, FVIII, FIX, FXI, Protein C, Protein S etc…
How can we trigger a haemostatic response in mice
Shine a laser through a microscope onto their blood vessels to initiate a haemostatic response.
See platelets arrive before fibrin deposition (BUT IN REALITY primary and secondary haemostasis occur simultaneously).
State the characteristics of platelets
Small (2-4µm)
Anuclear
Life span: ~10 days
Platelet count: 150-350 x 109/L
Can be stained by wright-Giemsa- will become purpley
Outline the differentiation pathway of platelets
Haematopoietic stem cell – promegakaryocyte
Promegakaryocytes – megakaryocytes (proliferation of DNA 2N-16N)
Megakaryocytes- -proplatelets
Proplatelets- - platelets
Describe how megakaryocytes give rise to 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.
model MK migrate from endosteal niche to the vascular niche where they do not pass the EC of sinusoidal blood vessels and remain in vascular niche. They form pseudopodia-like extensions (proplatelets) that extend in the lumen plt are released from tip of these long extensions by shear forces.
Other model is following migration from endosteal niche to vascular niche mature MK pass the E.C barrier and enter the circulation. Due to large size of MK they get trapped in the microvasculature of lungs mechanical force induce fragmentation of MK
Describe the key properties of megakaryocytes
Haemopoietic stem cells give rise to megakaryocyte precursors which undergo nuclear replication without cytoplasmic division, then maturation before migrating to the marrow sinusoids, extending proplatelets through the endothelial wall and fragmenting into platelets in the circulation.
Can have 8-16 nuclei- multipleudic
Each megakaryocyte produces ~4000 platelets.
- 1011 platelets are produced each day
- Lifespan ~10 days
Describe how platelets are responsive to agonists in their micro-environment
TP receptors- respond to thromboxane
PAR receptors- respond to thrombin
P2Y1/12- responds to ADP
State the two ways in which platelets can bind to collagen
It can bind via vWF to collagen (via the GlpIb/V/IX receptor)
It can bind directly to the collagen (via the GlpIa receptor or to the GPVI or to Alpha2Beta1 receptor)
How can the platelet bind to fibrinogen
Via the alpha2bBeta3 receptor
alpha beta- are integrins
Describe the other key features of platelets
Alpha granules which include growth factors (cytokines to be release upon activation), fibrinogen, FV and VWF
Dense granules- ADP, ATP, serotonin, Ca2+ and polyphosphates
Phospholipid membrane- quiescent in resting state of platelet. However, when platelet becomes activated, we see a flip-flop reaction- where the negative phospholipids normally on the I.C leaflet are now expressed on the E.C leaflet- which makes the surface of the platelet attractive for clotting factors.
Cytoskeleton- dynamic microtubules and actin cytoskeleton, helps the platelet change shape rapidly from quiescent to active upon activation.
What else do alpha granules contain
Alpha granules also contains extra pool of alphaIIbb3 also in the open canalicular system
Describe the platelet cytoskeleton
Important for platelet morphology, shape change, pseudopods,
contraction and clot retraction.
Platelet activation:
conversion from a passive to an interactive cell
inactive (flat) - rounded- flattened egg-shaped active cell
Summarise the roles of platelets
Haemostasis and thrombosis
Cancer
Atherosclerosis
Infection and inflammation- interact with leukocytes to clear infections
Vastly dynamic cells which will have different roles depending on the agonists that they are exposed to.
Describe the normal circulation of VWF (i.e when the vessel wall is intact)
Multimeric VWF circulates in plasma in a globular conformation. Binding sites are “hidden” from platelet GpIb
Describe the consequences of vascular injury on VWF
Vascular injury damages endothelium & exposes sub-endothelial collagen
several components of the subendothelium (e.g. collagen, fibronectin, laminin) become exposed, to which platelets will get recruited
Exposed sub-endothelial collagen binds globular VWF (tethering it to the endothelium)
Tethered VWF unravelled by rheological shear forces of flowing blood
VWF unravelling exposes platelet binding sites
(GpIb) - platelets get tethered
Binding of VWF to platelet GpIb recruits platelets to site of vessel damage
How else can platelets be recruited
Platelets can also bind directly to collagen via GPVI & α2β1
(only at low shear – i.e. not in arteries/capillaries)
Describe the importance of collagen for platelet activation in primary haemostasis
Activate platlets via GPVI and A2b1
Platelets change shape, release their granule contents and undergo the membrane flip-flop reaction (essentially, they become activated).
Thrombin can also activate platelets
Activated platelets release agonists (ADP and thromboxane) that will further activate platelets and allow recruitment of additional platelets
Via which domain does VWF bind to collagen
VWF can bind to collagen via its A3 domain
Describe platelet aggregation
Activated platelets (αIIbβ3) recruit additional platelets
αIIbβ3 also binds fibrinogen
Platelets will bind to each other via fibrinogen on activated aIIbb3 integrin. Platelet aggregation
αIIbβ3 also binds fibrinogen– platelet plug develops
Helps slow bleeding & provides surface for coagulation
Describe the importance of platelet aggregation
They will also provide support for initiation of the coagulation cascade
Provides a surface for coagulation
Allowing for generation of thrombin which will produce fibrin which will lead to clot stabilisation and stop the bleeding
Platelet activation enhances coagulation
Phosphatidylserine important in this surface
Describe how the shape of the platelet changes throughout primary haemostasis
Dormant- flowing disc-shaped platelet
Tethered to VWF and collagen- rolling ball-shaped platelet
Touches down on endothelium- hemishpere-shaped (firm but reversible adhesion)
Activation- spreading platelet which will release granules and aggregate.
Why is it important that there are multiple pathways for platelet adherence
We have 2 different mechanisms of platelet adhesion to ensure haemostasis works in all environments of the body.
What is alpha2beta3 also called
GPIIb/IIIa
Describe VWD
Mutations in VWD- can’t bind to collagen- therefore no recruitment of platelets- loss of primary haemostasis.
Summarise platelet disorders
Deficiencies in mutations or mutations in platelet activating processes or funcitons
Preventing platelets from binding to VWF and thus preventing primary haemostasis.
What is the normal range for platelets
150 x 10^9 0— 400 x 10^9 /L
Most of us have more platelets than necessary for normal haemostasis.
Needed for trauma and childbirth.
therefore:
<100 x 109/L
No spontaneous bleeding, but bleeding with trauma
Describe auto-ITP
<40 x 109/L
Spontaneous bleeding common
Immune Thrombocytopenia (ITP):
purpura,
multiple bruises,
ecchymoses
Platelets develop immune reaction against own platelets- resulting in destruction and clearance of platelets.
What can treatment of leukaemia result in
<10 x 109/L
Severe
spontaneous bleeding
Purpura in a male (53 yr) patient with acute
myeloblastic leukaemia
Petechiae too
Summarise thrombin
Extrinsic/Tissue Factor pathway
FVII exposed to tissue factor triggering a cascade that results in the production of a serine protease, thrombin, which catalyses the breakdown of fibrinogen (soluble)- to fibrin (insoluble) which is deposited on the platelets to form the clot.
What are the § Sites of synthesis of clotting factors, fibrinolytic factors and inhibitors:
- The liver – most plasma haemostatic proteins
- Endothelial cells – VWF, TM (anti-coagulant), TFPI (anti-coagulant)
- Megakaryocytes – VWF, FV
What do the clotting factors circulate as
Clotting factors circulate as inactive precursors
Either serine protease zymogens or cofactors
Activated by specific proteolysis
What are the zymogens and their corresponding serine proteases
prothrombin (zymogen) --- thrombin (serine protease) FVII -- FVIIa FIX- FIXa FX- FXa FXI - FXIa FXII- FXIIa FXIII --- -
Prothrombin, FVII. FIX and FXand their corresponding serine proteases (except for thrombin) contain a G1a domain.
Protein C and S also have the G1a domain.
What are the cofactors for prothrombin, FVII and FIX
TF
FVa
FVIIIa
What are the inhibitors in the coagulation cascade
TFPI for prothrombin (Kunitz-type)
FVII- Protein C (serine protease)
FIX- Protein X (cofactor for APC)
FX – Antithrombin (serpin)
List the serine protease domain-containing proteins
FVII FX Prothrombin FIX FXI Protein C
Describe the action of serine proteases
Once activated, serine protease domain catalyses proteolysis of target substrate
Serine protease contain a catalytic triad His/Asp/Ser
These serine protease cleave substrates after specific Arg (and Lys residues)
Essentially, how is coagulation initiated
Vessel injury
Tissue factor present on extravascular cells (e.g VSMC) is exposed to clotting factors in the circulation (FVII, FX, Prothrombin (FII), FV, FVIII, FIX, FXI, Protein C, Protein S etc…)
What is tissue factor
Cellular receptor and cofactor for FVII/VIIa
Only procoagulant factor that does not require proteolytic activation
Forms a complex with FVII/FVIIa once bound
How does tissue factor interact with FVII/FVIIa
FVII/FVIIa bind cell surfaces via Gla domain
All domains of FVII/FVIIa interact with TF
TF makes FVIIa 2x106 times more active
What are the key features of tissue factor
primary initiator of coagulation
47kDa integral membrane
Where is tissue factor located
normally located at extravascular sites
i.e not usually exposed to the blood (VSMC, fibroblasts etc…)
TF expressed higher in certain organs
(i.e. lungs, brain, heart, testis, uterus, and placenta)
TF in these locations provide further haemostatic protection in these organs.
What is factor VII and where is it produced
serine protease zymogen
48kDa plasma glycoprotein
expressed/secreted by the liver
Describe the structure of FVII
domain structure; Gla Domain (important for interacting with phospholipid and TF)- contains Ca2+ 2x EGF-like domains Serine protease domain
Describe the circulation of FVII
circulates in plasma at ~10nM
~1% of plasma FVII circulates in its activated form (FVIIa)- without this small amount of FVIIa circulating- our haemostatic system would not funciton
What do FVII, FIX, FX and PC share
a homologous modular structure (4 domains)
Gla domain - binding to phospholipid surfaces
EGF domain is involved in protein-protein interactions
all circulate in plasma in zymogen form
activated by proteolysis
List the Gla domain containing proteins and describe its importance
FVII
FX
Prothrombin
FIX
Protein C
Protein S
Important in binding to the negatively charged phospholipid membranes of activated platelets.
Describe the formation of secreted Gla-domain containing proteins
Nascent Gla domain-containing proteins have a glutamic residue at their N-terminal
Vitamin K carboxylase then adds more carboxylic acid groups to the N-terminal to form gamma-carboxyglutamic acid (Gla)- this is a post-translational modification which gives these Gla domains an affinity to bind Ca2+- which causes them to fold up into a conformation that can bind negatively charged phospholipids.
Consists of Gla domain (with Ca2+) and aromatic stack.
What do Gla domains contain
Gla domains contain
9 - 11 –carboxyglutamic acid residues
Gla domains bind 6/7x Ca2+ ions which causes a structural transition
Describe the action of warfarin
No direct anti-coagulant role
Vitamin K antagonist
Prevents gamma-carboxylation of clotting factors
Thus clotting factors cannot bind to negatively charged phospholipid surfaces
Diminishes the function of clotting factors.
When is warfarin indicated and explain its mechanism of action
In the liver, vitamin K mediates production of factors II, VII, IX and X which have extra carboxyl groups on.
The extra carboxyl groups (gamma-carboxyglutamic acid) allow the clotting factors to adhere to platelet membrane PLs.
Warfarin inhibits vitamin K epoxide reductase and thus reduces creation of secreted clotting factors reduces the clotting factors that bind to platelet PLs (phospholipids).
Thus warfarin inhibits the platelet surface interactions as the clotting factors cannot bind to the surface with Ca2+.
Warfarin = long-term anticoagulation following venous thrombosis AND treatment of atrial fibrillation.
How does FVIIa bind to TF
Via the Gla domain.
What initiates coagulation in the extrinsic/tissue factor pathway
TF binds to FVII- forming a TF/FVIIa complex
TF-FVIIa proteolytically activates FX & FIX (forming FXa and FIXa respectively).
Removes activation peptide to yield active enzyme (cleaves activation peptide atttached to the serine protease domain of the clotting factor).
What is the role of FXa
FXa can activate prothrombin to generate thrombin
Activation is inefficient - only small quantities of thrombin are generated
What does the small quantity of thrombin generated by FXa then go onto do
Thrombin can then activated FV and FVIII to FVa and FVIIIa respectively.
Describe the roles of the FVa and FVIIIa generated by the thrombin.
FVIIIa acts as a cofactor for FIXa- which can activate more FX to form FXa
FVa acts as a cofactor for FXa to generate more thrombin from prothrombin.
This generates more than 3000 times more thrombin than FXa alone.
Summarise haemophillia
Patients deficient in procoagulant factors can develop haemophilia
Haemophilia A (FVIII deficiency) Haemophilia B (FIX deficiency)
HA/HB are X-linked
(i.e. primarily effects boys)
Summarise the regulation of coagulation
TFPI can inhibit the initaiton of coagulation (i.e the binding of FVIIa to TF)
Anti-thrombin can inhibit thrombin, FXa and FIXa
APC and protein S can inhibit FVa and FVIIIa
What is the role of thrombin
To convert fibrinogen (soluble) into fibrin (insoluble)- which is deposited to form the clot.
Describe TFPI
TFPI-FXa can bind/inactivate TF-FVIIa active site via Kunitz domain 1 (K1)
Then squestesrs FXa with K2 domain
locks TF, FVIIa and FXa together to prevent action - dampens procoagulant response to small injury
tries to prevent initiation of coagulation
Describe the protein C pathway
Protein C pathway – Protein C is activated by thrombin-TM complex on EC
Activated protein C (APC) inhibits thrombin generation by proteolytically inactivating procoagulant cofactors FVa and FVIIIa
FV leiden- can’t inactivate FVA- arg – glut
Thus increased risk of thrombosis.
Describe protein C activation
Thrombin binds to TM with high affinity- thrombomodulin is expressed on intact endothelium- i.e the edge of the clot- so ring-fences thrombosis to site of damage
Thrombin then activated protein C- so becomes anti-coagulant
Protein C localised to endothelial surface (where thrombin/TM are)
Thrombin cleaves protein C to release activation peptide
Activates protein C zymogen to APC (serine protease)
Protein S acts as a co-factor for protein C
Haemostatic plug prevented from spreading beyond site of injury
What is important to remember about regulation of the coagulation cascade
TFPI regulates the initiation of coagulation
Protein C pathway regulates the propagation phase of coagulation by down-regulating thrombin generation – it does not inhibit thrombin
What happens to all the thrombin??
What stops coagulation occurring elsewhere??
Describe antithrombin
Antithrombin (AT) is a 58kDa serine protease inhibitor (SERPIN)
AT inactivates many activated coagulation serine proteases (FXa, thrombin, FIXa, FXIa)
AT “mops up” and free serine proteases that escape the site of vessel damage.
Antithrombin inhibits any thrombin or FXa that “escapes”
Describe the inhibitory coagulation mechanisms
Inhibitory coagulation mechanisms
(i) TFPI (tissue factor pathway inhibitor)
(ii) The protein C anticoagulant pathway (APC & protein S)
(iii) Antithrombin
Deficiencies of antithrombin, protein C and protein S
are important risk factors for thrombosis!
Summarise vessel repair and dissolution of clot
Vessel repair and dissolution of clot
Cell migration/proliferation & fibrinolysis
Restores vessel integrity
Describe firbonolysis
tPA binds to thrombin allowing tPA to convert plasminogen into plasmin
plasmin then degrades fibrin Fibrin degradation products, FDP - elevated in DIC
Describe a use of tPA
tPA can be used in therapeutic thrombolysis for Myocardial Infarction, ischaemic stroke etc.. (Clot busters)
Summarise clinical haemostasis
‘Clinical’ haemostasis
Drugs
anticoagulants - heparin, warfarin, DOACs (directly target coagulation factors)- used for venous thrombosis
antiplatelet agents – aspirin, P2Y12 blockers- used for arterial thrombosis
Tests
coagulation (PT, APTT)
platelet function tests
d-dimer
Describe the actions of heparins
Heparin is a linear negatively charged polysaccharide
Once bound to anti-thrombin it changes the position of the reactive loop and makes the inhibition occur faster
When inhibiting factor 10a, a relatively SHORT chain of heparin is sufficient (low molecular weight heparin)
When inhibiting thrombin, you require a LARGE chain (standard/unfractionated heparin)
NOTE: standard/unfractionated heparin inhibits either Factor 10a or thrombin
Describe the roles of platelets in accelerating blood coagulation
F5 and F8 are found on the platelet membrane.
F9a activates F10F10a on F8aPl.
o F8aPl acts to pull F9a and F10 together.
F10a then passes to F5aPl and activates F2 (prothrombin thrombin).
The thrombin created can then go onto drive more platelet activation and expression of F8aPl and F5aPl.
F8aPl and F5aPl accelerate thrombin generation x10,000.
When is heparin indicated
Heparin is used for – immediate anticoagulation in PE and DVT
How does antithrombin work
Antithrombin acts by irreversibly inhibiting the factors by altering the tertiary structure (such as the active site).
Summarise the lab tests for coagulation
APTT – Activated Partial Thromboplastin Time.
o Initiates coagulation through F12 and detects abnormalities in INTRINSIC and COMMON pathways.
PT – Prothrombin Time.
o Initiates coagulation through tissue factor and detects abnormalities in EXTRINSIC and COMMON pathways.
TCT/TT – Thrombin Clotting Time.
o Add thrombin shows abnormalities in fibrinogen to fibrin conversions.
Describe different uses of the lab tests for coagulation
APTT and PT used together to test for bleeding disorders.
APTT used to monitor heparin therapy in thrombosis – intrinsic pathway.
PT used to monitor warfarin treatment in thrombosis – extrinsic pathway.
acquired disorders such as disseminated intravascular coagulation (both tests).
