WEEK 5-7 - haemostasis Flashcards
haemostasis
the stopping of bleeding
haem = blood
stasis = halt
haemostasis and the circulatory system
haemostasis prevents excessive blood loss upon damage
haemostasis
components
1) blood vessel vasoconstriction
2) platelets
3) coagulation
4) fibrinolysis
haemostasis components:
1) blood vessel vasoconstriction
Vasoconstriction – reduces flow, limits volume of blood, promotes platelet and coagulation factor activation
nervous reflex (happens within seconds)
release of serotonin and thromboxane A2 from activated platelets
fibrinopeptide’s released during coagulation
haemostasis components:
2) platelets
small cell with no nucleus
lifespan 10 days
150-400 million per ml of blood
we produce ~1.2 million every second
haemostasis components:
2) platelets
made by:
made by megakaryocytes in bone marrow
haemostasis components:
2) platelets
megakaryocyte development
Matures by endomitosis – DNA replicates without nuclear or cell division – enlargement of cytoplasm
myeloid stem cell –> megakaryocyte development –> platlets
megakaryocyte:
Keep replicating nucleus but don’t divide
Gearing up to produce lots of platelets (each meg 1000 platelets)
haemostasis components:
2) platelets
platelet production in vivo
Thrombopoietin from the liver and kidney increases meg number and maturation
Demonstrated in fluorescent microscopy imaging in mice
haemostasis components:
2) platelets
platelet structure
surface glycoproteins important for activation
open membrane (canalicular) system provides large surface to which coagulation proteins are absorbed
storage granules - discharged on activation
- Alpha -> large molecules
- Dense -> Ca2+, serotonin
rich in signalling and cytoskeletal proteins
- Can change shape –> key aspect of platelet function
haemostasis components:
2) platelets
platelets monitor…
blood vessel integrity
haemostasis components:
2) platelets
action
damage exposes the subendothelial layer
- site of vessel injury (collagen, laminin etc exposed)
platelets plug hole at site of injury
- formation of platelet clot or thrombus
haemostasis components:
2) platelets
what is the clot called
thrombus
haemostasis components:
2) platelets
morphology change
resting - circular
activated - has membrane protrusions (pseudopodia)
When a platelet adhere to a substrate
- Lamellipodia and filopodia
haemostasis components:
2) platelets
platelet tethering is mediated by the…
GPIb-VWF interaction which enables binding of GPVI to collagen.
haemostasis components:
2) platelets
what is essential for firm platelet adhesion
Cellular activation and inside-out upregulation of β1- and β3 integrin affinity is essential for firm platelet adhesion.
haemostasis components:
2) platelets
what mediates thrombus growth
Released ADP and TxA2 amplify integrin activation on adherent platelets and mediate thrombus growth by activating additional platelets.
the platelet to release ADP and thromboxane A2, which activates other platelets
haemostasis components:
2) platelets
what locally triggers thrombin generation and contributes to platelet activation
exposed tissue factor
haemostasis components:
2) platelets
what stabilises the growing thrombus
stabilised by signalling CLEC-2
whose ligand/counter receptor remains to be identified
haemostasis components:
2) platelets
STEP 1: tethering and rolling
via GPIb-vWF
GP receptor on platelet attacked to vWF on collagen
Catching and release so slows down slowly (binding characteristics)
- Rolls and slows down then comes to rest on site of damage
haemostasis components:
2) platelets
STEP 1: tethering and rolling
- evidence for important of GPIb-V-IX and von willebrand factor (vWF)
GPIb-deficient humans have bernard-soulier bleed disorder (affects one per million)
–> platelets cannot catch to collagen
vWF-deficient humans have van Willebrand disease bleeding disorder (one per 100)
–> platelets aret tethering properly to sites of damage
haemostasis components:
2) platelets
the main mediator of membrane conformation
von willebrand factor
4% of blood
haemostasis components:
2) platelets
STEP 2: platelet activation and secretion
ADP T x A2
- secretion and positive feedback signalling
inside out integrin activation
TF (can also activate platelets) -> thrombin
- coagulation cascade
haemostasis components:
2) platelets
STEP 3: firm adhesion and thrombus growth
Platelets stick together by fibrinogen bridges
haemostasis components:
2) platelets
STEP 3: firm adhesion and thrombus growth
evidence for importance of integrins
humans deficient fro the major platelet integrin alpha-llb-beta3 have Glanzmann’s disease bleeding disorfer (one per 200,000)
haemostasis components:
2) platelets
what would you see through microscope
Initial rolling of platelets eventually Forming large aggregates
haemostasis components:
2) platelets
major platelet surface glycoproteins
- GPIb-IX-V
- binds vWF
- initial platelet rolling on von willebrand factor bound to collagen on damaged blood vessel wall
haemostasis components:
2) platelets
major platelet surface glycoproteins
GPVI/FcRy -
- powerful activation via tyrosine kinases and calcium signalling leading to integrin activation and granule secretion
collagen
haemostasis components:
2) platelets
major platelet surface glycoproteins
integrins
firm adhesion and spreading
fibrinogen
collagen
haemostasis components:
2) platelets
major platelet surface glycoproteins
G protein coupled receptors
positive feedback activation
thrombin
ADP
TXA2
haemostasis components:
2) platelets
activated platelets fight…
infection and promote repair
chemokines
cytokines
growth factors
haemostasis components:
2) platelets
activated platelets promote…
coagulation
phosphatidylserine flipped from inner leaflet to outer leaflet of plasma membrane (Not normally exposed to the environment) to create a negatively charged surface to recruit coagulation factors (that are +vely charged)
can ultimately produced net
haemostasis components:
3) coagulation
basics
vessel injury
–> activation of circulating coagulation factors (mostly serine proteases + cofactors + calcium) on tissue factor, collagen and platelet surface
–> dramatic amplification cascade
–> thrombin generation
–> converts soluble plasma fibrinogen into fibrin
–> fibrin enmeshes the platelet aggregate, stabilising the clot
haemostasis components:
3) coagulation
occurs on exposed…
collagen, platelet surface and tissue factor
haemostasis components:
3) coagulation
one factor IX generates…
two hundred million fibrin
haemostasis components:
3) coagulation
aims to ensure…
localised and limited production
haemostasis components:
3) coagulation
what initiates coagulation
tissue factor
When exposed to blood activated coagulation cascade
haemostasis components:
3) coagulation
tissue factor
transmembrane protein
- smooth muscles
- fibroblasts
- activated endothelial cells
Tissue factor on cells not normally exposed to blood
haemostasis components:
3) coagulation
thrombin has a dual role in
platelet activation and the coagulation cascade
haemostasis components:
3) coagulation
initiation (or extrinsic) pathway
Small amount of thrombin activates factors V, VIII and XI, as well as platelets.
Extrinsic because activated by external trauma.
This extrinsic pathway does not generate enough thrombin insufficient to generate significant fibrin (for a sufficient a stable fibrin net)
haemostasis components:
3) coagulation
initiation (or extrinsic) pathway:
factor X
Factor x usually inactive
Activated when is cleaved by TF VIIa
haemostasis components:
3) coagulation
initiation (or extrinsic) pathway:
activity of serine proteases
When cleaved exposes active site of factor x
So can go on to cleave other targets
example: activation of factor X by factor IX
haemostasis components:
3) coagulation
intrinsic pathway
explosively generates thrombin
Primed by small amounts of thrombin from extrinsic pathway
Requires calcium
occurs on platelet surface factor IX
Sets up positive feedback loop
- So can get enough thrombin and can ultimately get the fibrin network
haemostasis components:
3) coagulation
physiological coagulation
no role for factor XI
haemostasis components:
3) coagulation
contact pathway
No bleeding problems in individuals with contact factor deficiencies
Supplementary role in factor IX activation?
refers to contact between:
- factor XII (protease)
- kallikrein (protease)
- high molecular weight kininogen (cofactor)
haemostasis components:
3) coagulation
formation and stabilisation of fibrin
Fibrinogen hydrolysis releases fibrin monomers
Fibrin monomers initially link loosely via hydrogen bonds
factor XIII is a transglutaminase which covalently cross-links fibrin
Thrombin also activates factor 13
Covalently cross links fibrin
-> polymerisation and stable fibrin via action of factor 13
summary of haemostatic plug formation
initial platelet activation and aggregation temporarily controls bleeding
this clot or ‘haemostatic plug’ is stabilised by a fibrin meshwork produced by the coagulation cascade
AND FINAL STEPS:
clot retraction is mediated by the major platelet integrin , alpha-IIB-beta3 which linkes firbin to the platelet actin cytoskeleton
plug beings to auto digest by the process of fibrinolysis
summary of haemostatic plug formation
clot retraction mediated by
the major platelet integrin , alpha-IIB-beta3 which linkes firbin to the platelet actin cytoskeleton
summary of haemostatic plug formation
4) fibrinolysis
plug beings to auto digest by the process of fibrinolysis
Back to healthy blood vessels with no sign of damage
physiological limitation of coagulation
important to limit effect of thrombin to the site of injury
Unchecked coagulation would lead to dangerous blocking of vessels
physiological limitation of coagulation
tissue factor pathway inhibitor (TFPI)
inhibits initiation pathway
tissue factor pathway inhibitor (TFPI)
- endothelial cells, plasma, platelets
TFPI forms complex with VIIa, TF, Xa
TFPI expressed by endothelial cells
physiological limitation of coagulation
protein C and protein S
inhibit intrinsic pathway
protein C
- serine protease
- released by endothelial cells and platelets
- activated by thrombin and enhanced protein S
physiological limitation of coagulation
protein C and protein S
- mechanism
Thrombin binds to thrombomodulin on Ecs
This complex activates the serine protease protein C
Activated protein C inhibits cofactors VIII and V
Protein C action is enhanced by cofactor protein S, which binds protein C to the platelet surface
Protein C also enhances fibrinolysis
physiological limitation of coagulation
antithrombin inhibits…
thrombin and other serine proteases
antithrombin (plasma)
–> Negative regulator of thrombin
–> Clotting regulated to site of damage
Inhibits by interacting to form high weight complexes
inhibition of coagulation by blood flow
at the periphery of the damaged area
dilutes and disperses activated factors
activated factors destroyed by the liver
components of haemostasis
4) fibrinolysis
tissue plasminogen activator inhibitor (t-PA)
released by activated endothelium and platelets
- tPA binds fibrin
Negative regulatory pathway
most common coagulation disorders
haemophilia A - factor VIII deficiency
haemophilia B - factor IX deficiency
von Willebrand disease - vWF deficiency
most common coagulation disorders
haemophilia A
- factor VIII deficiency
30-100 cases per million
X-linked
bleeding tendency
treatment with recombinant factor VIII
Prolonged coagulation
More unstable clots
A third no family history
Bleeding into joints
Avoid contact sports
Anti-VIII antibodies
most common coagulation disorders
haemophilia B
- factor IX deficiency
6-20 cases per million
X-linked
bleeding tendency
treatment with recombinant factor IX
most common coagulation disorders
von Willebrand disease
- vWF deficiency
Variable severity
vWF is a carrier for factor VIII, protecting it from premature destruction
10,000 cases per million
bleeding tendency
treatment to increase vWF production, vWF, anti-fibrinolytics
most common coagulation disorders
haemophilia A
treatment
with recombinant factor VIII
most common coagulation disorders
haemophilia B
treatment
with recombinant factor IX
most common coagulation disorders
von Willebrand disease
treatment
treatment to increase vWF production, vWF, anti-fibrinolytics
