Haem 7 - Haemostasis Flashcards
Define haemostasis
The cellular and biochemical processes that enables both the specific and regulated cessation of bleeding in response to vascular insult
What is haemostasis for?
1) To prevent blood loss from intact and injured vessels
2) enable tissue repair
Describe normal haemostasis in terms of the balance between bleeding and thrombosis
See diagram - delicate balance
Bleeding - Increase in fibrinolytic factors + anticoagulant proteins. Decrease in coagulant factors + platlets
Thrombosis - Opposite.
Describe the outline of haemostatic plug formation
See diagram
1) Vessel constriction
2) Formation of an unstable platelet plug
3) Stabilisation of the plug with fibrin
4) Vessel repair and dissolution of clot
Describe the stage of vessel constriction
Vascular smooth muscle cells contract locally
Limits blood flow to injured vessel
- Mainly important in small blood vessels
- Local contractile response to injury
Describe the formation of an unstable platelet plug
platelet adhesion
platelet aggregation
Limits blood loss + provides surface for coagulation
Describe the stabilisation of the plug with fibrin
blood coagulation
Stops blood loss
Describe vessel repair and dissolution of clot
Cell migration/proliferation & fibrinolysis
Restores vessel integrity
What can the endothelial cell layer of a normal vessel wall be thought of as?
An anticoagulant - allow the blood to flow over that surface and irrigate the surface without clotting
What can the subendothelial layer of a normal vessel wall be thought of as?
A procoagulant: Basement membrane Elastin, collagen VSMC (vascular smooth muscle cells) - TF Fibroblasts - TF
Tissue factor - primary initiator of the coagulation cascade. Lies on on VSMC and fibroblasts
Describe platelets
Small (2-4µm)
Anuclear
Life span: ~10 days
Platelet count: 150-350 x 10^9/L
All platelets are derived from megakaryocytes
Describe megakaryocytes
Each megakaryocyte produces 4000 platelets
A megakarycyte when mature has finger like extensions into blood vessels where proplatelets bleb off.
Describe the ultrasound features of a platelet?
It has NOT got a nucleus but it is still a pretty active cell
The storage granules include granules containing ADP (which is very important for platelet function)
They have another type of storage granule called alpha granules
These are storage granules for proteins including Factor V and von Willebrand factor
When the platelet becomes activated, these factors are released
There are important glycoprotein receptors on the platelet surface which interact in the formation of a platelet plug
There are other receptors on the surface including a receptor for thrombin
See diagram/laz’s notes you don’t really need to know this that well.
Describe globular VWF?
Multimeric VWF circulates in plasma in a globular conformation. Binding sites are “hidden” from platelet Gp1b. In this globular form it will not interact with platelets
Think of it as a ball of string
Describe the change from globular VWF to tethered unravelled VWF
During vascular injury damage it exposes sub-endothelial collagen. The globular VWF then binds to the collagen before unravelling by rheological shear forces of flowing blood. Being unravelled all the platelet binding sites are then revealed allowing binding to occur. The Gp1b glycoprotein ensures the platelet binds.
Aside from the platelets binding to the VWF where else can they bind?
Platelets can also bind directly to collagen (and other matrix proteins) via GP1a and alpha2beta1 only at low shear forces (so not arteries or capillaries)
Once bound to collagen what happens to the platelets.
Collagen is a potent activator of platelets.
The platelets become activated and will further recruit platelets. Thrombin also activates platelets. This activation causes shape change, release of granule contents, change the composition of their phospholipid surface.
Activated platelets will recruit additional platelets as well as bind to fibrinogen causing aggregation. Mediated by (integrin alpha-II-beta-3 / GpIIb/IIIa)
What is inside the granules which are released from activated platelets?
Thromboxane and ADP (which further activate platelets that are around.)
What happens as platelets begins to accumulate?
The “primary” haemostatic plug develops which helps slow bleeding and provide surface for coagulation
Describe the platelet change in shape upon adhesion, activation and aggregation
See slides
1) Flowing disc shape
2) Rolling ball shape with filopodia coming out
3) Attach and flatten out to a hemisphere shaped platelet
4) Spreading out of the platelet
What is the most common inherited bleeding disorder?
Von-Willebrands disease - reduce the VWF levels or function. Meaning that platelets are not recruited as efficiently to the site of damage = bleeding disorder.
Platelet disease - similar bleeding phenotype as VWF disease.
What happens when the platelet count falls below 100x10^9/L?
No spontaneous bleeding but bleeding with trauma
What happens when the platelet count falls below 40x10^9/L?
Spontaneous bleeding is common Immune thrombocytopenia (ITP): purpura, multiple bruises, echhymoses
What happens when the platelet count falls below 10x10^9/L?
Severe bleeding
Purpura seen with acute myeloblastic leukaemia
What does thrombin do?
Cleave soluble fibrinogen to insoluble fibrin fibres
What is the liver responsible for?
For the production of most plasma haemostatic proteins - these clotting factors circulate in an inactive precursor form. They are either serine protease, zymogens or cofactors and are activated by specific proteolysis.
List the zymogens in the circulation
Zymogens = inactive
Prothrombin (FII) FVII FIX FX FXI FXII FXIII
List the serine proteases (activated)
The zymogens are converted to active proteases below:
Thrombin FVIIa FIXa FXa FXIa FXIIa
Serine proteases are a family of proteases that all have an homologous serine protease domain. The serine protease domain catalyses proteolysis of target substrate. Serine protease contain a catalytic triad of His/Asp/Ser. These serine protease cleave substrates after specific Arg (and Lys residues)
See slides
List the cofactors required for coagulation
TF (tissue factor)
FVa
FVIIIa
List the inhibitors which help regulate the process of coagulation
TFPI
Protein C
Protein S
Antithrombin
Describe the initiation of coagulation
TF is an integral membrane protein while FVII/FVIIa is a plasma protein.
Exposed TF interacts with FVII/FVIIa - TF makes FVIIa a lot more active.
TF is the only procoagulant factor that does not require proteolytic activation. It is only located at extravascular sites (so it is not usually exposed to blood)
The TF, FVIIa and FX form a complex allowing the activation of FX to FXa. It also activates FIX to FIXa.
Where is TF expressed higher?
Certain organs - lungs, brain, heart, testis, uterus and placenta - provides further haemostatic protection in these organs.
Describe the domain structure of factor VII?
From top to bottom:
1) serine protease domain
2) 2x EGF-like domains (2 and 1)
3) Gla domain - can bind to phospholipid surfaces - bind to the platelets
FVII, FIX, FX and protein C have exactly the same organisation. They all circulate in plasma in the zymogen form. They are activated by proteolysis
What kind of proteins are Gla domains?
Vitamin K dependent proteins.
glutamic acid –> Vitamin K Carboxylase –> Secreted Gla domain-containing proteins.
The carboxylase adds two carboxycylic acid groups on the end which are negatively charged. This enables it to bind to Ca2+ ion (6/7) resulting in the structural formation that enables phospholipid binding.
Warfrin - vitamin k antagonist. It screws up the production of Gla domains so they can’t bind to phospholipid surfaces and function correctly.
What does FVIIa do?
It proteolyses FX and FIX - removing the activation peptide to yield the active enzyme.
FXa and FIXa
TF also activates FV
What doe FXa go on to do?
It activates prothrombin to generate thrombin however this activation is inefficient meaning only a small quantities of thrombin are generated.
The small bit of thrombin produced cleaves FVIII to FVIIIa which is a cofactor to IXa. Together FVIIIa and FIXa activate more FXa.
The thrombin also activates FV resulting in the cofactors FVa and FXa activating prothrombin.
See diagram - it is essentially a feedback activation loop.
What is the relevance of factor VIIIa and IXa?
These are the two proteins associated with haemophilia a (FVIII deficiency) and b (FIX deficiency)
Both of these are X linked disorders (primarily affects boys)
What are the three natural anticoagulant pathways?
1) TFPI - Tissue factor protein inhibitor. Targets the initiation phase of coagulation (TF, FVII, FX). When FXa dissociates from the TF, FVIIa complex, TFPI binds and inhibits the active site of FXa before docking back onto the TF, FVIIa complex.
2) APC - activated protein C (pathway) and cofactor protein S. Protein C is activated by thrombin when it is bound to thrombomodulin on surface of endothelial cells. Protein C is converted to activated protein C by removing a coagulation peptide. It is converted to an active protease. This proteolyses FVa and FVIIIa (these two cofactors) - inactivating procoagulant cofactors. Protein C is generated around the clot.
3) AT - Antithrombin (SERPIN: Serine protease inhibitor). It mops up free serine proteases and inactivates coagulation serine proteases (FXa, thrombin, FIXa, FXIa).
Heparin - anticoagulant. It binds to antithrombin making it more effective at inhibiting thrombin and FXa.
What would deficiencies in AT, protein C and S cause?
Increases the risks of thrombosis
What does tPA do?
It is a tissue plasminogen activator. Converts plasminogen to plasmin which degrades the clot.
tPA is used therapeutically.
