Haemostasis Flashcards
State the steps involved in haemostatic plug formation from the time of injury.
- Vessel constriction
- Formation of an unstable platelet plug (platelet adhesion + platelet aggregation) PRIMARY haemo
- Stabilisation of plug with fibrin (blood coagulation) SECONDARY haemo
- Dissolution of clot and vessel repair (fibrinolysis)
What component found underneath the endothelium is involved in triggering the coagulation cascade?
Procoagulant subendothelial structures e.g. collagen Tissue factor is also expressed on the surface of cells under endothelium eg VSMC but it is NOT normally expressed within the circulation itself
State some important factors produced by endothelial cells.
Prostacyclin
Thrombomodulin
Von Willebrand Factor
Plasminogen Activator
What process during maturation of the megakaryocytes is important for the formation of platelets?
Granulation- granules like alpha granules which contain growth factors, vWF and factor V etc. Other granules include dense granules etc.
Platelets bud off from the megakaryocytes (ie take cytoplasm with them and the granules within the megakaryocyte cytoplasm). After budding, what is mainly left over is the nucleus of the megakaryocyte
How many platelets are produced by one megakaryocyte?
4000
What do the dense granules in platelets contain that is important for platelet function?
ADP
What do alpha granules in the platelets contain?
vWF
Factor V
various growth factors
State the two ways in which platelets can bind to collagen. Name the receptors involved.
It can bind via vWF to collagen (via the GpIb receptor)
It can bind directly to the collagen via GPVI & α2β1 (aka Gp1a from year 1)
What happens following the passive adhesion of platelets to subendothelial matrix (including collagen) via receptors (Gp1a/1b depending on whether with VWF or not)?
The receptors signal inside the platelet to release ADP from the storage granules and to synthesise thromboxane
These bind to receptors on the surface of the platelets (that released them) and activate them
Once activated, GpIIb/IIIa receptors become available, which can bind to fibrinogen and allows the platelets to aggregate
Which receptors on the platelets become available following activation of the platelets and what do they bind to?
GlpIIb/IIIa
These bind to fibrinogen
What else can activate platelets?
Thrombin (from secondary haemostasis)
What effect does aspirin have on this entire pathway? What effect does aspirin have on this entire pathway?
Aspirin is a COX1 inhibitor. COX enzymes are involved in thromboxane A2 synthesis. THromboxane and ADP are released by platelets to activate themselves to cause aggregation etc.
State some important drug targets in platelet aggregation.
COX
GlpIIb/IIIa
ADP Receptor
What is the most important test for monitoring platelets and their function?
Platelet count
What is a common cause of spontaneous bleeding? What are the clinical features?
Immune thrombocytopenic purpura (autoimmune antibodies clear platelets from the circulation) (aka idiopathic etc what u had)
This results in petechiae, purpura, easy bruises and ecchymoses
petechiae, purpura and ecchymoses are pretty much the same thing but petechiae are small dots and ecchymoses is the biggest one out of the 3
What is the normal range for platelet count?
150-400 x 109/L
Why do you get thrombocytopenia in leukaemia?
Leukaemic cells populate the bone marrow so it crowds out the megakaryocytes so the platelets aren’t produced in sufficient numbers
What is the bleeding time test used to observe?
This checks the platelet-vessel wall interaction
This isn’t used any more
Describe the platelet aggregation test.
The platelets are stimulated with ADP/thromboxane/collagen to study their function
This is used to diagnose platelet disease e.g. von Willebrand disease
Where is von Willebrand factor produced?
Endothelial cells and a little bit by megakaryocytes
What factors do megakaryocytes produce?
Factor V
Von Willebrand Factor
Tissue factor- factor 7a complex activates the clotting cascade by converting 9 to 9a and by converting 10 to 10a. What are the differences between these two pathways
9 to 9a – slower but produces more thrombin
10 to 10a – faster
State two accelerating factors. What are they activated by?
Factor VIII
Factor V
They are activated by trace amounts of thrombin
If you look at last year’s slides, these 2 were the cofactors in the green box
Which factors are activated on the surface of the platelet? Describe how this works.
10 to 10a
2 to 2a (prothrombin to thrombin)
For 9a to activate 10 it needs to come in close proximity with 10. They both bind to the surface of the platelet mediated by calcium ions, and factor 8a bring the two close together (F8a acts as cofactor) so that 9a can proteolytically cleave 10 to 10a
Factor Va does the same with 10a and 2 (prothrombin)
Which factors are affected by warfarin?
2, 7, 9, 10
What is common to all of these factors (2,7,9,10) and what is the significance of this common feature?
They have a cluster of glutamic acid
The glutamic acid is recognised by an enzyme in the liver and undergoes post-translational modification in the presence of vitamin K to Gamma-carboxyglutamic acid
Once this extra carboxyl group is added, calcium can facilitate the binding of gamma carboxyglutamic acid to the activated platelet membrane phospholipid
How does warfarin actually inhibit the post-translational modification of these factors?
Warfarin inhibits vitamin K epoxide reductase thus inhibiting the gamma carboxylation of factors 2, 7, 9 and 10
Which factors are inhibited by anti-thrombin?
2, 9, 10, 11
How does heparin work
Heparin potentiates the action of anti-thrombin
In what situation is heparin used?
Heparin is used for immediate anticoagulation in venous thrombosis and pulmonary embolism
Describe how anti-thrombin inhibits the clotting factors.
Anti-thrombin has a reactive loop that irreversibly inhibits the active site on the clotting factors
So anti-thrombin acts as a scavenger in stopping inappropriate action of clotting factors
What two proteins assemble on the surface of a clot to allow fibrinolysis to take place? Where are these proteins made?
Plasminogen
Tissue Plasminogen Activator (tPA)
Plasminogen is a plasma protein
tPA is produced by endothelial cells
What is produced from the break down of the fibrin clot and how does this level change in DIC?
Fibrin degradation products (FDP)
This is elevated in DIC
What factors are used in therapeutic thrombolysis of myocardial infarction?
tPA and bacterial activator streptokinase
Describe the function of the Protein C anticoagulant pathway.
Thrombin also has a role in anticoagulation
It binds to thrombomodulin on the surface of endothelial cells and by binding to thrombomodulin it activates protein C, which, along with co-factor protein S, INACTIVATES FACTOR Va and FACTOR VIIIa
Shape of normal VWF in circulation and how does it change when it binds to collagen
Normally globular, becomes linear when bound to sub-endothelial collagen after vessel injury. This VWF unravelling exposes platelet binding sites (GpIb) - platelets get tethered
What does thrombin cleave
Fibrinogen to fibrin (the meshwork that holds everything in a clot)
Clotting factors in secondary haemostasis circulate as inactive precursors of what 2 possible types of compounds
serine protease zymogens (ie when activated, they become serine proteases that catalyse other reactions)
OR precursors of cofactors
Which part of the serine proteases catalyse the proteolysis of target substance during secondary haemostasis
serine protease domain will catalyse the cleavage of activation peptide to activate other zymogens in the clotting cascade
What is Tissue factor and what is special about it
TF is a receptor on cell surface of the subendothelial VSMC cells and is the only procoagulant factor that does not require proteolytic activation. Ie it is active from the start
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.
Describe the first step in secondary haemostasis (NB this happens at the same time as primary haemostasis because endothelial damage will concurrently trigger platelet adhesion and secondary haemostasis at the same time)
Endothelial damage exposes subendothelial TF on VSMC. circulating F7 binds to TF by binding to VSMC membrane via gla domain and forms complex with TF. TF activates F7 to F7a (much more reactive than F7). TF-F7a complex then goes off and activates other serine protease zymogens within the clotting cascade
Where is TF located
extravascular sites i.e not usually exposed to the blood, but in cells under the endothelium (VSMC, fibroblasts etc…)
What is the function of Gla domains
Gla domain – allows coagulation proteins to bind to negatively charged phospholipid surfaces. eg when platelet is activated, its membrane becomes negatively charged. Coagulation proteins bind to platelet membrane via Gla domains
Describe the origin of Gla domain
slide 61
What two clotting factors are activated by TF-F7a complex
F9
F10
slide 64
(by proteolytically removing activation peptide)
What can F10a activate? What is the problem with this process initially
FXa can activate prothrombin to generate thrombin
However, initially, this activation is inefficient process - only small quantities of thrombin are generated
slide 65
What 4 things does thrombin activate? What is the purpose of activating the first 2 of the 3?
F5 and F8. Activation of these will speed up thrombin generation.
Fibrinogen to Fibrin
It also activates PLATELETS from primary haemostasis
slide 66
Which complex can further catalyse the activation of F10? What is the consequence of this
F8a-F9a complex
More F10 produced, so can catalyse the prothrombin to thrombin more
What complex can F10a form with which can also catalyse prothrombin to thrombin reaction?
F5a
Summarise the 3 ways of coagulation regulation
- TFPI tissue factor pathway inhibitor
- APC (activated protein C) and activated protein S
- AT (antithrombin)
What is the TFPI
slide 77
Normally, TF-F7a complex activates F10 to F10a and then F10a leaves. BUT when it leaves, TFPI binds to F10a via K2 domain. And then all of it binds to the F7a-TF complex via K1 domain to stop F10a from leaving to activate prothrombin
What does activated protein C inhibit
COFACTORS F5a and F8a
IT DOES NOT inhibit thrombin but it interferes with thrombin production due to cofactor inhibition of its synthesis pathway
Outline how protein C is activated
slide 87
look at animations from 81-92
Why do we need antithrombin
Protein C pathway helps to ringfence the site of clot so that procoagulant clotting factors and other stuff does not escape to other sites to cause clotting there.
However, if some do escape, AT inhibits them
AT inactivates many activated coagulation serine proteases (F2a (thrombin), 9a, 10a, 11a)
AT “mops up” and free serine proteases that escape the site of vessel damage.
summarise fibrinolysis
Plasminogen converted to plasmin via tPA
Plasmin breaks down fibrin clot into fibrin degradation products
