Haemostasis Flashcards
State the 4 steps involved in haemostatic plug formation from the time of injury.
Vessel constriction
Formation of an unstable platelet plug (platelet adhesion + platelet aggregation)
Stabilisation of plug with fibrin (blood coagulation)
Dissolution of clot + vessel repair (fibrinolysis)
What component found underneath the endothelium is involved in triggering the coagulation cascade?
Procoagulant subendothelial structures e.g. collagen
Tissue factor expressed on surface of cells underlying blood vessels but it is NOT normally expressed within the circulation
What process during maturation of megakaryocytes is important for formation of platelets?
Granulation
How many platelets are produced by 1 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
Growth factors
Fibrinogen
State the 2 ways in which platelets can bind to collagen. Name the receptors involved.
Via vWF to collagen (via GpIb receptor)
Bind directly to the collagen (via GpIa receptor)
What happens following the passive adhesion of platelets and engagement of receptors?
Receptors signal inside the cell to release ADP from storage granules + to synthesise thromboxane
These bind to receptors on the surface of platelets + activate them
Once activated, GpIIb/IIIa receptors become available, which can bind to fibrinogen + allows platelets to aggregate
Which receptors on the platelets become available following activation of the platelets and what do they bind to?
GlpIIb/IIIa
Bind to fibrinogen
What else can activate platelets?
Thrombin
What is the most important test for monitoring platelets and their function?
Platelet count
What is a common cause of spontaneous bleeding?
AI thrombocytopenia (AI antibodies clear platelets from the circulation) Results in purpura, multiple bruises + ecchymoses
What is the normal range for platelet count?
150-400 x 10^9/L
Why do you get thrombocytopenia in leukaemia?
Leukaemic cells populate the BM which crowds out megakaryocytes so platelets aren’t produced in sufficient numbers
What is the bleeding time test used to observe?
Checks the platelet-vessel wall interaction
Isn’t used any more
Describe the platelet aggregation test.
Platelets stimulated with ADP/thromboxane/collagen to study their function
Ued to diagnose platelet disease e.g. von Willebrand disease
Where is von Willebrand factor produced?
Endothelial cells + a little by megakaryocytes
What factors do megakaryocytes produce?
Factor V
Von Willebrand Factor
Tissue factor activates the clotting cascade by converting 9 to 9a and by converting 10 to 10a. What difference does this make?
9 to 9a: slower but produces more thrombin
10 to 10a: faster
State 2 accelerating factors. What are they activated by?
Factor VIII
Factor V
Activated by trace amounts of thrombin
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, + factor VIIIa bring the two close together 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 factors 2, 7, 9 + 10 and what is the significance of this common feature?
Have a cluster of glutamic acid
-recognised by liver enzyme + undergoes post-translational modification in the presence of vitamin K to Gamma-carboxyglutamic acid (Gla)
Once this extra carboxyl group is added, calcium can facilitate the binding of Gla to the activated platelet membrane phospholipid
How does warfarin actually inhibit the post-translational modification of factorsn 2, 7, 9 + 10?
Warfarin inhibits vitamin K epoxide reductase thus inhibiting the gamma carboxylation of factors 2, 7, 9 + 10
What is antithrombin? Which factors are inhibited by anti-thrombin?
Serine Protease Inhibitor (SERPIN)
2, 9, 10, 11
What effect does heparin have on anti-thrombin?
Heparin potentiates the action of AT
In what situation is heparin used?
Heparin is used for immediate anticoagulation in venous thrombosis + pulmonary embolism
Describe how anti-thrombin inhibits the clotting factors.
AT has a reactive loop that irreversibly inhibits the active site on the clotting factors
So AT acts as a scavenger in stopping inappropriate action of clotting factors
State three laboratory tests for blood coagulation.
Activated Partial Thromboplastin Time (APTT)
Prothrombin Time (PT)
Thrombin Clotting Time (TCT)
What do each of these laboratory tests represent?
APTT – abnormalities in INTRINSIC + COMMON pathways (coagulation is triggered by activation of factor 12)
PT – abnormalities in the EXTRINSIC + COMMON pathways (tissue factor is added to trigger the extrinsic pathway)
TCT – abnormality in the fibrinogen -> fibrin conversion (not important any more)
What are the main uses of these laboratory tests?
APTT and PT are used together for screening causes of bleeding disorders
APTT is used to monitor heparin therapy for thrombosis
PT is used to monitor warfarin treatment
What two proteins assemble on the surface of a clot to allow fibrinolysis to take place? Where are these proteins made?
Plasminogen (a plasma protein)
Tissue Plasminogen Activator (tPA) (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)
Elevated in DIC
What factor is used in therapeutic thrombolysis of myocardial infarction?
tPA “clot buster”
What is haemostasis? What is the purpose of haemostasis)
Cellular + biochemical process that enables both specific + regulated cessation of bleeding in response to vascular insult
Prevents blood loss from intact + injured vessels, enables tissue repair
What 2 things must be balanced for normal haemostasis?
Bleeding
Thrombosis
What can tip the balance towards bleeding?
Reduced platelet function + coagulant factors
Increased anticoagulant proteins + fibrinolytic factors
What can tip the balance towards Thrombosis?
Increased platelets + coagulant factors
Reduced anticoagulant proteins + fibrinolytic factors
Why does each step of haemstatic plug formation occur?
Vessel constriction: limits blood flow
Unstable platelet plug formation: limits blood loss + provides surface for coagulation
Fibrin stabilisation: Stops blood loss
Dissolution of clot: Restores vessel integrity
List 3 characteristics of platelets
Small (2-4um)
Anuclear
10 day lifespan
How does the phospholipid membrane of platelets change once activated?
Negatively charged phospholipids get exposed onto surface
Makes surface highly attractive to clotting factors
Why is the platelet cytoskeleton important for its function?
Allows rapid conversion from passive cell to interactive cell
Allows shape change from disc like to activated egg like structure
List 5 processes that platelets are involved in
Haemostasis + Thrombosis Cancer Atherosclerosis Immune response to infection Inflammatory response
How do clotting factors circulate?
As inactive precursors
Either serine protease zymogens or cofactors
Activated by specific proteolysis
Describe the mechanism of action for serine protease domain-containing proteins
Once activated, catalyse proteolysis of target substrate
Cleave substrates after specific Arg (+Lys) residues
What is the primary initiator of coagulation? Where is it located?
Tissue factor
Extravascular sites
Higher expression in certain areas e.g. brain provides further haemostat protection
What 5 features do factor VII, IX, X + PC share?
A homologous modular structure
Gla domain (ability to bind to phospholipid surface)
EGF domain involved in protein-protein interactions
All circulate in plasma in zymogen form
Activated by proteolysis
What produces FXa much more efficiently than the TF-FVIIa complex?
FVIIIa + FIX
What complex converts prothrombin to thrombin?
FVa + FXa
What does increased levels of thrombin allow?
Cleavage of fibrinogen to form fibrin fibres
Name the diseases resulting from deficiency of FVIII + FIX. Who is primary effected by these diseases?
FVIII: Haemophilia A
FIX: Haemophilia B
X-linked so primarily effects males
What are the 3 mediators of coagulation?
TFPI: Regulates initiation phase of coagulation
Protein C: Regulates FVa + FVIIIa
Antithrombin: Inhibits thrombin + FXa
How does TFPI regulate coagulation?
2nd Kunitz domain of TFPI inactivates FXa
Dampens pro-coagulant response to small injuries
How does Protein C regulate coagulation ?
Cleaves FVa + FVIIIa at various locations causing these cofactors to fall apart
What is FV Leiden disease?
Mutation of arginine 506 residue, which would have been a site of cleavage for Protein C
Thus FVa can’t be inactivated as efficiently
Describe activation of protein C
Thrombin binds to Thrombomodulin on healthy endothelial cells
TM converts thrombin into an anticoagulant enzyme which can activate Protein C (Removes activation peptide) to APC
APC + cofactor protein S ring fence CC
What are D-Dimer tests?
D dimer is a fibrin degradation product
Following coagulation, increased D-dimer in circulation
Indicates amount of clotting in recent past
What are commonly used to treat arterial and venous thrombosis?
Arterial: Anti-platelet agents
Venous: Anticoagulant drugs
