Basics of haemostasis: How to clot, but not too much Flashcards
What is primary haemostasis?
Primary clot formation which is the role of platelets
What is secondary haemostasis?
Formation of fibrin clot
What is blood homeostasis?
Stop significant blood loss at site of injury but switching off clotting so that blood doesn’t clot everywhere
What are the structures and systems involved in haemostasis?
Vascular endothelium
Platelets
Procoagulant and anticoagulant proteins
Fibrinolytic system
What 2 processes are in balance in a healthy person?
Clot formation and clot lysis
What are bleeding problems a result off?
Too much lysis too little clotting
What are clotting problems a result of?
Too much clotting too little lysis
What happens to blood vessels in response to injury?
Vasoconstriction
eg. pain receptors can cause reflex vasoconstriction
How does a blood clot form?
1- Initiation:
Following vascular injury there is reflex vasospasm of smooth muscle for a short time. Vascular endothelium stops producing NO and prostacyclin.
Injury exposes important subendothelial proteins like VWF and tissue factor.
2- Platelet adhesion and activation:
Circulating platelets bind to subendothelial matrix through platelet surface receptor (glycoprotein 1b/IX/V complex), via von-Willebrand factor.
Other interactions such as GP1a/IIa binding to collagen are important as well.
Platelets are activated by collagen, VWF, thrombin, and ADP.
Activated platelets change shape and release their granules. Activated platelets also express a new receptor which is a fibrinogen receptor allowing more agreggation of platelets.
ADP and TXA2 also recruit other platelets to help form platelet plug
How does intact endothelium prevent platelet adherence?
By producing NO and prostacyclin
What are the most important factors to platelet activation?
Platelets are activated by collagen, VWF, thrombin, and ADP
What happens to platelets when they are activated?
Activated platelets change shape and release their granules. Activated platelets also express a new receptor which is a fibrinogen receptor allowing more aggregation of platelets.
What do circulating platelets bind to?
Subendothelial matrix
How do circulating platelets bind to subendothelial matrix?
Though platelet surface receptor
What is the platelet surface receptor that binds to subendothelial matrix called?
Glycoprotein 1b/IX/V complex
How does von-Willebrand factor (VWF) help with platelet aggregation?
Important for binding and aggregation.
It combines with GPIb, GPIIb/IIIa and the collagen sunbendothelial microfibrils creating a link at many sites allowing the platelet to start spreading.
What receptor on the platelet binds directly to collagen?
GP1a/IIa
What do the granules of activated platelets contain?
ADP
Thromboxane 2
Fibrinogen
Von Willebrand Factor
What receptor do activated platelets express? What does it do?
Glycoprotein IIb/IIIa
What do ADP and TXA2 in granules do?
Recruit other platelets to help with platelet plug formation
What do ADP and TXA2 in granules do?
Recruit other platelets to help with platelet plug formation
What must happen before GPIIIa can be exposed to VWF?
GPIa must adhere to the collagen that the VWF is going to bind to
What must happen before GPIIb can be exposed to VWF?
GPIb must bind to VWF
What happens after GPIIb/IIIa binding to VWF?
Spreading of platelet
What are the 4 processes in platelet response?
Adhesion
Aggregation
Secretion
Pro-coagulant activity
What is the overall function of platelets?
Initial closure of vessel wall defect
Vasoactive substances result in platelet granule release (containing coagulation proteins and propagating messengers) which has a propagating effect activating clotting factors to bind to platelet surface and increase thrombin generation
What is purpura?
Autoimmune destruction of platelets
What happens during secondary haemostasis?
A fibrin clot is formed
What are the key steps to forming a fibrin clot?
Tissue factor combines with favtor VIIa to form FVIIa-TF complex.
The complex activates the other proteins that produce thrombin on top of the platelet surface
Thrombin then converts fibrinogen to fibrin.
What are the steps of coagulation?
Factor VIIa - TF complex activate small amounts of factor 9 to 9a and factor 10 to 10a
Factor 10a cleaves prothrombin to thrombin
Thrombin activates platelets which release further coagulation proteins.
Thrombin also activates proteins required to produce more thrombin
Thrombin is produced in large scale on the platelet surface.
At multiple locations there is positive feedback from thrombin (factor 11, factor 8, factor 5)
Factor 13 is not part of coagulation cascade and instead helps to cross-link fibrin
Simplification of thrombin formation so far
F7a-TF activates F9+F10 activates thrombin (aka F2) which feeds forward to factor 11, 8, and 5 (positive feedback)
What is another name for thrombin?
Factor II
What does factor 13 do?
Not part of coagulation cascade and instead helps to cross-link fibrin
What is thrombin important for?
Activation of coagulation factors
Activation of platelets
Activation of anticoagulant factors
Determining the balance of fibrinolysis
What is fibrinolysis?
Process by which clot is removed
What does tissue plasminogen activator do?
activates plasminogen and converts it to plasmin
What does plasmin do?
Enzymatically attacks fibrin molecule producing fibrin degradation products that are cleared from circulation by macrophages
What is produced after degradation by plasmin?
FDPs which form D-dimers (due to factor 13 cross-linkages)
Summary of haemostasis after disruption of vascular endothelium:
Platelets bind to subendothelium collagen to initiate primary closure of the defect in vessel wall
Tissue Factor in subendothelium combines with factor 7 (FVII) in blood to form the FVII-aTF complex which activates other clotting proteins leading to thrombin production.
Large scale production of thrombin takes place on platelet surface which converts fibrinogen to fibrin and then fibrinolysis is activated to localise clot to site of injury.
What is the purpose of thrombin?
To convert fibrinogen to fibrin
To activate factor 13 to form cross linkages
What pathways can trigger coagulation?
Intrinsic and Extrinsic
Where does the coagulation cascade happen?
On platelet surface (PL of platelet surface)
What does the coagulation cascade bring?
10, 8, 9, calcium, and phospholipid.
How is coagulation tested in the lab?
factor 12 is activated to test intrinsic pathway and is called APTT
Tissue factor is activated to test extrinsic pathway this test is called PT.
What is the APTT test?
Activated Partial Thromboplastin Time which uses Kallikroin to start the intrinsic pathway.
Where do the extrinsic and intrinsic pathway of coagulation meet?
At factor 10 activation and factor 2 activation
How are PT and APTT tests conducted?
Citrate added to blood to remove calcium.
Blood is centrifuged to create platelet poor plasma and 3 hings are added:
Activator
Platelet substitute
and calcium (time to clot formation is measured when variables are known)
What must be considered with screening tests?
Clinical history;
Personal bleeding history
Drugs
Family history
What are the limitation of screening tests?
They are in vitro which isn’t always reliable.
What are some important lab screening tests?
Platelet count
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
What are the contents of plasma?
Water
Albumin
Clotting proteins
Antibodies
Salts
Vitamins
Urea
Hormones
What is Prothrombin Time?
Measures time for clot to form after addition of Tissue Factor, phospholipid and calcium to decalcified, platelet poor plasma. It’s a good test for abnormalities in factors VII, X, V, II, and fibrinogen
What is the reference range for prothrombin time?
13 - 15 seconds
Which coagulation pathway does prothrombin time measure?
Extrinsic pathway
How does a factor deficiency affect PT?
Prolongs time exponentially at lower factor.concentrations
A change often must be more than 30% before it is noticeable.
What is PT used for?
Monitoring Vitamin K (and other stuff that we will learn later)
What is the International Normalised Ratio?
Ratio between PT of a test compared to a normal sample. This is corrected for the sensitivity of tissue factor used.
Why was the INR ratio developed?
To monitor patients on oral anticoagulants (warfarin)
What is warfarin?
An anticoagulant
What is in an APTT test?
Platelet poor plasma
Phospholipids
Contact activator (Kaolin)
Calcium
What does APTT measure?
Intrinsic and common pathways
What is the reference range of time for Activated Partial Thromboplastin TIme?
27 - 35s
What must deficient factor be to be detected?
<40% before the APTT is prolonged
What are the types of defects that fibrinogen can present?
Quantitative or qualitative defects
What is the reference range of fibrinogen in blood?
2 - 4g/L
How is the Clauss assay on fibrinogen conducted?
Clot diluted plasma with very high concentration of thrombin.
Use diluted plasma to remove effects of inhibitory substances such as warfarin
High thrombin concentration so clotting time independent of thrombin concentration for wide range of fibrinogen levels
What do immunological assays measure?
Concentration of fibrinogen not activity
What else is fibrinogen called?
fibrin
What do fibrin tests reveal?
Whether the cause of a problem is too little fibrinogen or not very functinal fibrinogen
