B6 - basics of haemostasis Flashcards
○ In the absence of injury
§ Intact endothelium prevents platelet adherence by production of Nitrous oxide and prostacyclin
§ Keeps blood flowing smoothly with laminar flow
Following vascular injury
there is reflex vasospasm of smooth muscle (transient)
§ Expose subendothelial matrix
○ Injury exposes important proteins in subendothelium that activate proteins that activate coagulation
§ VWF
§ Tissue factor
Circulating platelets bind to subendothelial matrix (at the site of vascular injury) through platelet surface receptor
(glycoproteins 1b/IX/V complex) via von-Willebrand factor (VWF)
surface receptor on platelets that binds to sub endothelial matrix
□ Glycoprotein 1B/IX/V complex
® Surface glycoprotein on the platelet allowing it to bind via von Willebrand factor
other interactions contributing to platelet adherence to sub endothelial matrix
GP1a/IIa, GPVI binding to collagen - less important than ib/IX/V complex
Activated platelets
change shape, release the contents of their granules (which contains substances such as ADP, TXA2, fibrinogen, VWF)
□ Leads to further platelet activation at the site through ADP
platelets are activated by
§ Platelets are activated by collagen, VWF, thrombin, ADP
fibrinogen receptor
Express glycoprotein IIb/IIIa in an active form on the platelet surface
Glycoprotein IIb/IIIa receptor
§ Express glycoprotein IIb/IIIa in an active form on the platelet surface (fibrinogen receptor)
□ Glycoprotein IIb/IIIa receptor is always on the platelet surface but only exists in an activated form when the platelet is activated
□ This activated form is the main binding process through which platelets bind fibrinogen
platelet shape change
□ In resting state, they are smooth disk shape
□ Become spikey cells when activated allowing for better cross binding
□ When clot matures they because flat and spread out
purpura
□ Immune thrombocytopaenia purpura
® Normal bone marrow production of platelets
® Platelets are rapidly consumed by antibody mediated mechanisms
® Surface bleeding, mucosal bleeding, bruising
- Clotting cascade (generation of fibrin clot)
○ Tissue factor is exposed in the subendothelial system
○ combines with factor VIIa (circulating factor in the blood) to form a small amount of FVIIa-TF complex
○ FVIIa-TF complex activates other clotting proteins leading to a small amount of thrombin production
○ Through feedback mechanisms large scale production of thrombin takes place on platelet surface
○ Thrombin converts fibrinogen to fibrin which forms the basis of the clot
○ Fibrin mesh at site of injury
initiation of clotting cascade
□ Initiation of clotting occurs when disruption of the endothelium exposes activated factor VII (VIIa) in blood to tissue factor on subendothelial cells (smooth muscle cells and fibroblasts)
□ FVIIa-TF complex activates other clotting proteins (esp. FX to FXa and FIX to FIXa)
□ Small amount of thrombin is formed
propagation of clotting
□ Thrombin (Factor IIa) activates platelets which release further coagulation proteins
□ Thrombin activates coagulation proteins required for it’s own production (feedback)
□ Large scale production of thrombin takes place on platelet surface
□ Thrombin catalyses fibrinogen to fibrin - forming structure of clot
□ FXIII because FXIIIa which allows for cross linking between fibrin stands
○ Clot stabilisation
§ Thrombin cleaves fibrinogen to fibrin
§ Fibrin strands provide the structural integrity to the thrombus
§ Factor XIII allows cross linking of fibrin monomers
§ Fibrinolysis is activated to localise the clot to site of injury and remove clot when healing has occurred
- Fibrinolysis (breakdown of the fibrin clot)
○ The process by which a clot is removed
○ Tissue plasminogen activator (tPA) activates plasminogen and converts it to active plasmin
○ Plasmin enzymatically attacks the fibrin molecule producing fibrin degradation products (FDP’s) that are cleared from the circulation by macrophages
summary of coagulation
○ Platelets bind to subendothelium collagen to initiate primary closure of the vessel wall defect
○ Tissue factor in subendothelium combines with FVII in blood to form the FVIIa-TF complex
○ FVIIa-TF complex activates other clotting proteins leading to thrombin production
○ Large scale production of thrombin takes place on the platelet surface
○ Thrombin converts fibrinogen to fibrin to form cross linked fibrin clot
○ Fibrinolysis is activated to localise the clot to the site of injury
biological amplification system
- Incredibly efficient: 1 mole XIa generates ~107 moles XIIa
- Clotting factors numbered I-XIII
- Binding of FVII to tissue factor (TF) initiates coagulation
§ Activates
□ FXI to XIa
□ FV to Va
□ FVIII to VIIIa
§ Massive but highly focussed burst of thrombin
□ Rapidly converts soluble fibrinogen to insoluble fibrin
□ Fibrin reinforces and stabilises the platelets ‘plug’
contact pathway/intrinsic pathways
plays a lesser role
eg. deficiencies of factor 12 cause no increases tendencies of bleeding
extrinsic/tissue pathway
main way of coagulation cascade activation in vivo
if PT is normal
but be a problem with APTT arm
If APTT is normal
must be a problem with factor 7
citrated blood
- citrate decalcifies the blood
- calcium is a cofactor for coagulation cascade to the blood stays liquid in the tube
- Centrifuge blood to take out platelets and cellular components
- Add activator and platelet substitute
- Add calcium
- Measure time to clot formation
Prothrombin Time (PT)
- Measures extrinsic and common pathways
○ Sensitive to abnormalities in factors VII, X, V, II and fibrinogen- Time for clot to form after addition of tissue factor (TF), phospholipid and calcium to decalcified platelet poor plasma
- Reference range usually ~13-15 seconds but will vary between labs based on exact technique used
- Basis for monitoring oral vitamin K antagonists (warfarin and warfarin like compounds)
- Relationship between PT and a factor deficiency is not linear
○ Prolongs exponentially at lower concentrations
○ For PT or APTT to be prolonged, factor should be at around 30%
○ Significant reduction required for prolongation - International Normalised Ratio
○ The INR is a ratio of the PT of a test compared to a normal sample. This ratio is corrected for the sensitivity of the tissue factor used in the PT assay
○ The INR was developed to monitor patients on oral anticoagulants (warfarin)
○ For other area e.g. assessing severity of coagulopathy in liver disease PT should be used
prothrombin time is sensitive to abnormalities in
Sensitive to abnormalities in factors VII, X, V, II and fibrinogen
prothrombin time measures
common and extrinsic pathways
what is added to decalcified platelet poor plasma
- Time for clot to form after addition of tissue factor (TF), phospholipid and calcium to decalcified platelet poor plasma
- Relationship between PT and a factor deficiency is not linear
○ Prolongs exponentially at lower concentrations
○ For PT or APTT to be prolonged, factor should be at around 30%
○ Significant reduction required for prolongation
- International Normalised Ratio
○ The INR is a ratio of the PT of a test compared to a normal sample. This ratio is corrected for the sensitivity of the tissue factor used in the PT assay
○ The INR was developed to monitor patients on oral anticoagulants (warfarin)
○ For other area e.g. assessing severity of coagulopathy in liver disease PT should be used
Activated Partial Thromboplastin Time
- Measures intrinsic and common pathways
- Platelet poor plasma + phospholipid + contact activator (e.g. Kaolin) + calcium
- Deficient factor <40% before APTT prolonged - requires significant deficiencies
- Normal range is longer than PT
- Reference range ~27~ 3.5s
APTT measures
intrinsic and common pathways
what is added to platelet poor plasma in APTT
Platelet poor plasma + phospholipid + contact activator (e.g. Kaolin) + calcium
is APTT or PT generally longer
APTT
Fibrinogen
- Reference range 2-4g/L
- Quantitative and qualitative defects
- Functional and immunological methods to measure
- Functional (Clauss assay)
○ Clot diluted plasma with very high concentration of thrombin
○ Use diluted plasma to remove effects of inhibitory substances e.g. heparin and FDPs
○ Measures conversion of fibrinogen to fibrin
○ High thrombin concentration so clotting time independent of thrombin concentration for wide range of fibrinogen levels - Immunological methods
○ Measure concentration not functional activity
- Functional (Clauss assay)
○ Clot diluted plasma with very high concentration of thrombin
○ Use diluted plasma to remove effects of inhibitory substances e.g. heparin and FDPs
○ Measures conversion of fibrinogen to fibrin
○ High thrombin concentration so clotting time independent of thrombin concentration for wide range of fibrinogen levels
if both PT and APTT are prolonged
deficiency in factor 2, 5, 10
reduced or abnormal fibrinogen
liver disease, fit K deficiency, heparin, warfarin
