Haemostasis Overview Flashcards
Definition of haemostasis
Mechanism which the body maintains blood in a fluid state within blood vessels
Upon injury the body can arrest bleeding with a blood clot
How the clot is removed after healing
In vivo it’s a balance between…
Not bleeding und not clotting
What is required for normal haemostasis?
Normal blood vessels (vasculature)
Number of functional platelets
Produce fibrin via the ‘coagulation cascade’
Normal fibrinolysis system
Levels of natural anticoagulants
Primary haemostasis
Occurs initially the formation of a platelet aggregate, Within 5 minutes of bleeding
Secondary haemostasis
Formation of stable fibrin clot within 10 minutes of bleeding
Primary haemostasis
Platelet adhesion
Platelet activation
Platelet aggregation
Definition of a platelet
Anucleated cell fragment derived from bone marrow MK’s
150-400 x 109/litre
7.7 -11.2 fl
Lifespan of 10 days 30% sequestered in spleen
They circulate as quiescent cells surveying integrity of vascularture
Undergo explosive activation upon vessel wall damage
Normal platelets are circles
A nuclear platelets are small red dots
What happens when a blood vessel is disrupted?
Collaged fibres and smooth muscle is exposed to blood. Platelets are attracted to this area, then attach to collages fibres by Von Willebrand factor (vWF). Platelets are activated = change shape, release granular contence = attracts further platelets
Blood can still flow through blood vessel
Surface for secondary hemostasis
Platelet adhesion
Exposior of collagen initiates binding of VWF which also binds to glycol protein 1b complex /IX/V
Locks platets to sight of injury = no blood clot in head
von Willebrand factor
Proteins synthesized by mega karyocytes (mks) and endothelial cells
Found in plasma, platelets, endothelial cells, subendothelium
Largest multimers = most effective in Haemostasis
260 kD
Undergoes conformational charge = can bind to Gp 1b
Primary haemostasis
Exposure of collagen fibres attract VWF and they bind. Then attracts platelets which bind to GPIb-IX receptor-complex
Release granular contents.
glycoproteins
vWF
fibrinogen
coagulation factors
ADP
serotonin
calcium
Secondary homeostasis
Blood forms a stable clot
What is a clot made from?
Mesh of fibrin and platelets which form a haemostatic plug at sight of injury in vessel wall
Coagulation
Process by which fibrin is produced
Fibrinolysis
Process by which fibrin is broken down
Coagulation factors - labile plasma proteins which are activated to become these proteins…
Serine proteases- hydrolyse peptide bonds - II, VII, IX, X
Cofactors- tissue factor, V, VIII
Transglutaminase- XIII
Coagulation Cascade
When 1 factor is activated, it acts on a factor further down the system = activation of that factor till thrombin is produced = converts fibrinogen to fibrin
Intrinsic pathway
Activated by, contact factors
Things which are exposed when blood vessels are disrupted
Extrinsic pathway
Damaged tissue release tissue factors = directly activates factor VII
= small amounts of thrombin produced
Cell based model of haemostasis
Initiation
Amplification
Propagation
Generates thrombin burst = haemostatic fibrin clot
Initiation phase
Tissue factor bearing cells
Binds to factor VII a = promotes activation of factor X to Xa in conjunction with cofactor V which converts prothrombin to thrombin
Factor IX is activated by tissue factor VII complex to produce small amounts of activated factor IX
Amplification
Thrombin produced activates further platelets, + releases factor VIII from its vWF complex. It’s bound to surface of platelets which combines with factor IX which was released in initial stage.
Propagation
Thrombin and activated platelets utilise factor IX to activate factor XI which acts of facto IIX = produces burst of thrombin
Clot formation
Once Fribrinogen has been converted to the fibrin monomer by the action of thrombin, these monomers polymerise with hydrogen bonding, but this fibrin polymer is still an unstable polymer, and it needs the action factor 13 to cross-link these fibrin polymers to cross linked fibrin to give an insoluble fibrin that will combine with a platelets to form a stable fibrin platelet clot
Fibrinogen - fibrin monomer - fibrin polymer - cross-linked fibrin
Fibrinopeptide release 2. Hydrogen bonding
3.Transamidase bonding
What is fibrinolysis?
Mechanism the fibrin clot is broken down into smaller fragments for disposal.
most important of these fibrin degradation products is the D-dimer
Diagnosis of DVT deep vein thrombosis
DIC (Disseminated intravascular coagulation)
Process of fibrinolysis
Plasminogen - plasminogen - fibrin - fibrin degradation products
Summary
Define coagulation and the process involved in clot formation,
The importance of coagulation factors
What are the stages/pathways involved in coagulation?
How do we best describe what happens in vivo?
Examine the role thrombin plays in secondary haemostasis.
Define fibrinolysis.
Laboratory tests
Samples must be suitable for parameter being measured
Test system be regulated
Quality control
Results compared to locally established normal ranges
Things required for abnormal haemostasis
History of abnormal haemostasis
Family history
Preventative screening
Monitoring of anticoagulant therapy
Abnormalities =
Vasculature
Platelets
Functional fibrin via coagulation cascade
Fibrinolysis
Functional natural anticoagulants
Vasculature
No specific tests
Imagining techniques
Markers of inflammation e.g. CRP, ESR + vWF
Platelets
Count number + size using automated blood cell analysers
Check morphology using blood film
Bleeding time
Platelet function analysers PFA
Platelet aggregation tests
Platelet nucleotides
Fibrin production
Prothrombin time PT
Activated partial thrombophlebitis time APTT
Thrombin time TT
Fibrinogen level Fib
Fibrinolysis
Clot lysis times
D-diner levels
Plasminogen levels
Alpha -2- antiplasmin levels
Natural anticoagulants
Function + absolute levels
Protein C, S
Antithrombin
Activated protein C resistance/factor V Leiden
PFA-100 platelet function analyser
Mimics primary haemostasis
Platelet aggregation
Centrifuge blood gently to obtain platelet rich plasma
Stirred in a cuvette at 37 oC between a light source and photocell
Addition of agonist = platelet aggregation = platelets clumps = transmission increases + absorbacne decreases
Addition of different agonists at a range of concentration allows detection of certain defects
Agonists bind to their own specific receptors resulting in activation. Shape change, granule release, aggregation
Fibrin formation
Measures secondary haemostasis
Routine test = coagulation screen (CS)
300-500 daily
Screening tests
Prothrombin time (PT)
Clotting pathway initiated with damaged tissue
How to make a reagent
Emulsify in a blender !
Wash with chloroform
Sieve !
Dilute in buffer
Standardise
Screening tests
100ul plasma (in sodium citrate, which binds calcium)
Warm to 37oC
200ul Tissue factor + Phospholipid (to replace platelets removed by centrifugation) + Calcium (removed above)
Time for clot formation
Establish local normal range
Screening tests PT abnormal in
inherited deficiencies of fibrinogen, prothrombin, FV, FVII, FX
Acquired deficiencies of above
Consumptive - bleeding- over activation- liver disease
Therapeutic - warfarin
Acquired inhibitors to factors
Screening tests international normalised ration INR
Used to standardise warfarin therapy around world
INR {patients Prothrombin time (seconds)/ control Prothrombin time (seconds)} isi
Isi= international sensitivity index
Screening tests
Activated Partial Thromboplastin Time (aPTT)
Demonstration of intrinsic pathway + common pathway
Activated using contact activation
Need source of phospholipid
Need calcium
Screening tests aPTT abnormal in
Inherited deficiencies
X Acquired deficiencies of above
Consumptive
Bleeding
Over activation (Disseminated Intravascular Coagulation, DIC)
Liver disease
Therapeutic – Heparin
Acquired Inhibitors to factors
Thrombin Time (TT)
Screening tests
Addition of diluted Bovine Thrombin to neat sample
Direct conversion of Fibrinogen Fibrin
Abnormal in
Patients with low fibrinogen
Patient with non-functioning fibrinogen
Patients with Inhibitors of Thrombin
Anticoagulant - Heparin
Fibrinogen estimation
Screening tests
Similar addition of Thrombin (concentrated) to diluted plasma (1:5, 1:10, 1:20)
Time taken to clot = Fibrinogen concentration in the sample
Reported from comparison of standard curve
Oxford range 1.5-4.0 g/l
Abnormal in Inherited fibrinogen deficiency
Acquired deficiency
Bleeding
DIC
Liver disease
D-dimer (DD)
Screening tests
Breakdown product of clot formation
Only found in cross-linked Fibrin breakdown
Marker of rate of clot formation/breakdown
Reported as ug/l FEU
Oxford <500 ug/l FEU
Summmary
What body does to prevent blood loss
Understand processes overall
Understand the difference between Primary and Secondary haemostasis
Understand which routine laboratory tests can be used to investigate Haemostasis
Understand test procedures
