Post on 21-Jul-2016
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Normal Haemostasis
Vascular Damage
Haemorrhage
Reduced Blood Loss
Platelet Adhesion
Vasoconstriction
Platelet Aggregation
Platelet Activation
Platelet Plug Formation Platelet Plug
Re-enforcement
Fibrin
Formation
Vascular Smooth
Muscle
Contraction
Coagulation
Activation
Blood Vessels
White cells tend to flow towards the centre and platelets closer to the
vessel wall
Under normal conditions the inner layer ‘intima’ is anti thrombotic but
when damaged the subendothelium is exposed and this is thrombogenic
Endothelin 1, a potent vasoconstrictor is released from damaged
endothelial cells
Blood flow contributes to effective
haemostasis
It is faster in the centre of the blood
vessel than at the edges near the
vessel wall
Vasoconstriction
The smooth muscle cells of the media contract and
reduce the diameter of the blood vessel
Reduces blood flow and therefore blood loss
Cells of the adventitia express tissue factor which
initiates reactions of the coagulation cascade
Coagulation - History Aristotle and Hippocrates hypothesized that blood clotting
was due to cooling
In the 1790s, John Hunter hypothesized that blood clotting was due to exposure of blood to air
1830s: fibrin and its hypothetical precursor (fibrinogen) were identified
In the late 1800s, fibrinogen was isolated and Alexander Schmidt discovered that the conversion of fibrinogen to fibrin was an enzymatic process
He named the responsible enzyme thrombin – with prothrombin being its hypothetical precursor
Based on these observations, Morawitz proposed the first model of coagulation in 1904
Early Model of Coagulation
Phase 1 thrombinase
calcium
Prothrombin Thrombin
Phase 2 thrombin
Fibrinogen Fibrin
Zahn had observed that bleeding was initially blocked by a ‘white thrombus’ and not by fibrin
Several investigators had observed a colourless cell in blood that was smaller than red cells or white cells
They deduced that platelets supply a factor required for coagulation
It was shown that the rate of clotting and the prothrombin consumption was low in platelet poor plasma and increased as the number of platelets increased
Therefore early observations established the importance of both platelets and plasma proteins in coagulation
Platelets form the initial haemostatic plug
Fibrin stabilizes the platelet plug
Bell & Alton in 1954 suggested that brain phospholipids be used in clotting assays as it was difficult to produce consistently satisfactory platelet suspensions
The use of phospholipids allowed the study of coagulation proteins to be carried out more easily and the results to be reproducible
More coagulation factors were discovered based on naturally occurring deficiencies and the information needed to be organised…..
Coagulation Cascade
FXIII Stabilises clot
Platelets
Cascade model – Intrinsic Pathway
Laboratory screening
test - APTT
FXIII (stabilises clot)
Cascade model – Extrinsic Pathway
Laboratory
screening test –
prothrombin time
FXIII (stabilises clot)
Cascade model
FXIII (stabilises clot)
Common Pathway
Samples for Coagulation
Always collected in sodium citrate which removes
calcium ions and prevents coagulation but does not
compromise any of the clotting factors
Samples taken early in venepuncture to avoid
activation
Samples are spun at 3000rpm for 15 mins
removes platelets from the plasma - platelet poor
plasma
keeps platelet intact - contents of platelets will
affect the clotting times
Activated Partial Thromboplastin Time
Coagulation time of platelet poor plasma after the
addition of an activator of the contact phase
(eg.ellagic acid, kaolin,silica), addition of
phospholipids as platelet substitute and addition of
calcium ions
Screens the intrinsic pathway of coagulation and will
be prolonged in deficiencies of
Kallikrein, High Molecular Weight Kininogen,
Factors XII,XI,IX,VIII,X,V,II and Fibrinogen
Sensitive to presence of Lupus inhibitors, heparin
and oral anticoagulants
Prothrombin Time
Coagulation time of a mixture of platelet poor citrated
plasma and brain extract of different animal origin
(thromboplastin) containing calcium ions
The complex formed by FVII and tissue factor in the
presence of the calcium ions activate FX
Checks the integrity of the extrinsic pathway
Prolonged in deficiencies of
FVII, FX, FV, FII and fibrinogen (FI)
Sensitive to oral anticoagulant
Prolonged clotting times
Can investigate prolonged clotting times using correction studies with
Normal plasma
Adsorbed plasma
Normal serum
Addition of these (50:50 mix) will enable the determination of
missing/deficient factors or the presence of an inhibitor. If the missing
factor is added there will be a correction in the clotting time
Normal plasma will contain all clotting factors at normal levels – is
used to detect inhibitors
Adsorbed plasma contains FI,FV,FVIII,FXI,FXII
Normal Serum contains FVII,FIX, FX,FXI, FXII
Correction Studies
If a Prothrombin Time or APTT are prolonged, mixing the patients plasma
with adsorbed plasma or normal serum can help us deduce the factor
deficiency
Example:
Prothrombin Time is prolonged, APTT is also prolonged
Therefore deficiency is in ‘common pathway’ ie FX, FV, FII or FI
(fibrinogen)
Prothrombin Time doesn’t correct with addition of adsorbed plasma
(FI,FV,FVIII,FXI,FXII)
Therefore, deficiency cannot be FI or FV and must be FX or FII
Prothrombin time corrects with addition of normal serum (FVII, FIX,
FX, FXI, FXII)
Therefore deficiency must be FX
Correction Studies
Mixing with normal plasma can show if there is a
factor deficiency or an inhibitor
Prolonged APTT
if corrects with normal plasma it indicates a
factor deficiency
if it doesn’t correct on addition of normal
plasma it indicates an inhibitor is present eg
Factor inhibitor or Lupus anticoagulant
Other routine coagulation tests
Fibrinogen level (Clauss) – plasma is diluted &
clotted with excess thrombin. The fibrinogen
concentration is inversely proportional to the clotting
time. Detects deficiencies of fibrinogen and any
alterations in the conversion of fibrinogen to fibrin
Thrombin time – thrombin is added to plasma to
convert fibrinogen to fibrin. Sensitive to presence of
heparin.
Vascular Damage
Haemorrhage
Reduced Blood Loss
Platelet Adhesion
Vasoconstriction
Platelet Aggregation
Platelet Activation
Platelet Plug Formation Platelet Plug
Re-enforcement
Fibrin
Formation
Vascular Smooth
Muscle
Contraction
Coagulation
Activation
Platelet Production
• Platelets are produced by fragmentation of the
cytoplasm of megakaryocytes
• Most text books state that this takes place in
the Bone marrow but some haematologists
argue that there is growing evidence in favour
of this process occurring within the
pulmonary microvasculature ie lungs
Megakaryocytes are very different from other blood cell precursors
They are polyploid and unique to mammals
Each megakaryocyte may produce about 3000 essentially similar cells (compared to 2 daughter cells in other lineages)
Platelet formation is far more complicated than the production of white cells
Megakaryocytes
Dense tubular system (platelet Ca2+ store)
Dense granules
a-granules
Open canalicular system (OCS) Mitochondrion
Microtubules
Actin filaments
Lysosomal-granules
Glycogen stores
Platelet structure and organelles
• very small: 1x3 mm (7 fl) • no nucleus
• disc-shaped • plentiful: 150-400 x 109/ml
Plasma membrane Glycocalyx
Biological Role of Platelets
Form haemostatic plug at sites of vascular injury
Implicated in occlusion of blood flow by
formation of a thrombus, triggered by alterations
in the vessel wall
Involved in tissue injury, inflammation and
wound healing by attracting and binding
leucocytes
Platelet
Adhesion / Aggregation
http://www.le.ac.uk/by/je14/integrin5.htm http://www.akh-wien.ac.at/biomed-research/htx/anatomy.htm
Platelets are activated by the chemicals released at the point of tissue
damage, they then adhere (von Willebrand Factor needed) to the collagen
in the damaged vessel wall and aggregate (fibrinogen needed) to stop
bleeding.
VWF
‘Bridging’ of glycoprotein IIb/IIIa receptors on platelets
via VWF & Fibrinogen molecules
Platelet Plug Formation
& Clot Retraction
Clots are formed from aggregated platelets
It is strengthened by a mesh of fibrin with white cells
and red cells also becoming part of the mesh
This ultimately results in a blood clot
The clot retracts
helping the platelet rich thrombi to withstand the high shear
forces caused by blood flow
to facilitate normal blood flow while the damaged blood
vessel heals
Retraction is facilitated by the platelets and FXIIIa
Laboratory Investigation of
Platelets • Quantitative Measurement
Platelets can be measured on all modern Blood
Count analysers
Normal count is 150-400 x 109 / L Low platelet counts can lead to bruising and bleeding
in patients Platelets can be examined for numbers, shape & size
using microscopy
Platelets in Peripheral Blood
Platelet count can be estimated
from a blood film
This would have given a low
platelet count on the blood count
analyser-the platelet count is
probably normal
This phenomenon can be due to
the platelets being sensitive to
EDTA
Main causes of Thrombocytopenia
Drug induced - Recent or present drug ingestion
Acute idiopathic - Commonly after recent infection
Chronic idiopathic - ITP
Acute leukaemia - often due to treatment
Aplastic anaemia - sometimes due to drug ingestion or exposure to toxic agents eg. Chemicals, radiation
SLE - not always present in onset but will develop in ongoing illness
Hypersplenism - symptom of disorders causing hypersplenism
Neoplastic bone marrow infestation - myelofibrosis & malignant lymphomas
HIT- caused by IV heparin activating platelet factor 4.
DIC - increased consumption of platelets
Qualitative Measurements Adhesion- Bleeding Time
A blade is used to
make a
standardised
incision on the
patients forearm
A pressure cuff is
put on the patient
at 40mmHg to
standardise the
pressure of the
blood flow
Blood is mopped
up taking care not
to disturb the plt
plug. The time for
the bleeding to
stop is noted-
Bleeding Time
The bleeding time is operator dependent, poorly reproducible and neither
objective or sensitive
Platelet Aggregation Studies
Performed either on whole blood or more often on
Platelet Rich Plasma (PRP)
A series of agonists (platelet activators ) are added to the PRP and a dynamic measurement of platelet aggregation is recorded
The percentage aggregation is calculated
ATP release can be measured simultaneously using a luminescent marker
Platelet Aggregometry
Naturally occurring anticoagulants
Clotting cannot carry on indefinitely and needs to be
slowly stopped
Naturally occuring anticoagulants will slow down the
clotting process
Naturally occuring anticoagualnts are
Protein C- inactivates FVa and FVIIIa
Protein S – co-factor to Protein C
Antithrombin – complexes with FXa
Mode of action of naturally occurring anticoagulants
Thrombin (T) acts with thrombomodulin to activate Protein C (PC).
The activated Protein C with Protein (PS) as a cofactor inactivates Factor Va and
Factor VIIIa
Protein C Anticoagulant
Pathway
Heparin
Mechanism of Antithrombin
Antithrombin very slowly binds to Factor Xa and reduces the amount of Fxa in the circulatory system which reduces the rate
of fibrin clot formation
On addition of heparin to the system a heparin- antithrombin complex is produced and the action of this complex is much
quicker than antithrombin on its own
This is the basis of Heparin anticoagulation
Fibrinolysis
A normal response to vascular injury
Clots need to be broken down and this process is
called fibrinolysis
Plasminogen activators convert plasminogen to
plasmin which cleaves the peptide bonds in fibrin
and fibrinogen producing fibrinogen degradation
products (FDPs)
Fibrinolytic Pathway
Fibrinogen degradation products
(FDPs)
Plasmin degrades fibrin FDPs: X&Y,
D&E
D-dimer is produced by the factor XIIIa-mediated
crosslinking of fibrin
Can be detected by immunological based
assays
Raised plasma D-dimer levels indicate
thrombolysis
The D-Dimer level (raised) is used in
emergency situations as an indication that
someone has had a DVT
D-Dimers
D-dimer test is more specific for fibrinolysis than FDPs
requires the action of thrombin (to activate factor XIII) to produce crosslinked fibrin
cleavage of this fibrin by
plasmin FDP assays cannot distinguish
between plasmin action on fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis), therefore FDPs can be raised when there is no clot present (and plasmin is just cleaving fibrinogen).
Clearview Simplify D
Dimer (Inverness Medical)
Glossary
PT – Prothrombin Time
APTT – Activated Partial Thromboplastin Time
PK – Prekallikrein
HMK – High Molecular Weight Kininogen
vWF – von Willebrand factor
ITP – Idiopathic thrombocytopenia purpura
SLE – systemic lupus erythrematosis
HIT – heparin induced thrombocytopenia
DIC – disseminated intravascular coagulation
DVT – Deep Vein Thrombosis