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 doesnt 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 doesnt 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 Ca
2+
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 10
9
/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 10
9
/ 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