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