Platelet function tests

1. PLATELET FUNCTION TESTS
Presenter : DR. G Santhi priya
Moderator : DR. Divyasree B N
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2. CONTENTS
• Introduction
• Platelet formation
• Platelet structure
• Platelet functions
• Platelet function tests
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3. INTRODUCTION
• Platelets are 2nd major component of the hemostatic
system
• There role is first established by Bizzozero in
1892
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4. PLATELET FORMATION
• From megakaryocytes in the bone marrow
• Endomitosis : involves nuclear replication without
cellular division
• To generate 2000 – 3000 platelets per magakaryocyte
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6. PLATELET STRUCTURE
• Platelets are small, anucleate cells
• Discoid in shape
• Diameter : 1.5-3.5 µm
• Normal count : 150 – 440 x 109/L
• Storage granules : α, dense, lysosomes
• Primary function : rapid formation of a vascular plug
following vessel injury
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7. Peripheral zone
Structural zone
Organelle zone
Membrane systems
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8. α Granules
Group 1- hemostatic proteins
• Fibrinogen
• Factor v
• Von willebrand factor
• Plasminogen
• Plasminogen activator
inhibitor(PAI-1)
• α2antiplasmin
Group II- non hemostatic proteins
• Platelet specific
 Β thromboglobulin
 Platelet factor 4
 Platelet derived growth factor
• Non platelet specific
 Albumin
 Thrombospondin
 Fibronectin
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9. PLATELET FUNCTIONS
• Surveillance of blood vessel continuity
• Formation of primary haemostatic plug
• Surface for coagulation factor to make secondary
haemostatic plug
• Aid in healing of injured tissue
1. Adhesion
2. Shape change
3. Aggregation
4. Secretion
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10. ADHESION
• The initiating event following vascular damage
• to exposed subendothelial matrix proteins
• The platelet glycoprotein (GP) receptors which
mediate adhesion are dependent on the rate of shear
• Intermediate to high shear (arterioles) -strictly
dependent on von Willebrand factor (vWF) and its
receptor, the GPIb–IX, V complex
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11. • lower rates of shear (venous circulation) and in the
static conditions (experimental purposes)
• Adhesion - subendothelial matrix proteins such as
collagen and fibrinogen
• Adhesion is strengthened –activation of platelet
surface integrins, which leads to an increase in
affinity for their adhesive ligands
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14. SHAPE CHANGE
• Upon activation, platelets become spherical and
extend pseudopodia
• This enables to attach to other platelets and to the
vessel wall
• Shape change is mediated by phosphorylation of
myosin light chains
• As a consequence of elevation in intracellular Ca2+
ions, which activate myosin light chain kinase
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15. • Inhibition of myosin light chain phosphatase
• As the platelet flattens and spreads, granules and
organelles are squeezed into the centre of the cell,
resulting in a characteristic fried egg appearance
• Granules are secreted from the centre of the cell
directly into the surface open canalicular system
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16. • Dense granules - adenine nucleotides ADP and ATP,
the amine 5-HT and Ca2+
• Rapid release of ADP - plays a major positive
feedback role in promoting platelet activation
• α - granules contain the adhesive proteins fibrinogen
and vWF
• α-Granule release of vWF is critical for normal
thrombus formation at intermediate and high rates of
flow
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17. AGGREGATION
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18. PLATELET FUNCTION TESTS
• ADHESION TESTS
 Retention in a glass-bead
column
 Baumgartner’s technique
 PFA-100
• AGGREGATION TESTS
 Turbidometric technique using
 ADP
 Collagen
 Ristocetin
 Adrenaline (epinephrine)
 Thrombin
 Arachidonic acid
 Endoperoxide analogues
 Calcium ionophore
• INVESTIGATION OF
GRANULE CONTENT AND
RELEASE
 Dense bodies
 Electron microscopy
 ADP and ATP content
(bioluminescence)
 Serotonin release
• GRANULES
 b-thromboglobulin
 Platelet factor 4
 VWF
 Fluorescence by flow
cytometry
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19. • PROSTAGLANDIN
PATHWAYS
 TXB2 radioimmunoassay
• PLATELET COAGULANT
ACTIVITY
 Prothrombin consumption
index
• FLOW CYTOMETRY
 Glycoprotein surface
expression
 Activation
 P selectin (CD62) surface
expression
 FIBRINOGEN BINDING
 Annexin binding (to
phosphatidyl serine)
 Conformational changes in
Gp IIbIIIa
 Platelet granule
fluorescence
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20. PFA-100 [Platelet Function Analyser]
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21. • New generation of rapid whole-blood analyzers
• Designed to screen for platelet function defect
• Whole blood is passed through a hole in a membrane
coated with collagen and either ADP or epinephrine
• Combined influence of high shear rates and VWF,
platelets encountering this membrane spontaneously
adhere to collagen
• And aggregate because of stimulation by ADP or
epinephrine
• Measures closure time (CT), the amount of time
required for a platelet plug to occlude the aperture
and stop flow
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22. • CT is prolonged in - severe congenital and acquired
platelet dysfunction
• Defects in platelet glycoprotein receptors GPIIb/IIIa
(Glanzmann thrombasthenia) and GPIb (Bernard-
Soulier syndrome)
• Monitoring of patients taking aspirin – aspirin
resistance
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23. • Hematologic variables - influence PFA-100 CT
independent of platelet function are
 Severe thrombocytopenia
 Anemia
• CT seems to be sensitive to VWF levels, as indicated
by prolonged CT in patients who have von
Willebrand disease
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24. ADVANTAGES
• Whole blood test
• High shear
• Small blood volumes
• Simple
• Rapid
DISADVANTAGES
• Inflexible
• vWf dependent
• Insensitive to
clopidogrel
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25. OTHER ADHESION TESTS :
• Retention in a glass bead column
• Baumgartners technique
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26. Retention in a glass-bead column
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27. AGGREGATION TESTS
• Measures the ability of various agonists to platelets to
induce in vitro activation and platelet-to-platelet
activation
• Change in their shape from discoid to spiny spheres
• Associated with a transient increase in optical density
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28. STRONG AGONISTS
• Directly induce platelet
aggregation, TxA2
synthesis and platelet
granule secretion
• Eg : Collagen
Thrombin
TxA2
WEAK AGONISTS
• Induce platelet
aggregation without
inducing secretion
• Eg : ADP
Epinephrine
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Mechanism
Platelets are non- thrombogenic in their resting
state
The expose receptor on their surface on
activation by agonists and becomes sticky
Activated platelets adhere on the surfaces of the
Sensor and aggregates form a insulation layer
The impedance between the electrodes rises
The increase of impedance is transformed into
aggregation units and plotted against time

30. Normal aggregation curve
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31. Platelet aggregation curves in von Willebrand disease
and Bernard-Soulier syndrome (absent aggregation with
ristocetin, normal aggregation with ADP, epinephrine,
and arachidonic acid)
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32. Platelet aggregation curves in storage pool defect
(absent second wave of aggregation with ADP and
epinephrine, absent or greatly diminished aggregation
with collagen, and normal ristocetin aggregation)
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33. Platelet aggregation curves in Glanzmann’s
thrombasthenia (absent aggregation with all agonists
except ristocetin)
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35. SECRETION ASSAYS
• Measured by 2 different ways
 Measuring ATP secretion from platelets
 14C-labelled serotonin
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36.  ATP secretion from platelets
• PRP or whole blood is incubated with firefly
luciferase and its substrate luciferin
• Addition of platelet agonists causes release of ATP, a
cofactor in a light-producing luciferin-luciferase
assay
• Resulting light can be measured with a photodetector
system
• Often performed simultaneously with a platelet
aggregation assay
 14C-labelled serotonin : measured by allowing
platelets to take up 14C-labelled serotonin
• Its release is then measured in response to agonists20/08/2018 SEMINAR

37. FLOW CYTOMETRY
• Method used to measure multiple properties of single
cells in suspension
• Using specific monoclonal antibodies, it can be used
to gauge the expression of proteins in these cells
• Antibodies are conjugated with fluorescent tags that
are activated as the cells pass in a single-file stream
through the flow cytometer
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38. • Light emitted from each fluorochrome can be
individually detected and its intensity measured for
each cell
• Diagnosis of inherited defects in platelet surface
glycoproteins
• Decreased expression or absence of GPIb - Bernard-
Soulier syndrome
• GPIIb/IIIa - Glanzmann thrombasthenia
• Multiple antibodies against different GPIIb/IIIa
epitopes - acquired Glanzmann thrombasthenia
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41. REFERENCES
• 1.Hoffbrand A V, Catovsky D, Tuddenham E G D, editors.
Postgraduate Hematology.5th ed. Massachusetts:Blackwell
Publishing Ltd; 2005.P.808-24.
• 2. Seegmiller A, Sarode R. Laboratory Evaluation of Platelet
Function. Hematol Oncol Clin N Am 2007;21:731–42.
• 3. Quinn M, Fitzgerald D, Platelet function, assesment,
diagnosis and treatment. New Jersy:Humana press;2005:P.202-
328.
• 4. Platelet Function Testing: Light Transmission Aggregometry
[LTA], 29-4-2012, Available from http://www.platelet-
research.org/3/aggregometry.htm.
• 5.Laffan M A, Manning R , Investigation of hemostasis, Bain
B.J, Bates I, Laffan M.A, Lewis S.M, Dacie and Lewis
Practical Haematology. 11th ed. London: 2012. P.424-39.
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42. Thank you!!!
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