Transcript Platelets Aggregation

General Approach in
Investigation of Haemostasis
Platelets aggregation
Studying Platelets Disorders
 Numbers

CBC
 PLT count
 PLT morphology
 Function
Bleeding Time (BT)
 Platelet Aggregation
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Whole blood aggregation
Platelet-rich plasma aggregation
Platelets contribute to Hemostasis in two
main ways:
 Primary haemostatic plug :
 Adhesion
 Aggregation
 Secretion
 Secondary Haemostatic plug:
 Procoagulant activities are generated
Platelet Plug Formation: Adhesion
 Platelets bind to exposed adhesive subendothelial
connective tissue
 Collagen
 vWF
 Fibronectin
 Mechanism components
 vWF: links PLT to endothelial binding site
 PLT receptor GPIb
 Collagen fibers
 Actin contracts & pseudopods form
 REVERSIBLE
 Facilitates activation
Platelets Aggregation
 Platelet-Platelet interaction
 Mechanism components
 ATP
 Ionized calcium
 Fibrinogen
 PLT receptor GPIIb/IIIa
 Initial aggregation – REVERSIBLE
 Secondary aggregation – IRREVERSIBLE* = white clot, also
know as platelet plug formed.
* The transformation of irreversible aggregated platelets into a mass of degenerative platelet material
without membranes is termed viscous metamorphosis. (by platelet lysosomes)
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Why Platelet aggregation testing
Evaluation of suspected hereditary and acquired disorders of platelet
function.
 Platelets normally contain three major types of granules .
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The alpha granules contain fibrinogen, PF4, factor V, von Willebrand factor….
 The dense granules contain ADP or ATP, calcium and serotonin.
 lambda granules – similar to lysosomes and contain several hydrolytic enzymes.
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 Hereditary platelet function disorders includes
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Rare defects of adhesion (Bernard Soulier syndrome)
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Rare defects of aggregation (Glanzmann thrombasthenia)
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More common defects of secretion (alpha or dense granule deficiency, aspirin-like
defects, or other primary secretion defects).
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Acquired platelet function disorders are more common than the
hereditary disorders and include drug-induced platelet dysfunction
(including aspirin, NSAID’s, clopidogrel, antibiotics, various cardiovascular and
psychotropic drugs), uremia, and myeloproliferative disorders.
Principle
 Using Aggregating agents to induce platelet aggregation
or cause platelets to release endogenous ADP, or both.
 Platelet aggregation is studied by means of a platelet
aggregometer, Used Principle:
1. Photo-optical Method
2. luminescence technology (Platelet Lumiaggregometry)
3. Electrical Impedance Method
AGGREGATING AGENTS
Electrical Impedance Method
 These types of analyzers may use citrated whole blood, as
the test sample.
 As platelets aggregate, the coat an electrode, impeding the
electrical current through the analyzer.
http://www.platelet-research.org/3/pfa.htm
Luminescence technology (Platelet Lumi aggregometry)
 The lumi-aggregometer may be used to simultaneously
measure platelet aggregation and secretion. The instrument
records both aggregation and secretion of dense-granule ATP.
 The ATP is measured by its reaction with firefly luciferin to
give chemiluminescence. The resulting light emission is
detected, amplified, and recorded by the instrument.
 Performed by using whole blood or PRP.
 This modification of aggregation is particularly sensitive to
ATP release, and is as sensitive measure of platelet activation.
[A] + [B] → [AB◊] → [Products] + light
Photo-optical Aggregometer (PLT Aggregometry Using PRP)
 The Platelet-rich plasma, which is turbid in appearance, is placed in a
cuvettes, warmed to 37°C in the heating block of the instrument,
and stirred via a small magnetic bar.
 Baseline light transmittance through the platelet-rich plasma is
recorded. The addition of an aggregating agent causes the formation
of larger platelet aggregates with a corresponding increase in light
transmittance, because of a clearing in the platelet-rich plasma. The
change in light transmittance is converted to electronic signals and
recorded as a tracing by the chart recorder.
 Patient Sample – 3.2% citrated WB
 Approximately 20 ml of blood is needed for a full aggregation study.
 Test Sample – PRP ( Platelets count fall (200–600 × 109/L)
 Principle – photometry: optical density of PRP warmed to 37° C is
determined before and after the addition of various aggregating agents
 Issues
Sample quality is critical
Fibrinogen levels are important
Agonists must be prepared fresh daily
Thrombocytopenia makes result interpretation difficult
Complete patient history is essential
http://www.platelet-research.org/3/aggregometry.htm
Procedure:
1. Switch on aggregometer to warm to 37°C. Select AGGR TEST
2. Select the type of agonist (ADP, AA, RISTO, THR, COLL, ADR)
3. Place 0.27 ml PRP in a test cup and put in the incubation channels.
Put beads into the cups, wait 1 minute to warm.
4. Transfer the cups to testing channels.
5. Press PPP, it will appear the status <PPP>
6. Add 0.03 ml of agonist to bottom of cuvettes and press start key then
monitor optical density changes for three minutes.
7. Repeat this procedure for each agonist
8. Curve Display.
* ADP should placed in ICE.
 ADP (at appropriate concentration)
Biphasic curve: 1o and 2o waves
(requires intact prostaglandin pathway)
Note: if ADP is added at too low or too
high concentration, it will not get
biphasic response
 Epinephrine
Biphasic curve; requires intact
prostaglandin pathway
 Collagen
Lag phase followed by 2o wave only
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Ristocetin
A biphasic however, often only a single
broad wave Binds to vWF/GPIb/IX
complex and results in agglutination Evaluates
adhesion reaction
 Thrombin
Biphasic curve. Irreversible aggregation only (does
not require cyclooxygenase)
 Arachidonic acid
2o wave only; assesses cyclooxygenase pathway
 Serotonin
A primary wave of aggregation with a maximum of
10% to 30% transmittance followed by
disaggregation.
Interpretation
 Platelet aggregation occurs as a two-step process, known as
primary and secondary waves of aggregation.
 The primary wave of aggregation is observed when platelets
adhere to one another in the presence of an external agent
(agonist) such as ADP, epinephrine, or ristocetin.
 Secondary aggregation is characterized as the aggregation that
occurs after the platelets have been stimulated to secrete the
substances contained in their organelles.
 It should be noted that some agonists will stimulate primary
aggregation and some will stimulate secondary aggregation.
Others will stimulate both primary and secondary
aggregation, yielding a "biphasic" aggregation curve.
 In addition, different concentrations of the same agonist can
produce varying patterns of primary and secondary
aggregation. For example,
 Low concentrations of ADP induce biphasic aggregation (i.e., both a primary and a
secondary wave of aggregation);
 Very low concentrations of ADP (l.5 ug/ml. final concentration) induce a primary
wave followed by disaggregation;
 And high concentrations of ADP (10 ug/ml, final concentration) induce a single,
broad wave of aggregation“
A biphasic aggregation response to ADP will not be seen in
patients with platelet release disorders.
 Patients with Glanzrnann's thrombasthenia show incomplete
aggregation with ADP regardless of the final concentration.
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 In patients with severe von Willebrand disease, aggregation
to ristocetin is characteristically absent. Decreased to
normal aggregation to ristocetin can be seen in patients with
mild von Willebrand disease. Correction of the abnormal
ristocetin aggregation curves can be seen by the addition of
normal, platelet-poor plasma to the patient's platelet-rich
plasma.
 Abnormal ristocetin-induced platelet aggregation may also
occur in patients with
1. Bernard-Soulier syndrome,
2. Platelet storage pool defects
3. Idiopathic thrombocytopenia purpura (ITP).
Glanzmann thrombasthenia
o Normal PLT count, but abnormal clot retraction
o Absence of secondary aggregation to ADP, epinephrine, collagen,
(thrombin)
o Normal response to ristocetin
Bernard-Soulier syndrome
o Platelet aggregation test
o Failure to aggregate in the presence of ristocetin
o Aggregation by other agonists (ADP, collagen, epinephrine): normal
o Response to low-dose thrombin: may be delayed
Platelet storage granule defects
o Dense (δ) granule defects ~ storage pool deficiency
o α granule defects ~ gray platelet syndrome
o Heterogeneous group of disorders
o Mild to moderate bleeding diathesis
o Abnormalities in platelet aggregation
Precautions Prior To Studying Platelet Aggregation
 Aspirin-containing compounds should be excluded for at least 10
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days prior to testing, as Aspirin interferes with the release reaction.
Ingestion of other drugs known to influence platelet function should
also be avoided for at least the time required for their elimination
from the circulation. These include certain antihistamines,
antibiotics, and anti-depressants. A check should be made of any
drugs being prescribed before performing platelet function testing.
Chylomicrons can interfere with the measurement of platelet
aggregation, studies should not be carried out shortly after a fatty
meal.
Many other “normal” dietary constituents, including alcohol, onions,
garlic, peppers, and ginger, may also inhibit platelet aggregation.
Chilling activates platelets, and so the blood is processed at 20°C –
25°C.
Comment
 In evaluating patients with suspected platelet disorders, the
aggregating agents most commonly used are ADP in varying
concentrations, collagen, epinephrine, and ristocetin.
 Aspirin, aspirin compounds, and anti-inflammatory drugs
inhibit the secondary wave of aggregation by inhibiting the
release reaction of the platelet.
 The intensity of platelet aggregation may be estimated by
recording the change in absorbance as a percentage of the
difference in absorbance between platelet-rich and plateletpoor plasma.
 This has limited usefulness because absorbance is dependent
on the size and density of platelet clumping and the number of
platelets that aggregate.
Drugs and PLT Function
 Aspirin:
Acetylsalicyclic acid  Irreversibly inhibits Cyclooxygenase
 Clopidogrel :
Plavix Irreversibly inhibits P2Y12
 Dipyridamole:
 inhibits Thromboxane synthase
 Abciximab
ReoPro  inhibits GP IIb/IIIa