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Platelet Aggregation

Mrs. Ibtisam H. Alaswad


Mr. Mohammed A. Jaber
Lab Investigation of Primary Hemostasis

Platelets
Numbers
CBC
PLT count
PLT morphology
Function
Bleeding Time (BT)
Platelet Aggregation
Whole blood aggregation
Platelet-rich plasma aggregation
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Platelet Structure

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PLT Granules’ Content
Granule Function
Alpha
Thromboglobulin(β-TG) Inhibit heparin; vessel repair
PF4 Inhibit heparin
PDGF Vessel repair
Fibrinogen, Factors V & VIII Fibrin formation
vWF PLT Adhesion
Plasminogen Precursor of plasmin (fibrinolysis)
a1-antiplasmin Plasmin inhibitor
HMWK Contact activation: intrinsic coagulation path
Fibronectin Promotes PLT spreading
Dense
ADP/ATP PLT agonist
Calcium Regulates PLT activation
Serotonin Promotes vasoconstriction
Lysosomes
Proteolytic, hydrolytic enzymes Digest vessel wall matrix and debris

Primary hemostasis, Secondary hemostasis


Overview: Platelet Function

• PRIMARY HEMOSTASIS
• Form platelet plug:
damaged endothelia
• Nurture endothelia

• SECONDARY HEMOSTASIS
• Reaction surface for
coagulation

Graphic accessed at URL


http://www.medicine.mcgill.ca/physio/209A/Blood/blood6a.htm, 2007.
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
Platelet Activation

After PLT adhesion


A change in PLT shape
Generation of biologically active mediators
Degranulation
The specificity of PLT activation and signal
transduction is maintained by the presence of PLT
receptors that recognize the appropriate PLT
agonists.
Thrombin
ADP
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Platelet Plug Formation: Aggregation

Platelet-Platelet interaction
Mechanism components
ATP
Ionized calcium
Fibrinogen
PLT receptor GPIIb/IIIa
Initial aggregation – REVERSIBLE
Secondary aggregation – IRREVERSIBLE* =
white clot, a.k.a platelet plug formed.
Platelet Plug Formation: Secretion

Discharge of
granules’ contents
Markers of PLT
activation*
 PF4
 PDGF
 Thromboglobulin (β-TG)
Promote & Amplify PLT
activities
 Primary hemostasis
 Secondary hemostasis
Inherited Platelet Disorders- Qualitative

Qualitative disorders
 Adhesion
Bernard-Soulier syndrome ( GP Ib-IX )
Platelet-type (Pseudo-) von Willebrand disease
( GP Ib receptor) *
Collagen receptor deficiency (GP VI)
 Aggregation
Glanzmann thrombasthenia (Gp IIb-IIIa)
 Secretion
Dense (δ) granule defects (storage pool
deficiency)
α granule defects (gray platelet syndrome)
 Platelet procoagulant activity
Scott syndrome PF3
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Platelet Activation (signaling)

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Platelet Aggregometry
 Platelet aggregation is an essential part of the
investigation of any patient with a suspected platelet
dysfunction.
Principle
 We are 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. Electrical Impedance Method
3. luminescence technology (Platelet Lumiaggregometry)
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Aggregating Agents (agonist)

 Collagen*
 ADP*
 Epinephrine*
 Arachidonic acid*
 The antibiotic ristocetin*
 Thrombin
 Serotonin
 Snake venoms, antigen-antibody complexes, soluble
fibrin monomer complexes, and fibrin(ogen) degradation
products (FDPs).

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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.
luminescence technology (Platelet
Lumiaggregometry)

• The lumiaggregometer 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.
Photo-optical Aggregometry

 Patient Sample – 3.2% citrated


WB
 Test Sample – PLT-rich Plasma
 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 Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is
approximately 200 × 109/L, and platelets are maintained in suspension by a magnetic stir
bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.)

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Figure 2 – Five possible phases of PLT aggregation: 1) baseline, 2) agonist addition and
shape change, 3) primary wave, 4) secretion, and 5) secondary wave.

Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.


o Sample
o Platelet-Rich Plasma
(PRP)
o PRP is prepared and
adjusted, if
necessary, to a count
of 200-300 X 109/L
by mixing with PPP.
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Graphics accessed URL http://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpg, http://www.accumetrics.com/images/img_product_overview.jpg, & http://cmed-tech.com/graphics/platelet2.jpg, 2009.


PRP Aggregometry Agonist & Patterns

ADP (at appropriate concentration)


 Biphasic curve: 1o and 2o waves
(requires intact prostaglandin
pathway)
Note: if ADP is added at too low a
concentration or too high a
concentration, will not get
biphasic response
Epinephrine
 Biphasic curve; requires intact
prostaglandin pathway
Collagen
 Lag phase followed by 2o wave
only
Ristocetin
 A biphasic however, often only a
single broad wave
 Binds to vWF/GPIb/IX
complex and results in
agglutination
 Evaluates adhesion reaction
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PRP Aggregometry Agonist & Patterns

 Thrombin
 Biphasic curve. Irreversible aggregation only (does not
require cyclooxygenase)
 Arachidonic acid
 2o wave only; assesses cyclooxygenase pathway
 Serotonin
o A primary wave of aggregation with a maximum of 10% to
30% transmittance followed by disaggregation.

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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.
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Interpretation
 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" (Fig.)
 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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Interpretation

 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).

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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

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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

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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

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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.
 Reduced or absent aggregation as well as disaggregation curves
may be observed in patients taking medication containing aspirin.
Other medications or substances have also been identified as
inhibiting platelet function, such as ibuprofen, red wine, and a
variety of herbs. Patients should be questioned carefully about
possible ingestion of these substances before interpreting abnormal
aggregation results.

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Comment

 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 platelet-poor plasma.
 This has limited usefulness because absorbance is
dependent on the size and density of platelet
clumping and the number of platelets that
aggregate.

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Drugs and PLT Function
Aspirin
 Acetylsalicyclic acid
 Irreversibly inhibits
Cyclooxygenase
Clopidogrel
 Plavix
 irreversibly inhibits
P2Y12
Dipyridamole
 inhibits Thromboxane
synthase
Abciximab Brinkman WT, Terramani TT, Najibi S, Chaikof EL. Platelets: is aspirin sufficient or must we know how to pronounce abciximab? Semin Vasc Surg.

(Pronounce: ab-SIKS-ih-mab)•
 ReoPro
 inhibits GP IIb/IIIa