Platelet Aggregation
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Transcript Platelet Aggregation
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)
PF4
PDGF
Fibrinogen, Factors V & VIII
vWF
Plasminogen
a1-antiplasmin
HMWK
Fibronectin
Inhibit heparin; vessel repair
Inhibit heparin
Vessel repair
Fibrin formation
PLT Adhesion
Precursor of plasmin (fibrinolysis)
Plasmin inhibitor
Contact activation: intrinsic coagulation path
Promotes PLT spreading
Dense
ADP/ATP
Calcium
Serotonin
Lysosomes
Proteolytic, hydrolytic enzymes
Primary hemostasis, Secondary hemostasis
PLT agonist
Regulates PLT activation
Promotes vasoconstriction
Digest vessel wall matrix and debris
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
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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.
ReoPro
inhibits GP IIb/IIIa
(Pronounce: ab-SIKS-ih-mab)•