Mrs. Ibtisam H. Alaswad Mr. Mohammed A. Jaber
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Transcript of Mrs. Ibtisam H. Alaswad Mr. Mohammed A. Jaber
Platelet AggregationPlatelet AggregationMrs. Ibtisam H. AlaswadMr. Mohammed A. Jaber
Lab Investigation of Primary HemostasisLab Investigation of Primary Hemostasis
PlateletsNumbers
CBCPLT countPLT morphology
FunctionBleeding Time (BT)Platelet Aggregation
Whole blood aggregation Platelet-rich plasma aggregation
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Platelet Structure
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PLT Granules’ Content GranuleFunction
AlphaAlphaThromboglobulin(β-TG)
PF4PDGF
Fibrinogen, Factors V & VIIIvWF
Plasminogen1-antiplasmin
HMWKFibronectin
Inhibit heparin; vessel repairInhibit heparinVessel repairFibrin formationPLT AdhesionPrecursor of plasmin (fibrinolysis)Plasmin inhibitorContact activation: intrinsic coagulation pathPromotes PLT spreading
DenseDenseADP/ATPCalcium
Serotonin
PLT agonistRegulates PLT activationPromotes vasoconstriction
LysosomesLysosomesProteolytic, hydrolytic enzymesDigest vessel wall matrix and debris
Primary hemostasis, Primary hemostasis, Secondary hemostasisSecondary hemostasis
Overview: Platelet Function
• PRIMARY HEMOSTASISPRIMARY HEMOSTASIS
• Form platelet plug:platelet plug: damaged endothelia
• Nurture endothelia
• SECONDARY HEMOSTASISSECONDARY HEMOSTASIS
• Reaction surface for coagulation
Graphic accessed at URL http://www.medicine.mcgill.ca/physio/209A/Blood/blood6a.htm, 2007.
Platelet PlugPlatelet Plug Formation :Adhesion Platelets bind to exposed adhesive subendothelial
connective tissue Collagen vWF Fibronectin
Mechanism components vWF: links PLT to endothelial binding sitevWF: links PLT to endothelial binding site PLT receptor GPIbPLT receptor GPIb Collagen fibers
Actin contracts & pseudopods form REVERSIBLE
Facilitates activation
Platelet ActivationPlatelet Activation
After PLT adhesionA change in PLT shapeGeneration of biologically active mediatorsDegranulation
The specificity of PLT activation and signal transduction is maintained by the presence of PLT receptors that recognize the appropriate PLT agonists.
ThrombinADP
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Platelet PlugPlatelet Plug Formation :Aggregation
Platelet-Platelet interactionMechanism components
ATPIonized calciumFibrinogen Fibrinogen PLT receptor GPIIb/IIIaPLT receptor GPIIb/IIIa
Initial aggregation – REVERSIBLESecondary aggregation – IRREVERSIBLE** = white clot, a.k.a platelet plug formed.
Platelet PlugPlatelet Plug Formation: Secretion
Discharge of granules’ contentsMarkers of PLT activation**
PF4PDGFThromboglobulin (β-TG)
Promote & Amplify PLT activities
Primary hemostasisSecondary hemostasis
Inherited Platelet Disorders- Qualitative
Qualitative disordersAdhesion
Bernard-Soulier syndrome ( GP Ib-IX )Platelet-type (Pseudo-) von Willebrand disease
( GP Ib receptor) **Collagen receptor deficiency (GP VI)
AggregationGlanzmann thrombasthenia (Gp IIb-IIIa)
SecretionDense (δ) granule defects (storage pool
deficiency)α granule defects (gray platelet syndrome)
Platelet procoagulant activityScott syndrome PF3
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Platelet Activation (signaling)
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Platelet AggregometryPlatelet 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 Method2. 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 MethodElectrical 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 ana lyzer.
luminescence technology (Platelet luminescence technology (Platelet Lumiaggregometry)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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Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.
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.)
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.
oSampleSampleoPlatelet-Rich Plasma
(PRP)oPRP 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 pathwayCollagen
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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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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PRP Aggregometry Agonist & Patterns
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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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. 10/11/2010Islamic Unversity of Gaza24
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 defects3. Idiopathic thrombocytopenia purpura (ITP).
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Interpretation
Glanzmann thrombastheniaGlanzmann thrombasthenia
o Normal PLT count, but abnormal clot retractiono Absence of secondary
aggregation to ADP, epinephrine, collagen, (thrombin)
o Normal response to ristocetin
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Bernard-Soulier syndromeBernard-Soulier syndrome
o Platelet aggregation testo Failure to aggregate in the presence of
ristocetinoAggregation by other agonists (ADP,
collagen, epinephrine): normaloResponse to low-dose thrombin: may be
delayed
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Platelet storage granule defectsPlatelet storage granule defects
o Dense (δ) granule defects ~ storage pool deficiency
o α granule defects ~ gray platelet syndrome
o Heterogeneous group of disorderso Mild to moderate bleeding diathesiso 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 FunctionDrugs and PLT FunctionAspirin
Acetylsalicyclic acid Irreversibly inhibits
CyclooxygenaseClopidogrel
Plavix irreversibly inhibits
P2Y12 Dipyridamole
inhibits Thromboxane synthase
Abciximab ReoPro
inhibits GP IIb/IIIa
Brinkman WT, Terramani TT, Najibi S, Chaikof EL. Platelets: is aspirin sufficient or must we know how to pronounce abciximab?Platelets: is aspirin sufficient or must we know how to pronounce abciximab? Semin Vasc Surg. 2002
Dec;15(4):245-55. •))Pronounce: ab-SIKS-ih-mabPronounce: ab-SIKS-ih-mab((