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((