Platelet Function Testing

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Platelet Function Testing

• John Francis Ph.D.
• Florida Hospital Center
• for Hemostasis and Thrombosis,
• Orlando, FL, USA

www.fhitr.com
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Typical approach to platelet function testing

• personal and family bleeding history
• CBC and platelet count
• bleeding time or PFA-100

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Bleeding Time as a screening test of primary
hemostasis

• screening for congenital and acquired platelet
  dysfunction
• screening for von Willebrands Disease
• screening for aspirin (and similar) effects
• pre-operative risk assessment

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The Bleeding Time test lacks clinical benefit
CAP and ASCP position statement 1998

• in the absence of a history, BT does NOT predict
  bleeding associated with surgery
• a normal BT does NOT exclude possibility of
  surgical bleeding
• BT CANNOT reliably identify individuals who have
  recently ingested aspirin or who have a
  drug-induced platelet defect.

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Attempts to simulate the bleeding time in vitro

• Platelet retention test
• Machine Bleeding Time
• Platelet-Stat test
• Hemostatometer
• Thrombostat 4000
• Platelet Function Analyzer (PFA)-100

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Principle of the PFA-100

• Collagen/Epinephrine (CEPI) primary screening
  cartridge
• Collagen/ADP (CADP) differentiates dysfunction
  due to aspirin

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Comparison of Bleeding Time and PFA-100
20/23 had abnormal platelet aggregation
Francis et al. Platelets 10 132-136,1999
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Comparison of Bleeding Time and PFA-100

• overall agreement in 70-80 cases
• PFA (CEPI) more frequently abnormal
• PFA more sensitive to aspirin
• equivalent sensitivity to congenital platelet
  function defects
• PFA more sensitive to von Willebrand Disease
• PFA more cost effective

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Factors that determine closure time in the PFA-100

• platelet count
• hematocrit
• platelet function
• drug-induced defects
• other acquired defects
• congenital defects
• von Willebrand Factor

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Effect of aspirin on the PFA-100
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Relative sensitivity of PFA and BT to von
Willebrand Disease
Fressinaud et al 1998 Cattanep et al 1999 Dean
et al 2000
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Bleeding time vs PFA-100
Bleeding Time better
PFA-100 better
Von Willebrands Disease
Aspirin ingestion
Congenital platelet receptor disorders
Platelet secretion defects (CEPI)
Platelet secretion defects (CADP)

• PFA and BT agree in 70-80 cases
• PFA correlates more closely with aggregometry
• PFA-100 is more useful in clinical practice

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Pre-operative hemostatic testing
Pre-op patients (n5649)
Hemostatic workup Platelets, PT, APTT PFA-100
(EPI, ADP)
97 detectable by PFA-100
Koscielny et al Clin Appl Hemost Thromb 10
195-204, 2004
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Other tests of platelet function

• Platelet aggregation
• Optical
• Impedance
• VerifyNow
• Plateletworks
• Flow cytometry
• Thromboelastography

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Platelet aggregation

• Impedance (lumi) aggregometry

Optical aggregometry
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Optical aggregometry
Agonist
LIGHT
Light
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Optical aggregometry
Arachidonic Acid
Collagen
ADP
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Impedance aggregometry

• probe inserted in sample
• electrical current across electrodes
• platelets form monolayer on probe
• electrical resistance (impedance) proportional to
  increasing platelet recruitment and aggregation

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Lumi-aggregometry
Aggregation
Collagen
ATP Release
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Lumi-aggregometry vs optical aggregometry

• faster turnaround time
• less processing of blood sample
• release easier to assess
• requires smaller sample (pediatrics)
• technically easier
• affected by thrombocytopenia

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VerifyNow (Ultegra Rapid PFA)
Mixing chamber
Light source
T
Increasing light transmission
GpIIb/IIIa
Fibrinogen
Platelets activated by specific agonist
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
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Plateletworks
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Flow Cytometry
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Applications of flow cytometry

• platelet activation
• diagnosis of specific platelet disorders
• monitoring antiplatelet agents
• reticulated platelets (thrombopoiesis)
• platelet-associated antibodies
• research applications

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Detection of platelet activation by flow cytometry
P-Selectin (CD62P)
Annexin V
Fibrinogen binding to GpIIb/IIIa
Micro- particles
Activated platelet
RESTING
DETECTION
ACTIVATION
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Thromboelastography
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Thromboelastography
Max Amplitude Fibrinogen Platelets
Clotting Rate Fibrinogen Platelets
Clot Time Factor levels Anticoagulants
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ThromboelastographyEffect of platelets on clot
formation
R 100 90 ? 67.5 45.5 MA 64.0 22.5
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Anti-platelet effect of aspirin

• arachidonic acid converted to thromboxane A2 - a
  potent aggregating agent
• aspirin blocks cyclo-oxygenase-1 (COX-1)

Arachidonic acid
COX
PGG2
ASA
COX
PGH2
TXA2
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Assessing the anti-platelet effect of aspirin

• detect surreptitious aspirin intake
• transfusion medicine
• pre-op detection of bleeding risk
• surgery, spinal anesthesia, lithotripsy
• measure efficacy of aspirin therapy, control
  compliance, identify resistance
• cardiovascular medicine

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Aspirin Resistance

• widespread use of aspirin for prevention of MI
  and stroke (80 million tablets / day)
• aspirin resistance appears to be common
• aspirin resistance (or non-compliance) may be
  associated with greater risk of cardiovascular
  death
• how should aspirin resistance be defined and
  assessed?

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Frequency of aspirin resistance
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Possible causes of aspirin resistance

• inadequate dose
• non-compliance
• other routes of platelet activation bypassing the
  COX-1 (aspirin-sensitive) pathway
• interference with aspirin-binding sites on
  platelets by concomitant NSAID use
• genetic defect(s) affecting aspirin sensitivity
• elevated cholesterol
• method-dependent factors

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How should Aspirin Resistance be defined?

• clinical inability of aspirin to protect against
  arterial thrombosis ?
• failure of aspirin to inhibit platelet function?
• normal urinary concentration of thromboxane
  metabolites despite aspirin intake ?

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How should Aspirin Resistance be measured?

• Platelet function testing
• PFA-100
• platelet aggregation in whole blood
• platelet aggregation in platelet-rich plasma
• VerifyNow Aspirin Assay
• thromboelastography
• Urinary thromboxane A2 metabolites

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Aspirin resistance in normal volunteers by PFA-100
Francis (unpublished data)
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Discordance between PFA-100 and platelet
aggregation

• 325 patients with stable CVD - 325 mg/day
• platelet aggregation (ADP and AA)
• resistance (ADPAA) - 6
• semi-responders (ADP or AA) - 24
• PFA-100 (CEPI)
• resistance - 10
• low concordance between methods for
  aspirin-resistant subjects - 22

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Aspirin resistance by PFA-100

• 53 patients on aspirin (100 mg daily) for 20
  prevention of cerebrovascular accidents
• asymptomatic (no events for 2 yr) 18
• PFA-100 prolonged in all patients
• symptomatic (stroke or TIA) 35
• PFA-100 significantly shorter
• PFA-100 normal in 12/35

Grundmann et al. J. Neurol 250 63-66, 2003
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Aspirin resistance by PFA-100Real or due to
elevated vWF?

• 120 patients on aspirin (75-300 mg daily)
• 22 (18.3) aspirin-resistant
• median CADP significantly shorter in
  aspirin-resistant group
• vWF levels significantly higher in
  aspirin-resistant patients

Harrison et al. ISTH 2003
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Aspirin resistance by PFA-100Real or due to
elevated vWF?
Chakroun et al. Brit J. Haematol 124 80-85, 2004
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Aspirin resistance and outcome
Platelet Aggregation

• 326 patients with stable CAD on aspirin
• aspirin resistance assessed by platelet
  aggregation gt70 (ADP) and gt20 (AA)
• 5.2 - aspirin resistant
• hazard ratio of death, MI or CVA 3.12 (plt0.03)
• aspirin resistance associated with more than
  three-fold increase in major adverse event rate

Gumm et al JACC 41 961-965, 2003
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VerifyNow Aspirin Assay
Mixing chamber
Light source
T
Increasing light transmission
GpIIb/IIIa
Fibrinogen
Platelets activated by Arachidonic Acid
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
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AspirinWorks test for aspirin resistance

• 11-dehydro-TXB2 is a stable metabolite of TXA2
• excreted in urine
• normal levels in patients on aspirin indicate
  resistance
• measured by ELISA

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Aspirin resistance and outcome
Eikelboom et al Circulation 105 1650-1655, 2002
Relative Risk of CV death
Range
11-dehydro-TXB2
1.0
lt134
1st quartile
2.0
134 193
2nd quartile
2.5
194 298
3rd quartile
3.5
gt298
4th quartile
pg urinary 11-dehydro-thromboxane B2/mg
creatinine (normal value gt298)
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Thromboelastography for measuring aspirin
resistance
Heparinized blood Activated FXIII Reptilase Arachi
donic Acid
No aspirin
Aspirin
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Prevalence of aspirin resistance in coronary
artery disease

• PFA-100 gt20
• VerifyNow gt20
• Urinary dehydro-TXB2 gt20
• AA-induced aggregation 5
• TEG Platelet Mapping (AA) lt1

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Tests predict clinical outcome

• Laboratory test
• Bleeding time
• PFA-100
• Aggregation
• VerifyNow
• Flow cytometry
• Urinary 11-dehydro-TxB2

• Predictive of MACE
• No
• Yes
• Yes
• Yes
• No
• Yes

MACE Major Adverse Cardiac Events
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The ISTH Position

• Must develop a clinically meaningful definition
  of AR - linking aspirin-dependent lab tests to
  clinical outcome
• How do we treat AR? No data to show improved
  outcomes from changing therapy
• Not appropriate to test for AR or to change
  therapy based on current tests

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Action of clopidogrel (Plavix)
ADP receptors
P2X1
PLC
Ca
cAMP?
Ca ?

• blocks GpIIb/IIIa activation
• irreversible effect (7 days)
• inhibits aggregation to exogenous ADP
• prevents amplification by other agonists
• no effect on cyclooxygenase

Aggregation
Release
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Testing for clopidogrel

• Platelet aggregation to ADP
• PFA-100
• ADP cartridge relatively insensitive
• New cartridge in development
• VerifyNow P2Y12 Assay
• High correlation with PA (5 and 20 µM ADP)
• Vaosdilator-Stimulated Phosphoprotein (VASP)
  phosphorylation

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Clopidogrel resistance
Study n Patients Dose Time CR ()
Jeremo 2002 18 PCI 300/75 24 h 28
Gurbel 2003 92 PCI 300/75 24 h 31 - 35
Mueler 2003 105 PCI 600/75 4 h 5 - 11
Kesmarkey 2003 226 CVD 75 - 31
Total 441 5 - 35
Gurbel et al. Curr Pharm Design 12 1261, 2006
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Possible mechanisms of clopidogrel resistance

• Platelet count
• Concomitant medications
• Genetic polymorphisms
• Cytochrome P450 (CYP344)
• P2Y12

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SummaryPlatelet Function Testing

• multiple methods available
• PFA-100 has advantages over bleeding time
• lumi-aggregometry is more rapid and convenient
  than the optical method
• near-patient aggregation methods may be
  advantageous in specific situations
• TEG provides a global assessment of platelet and
  coagulation function

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SummaryAspirin Resistance

• aspirin resistance (AR) can be assessed by
  several methods
• correlation between methods is generally poor
• AR by PFA-100 partly related to increased vWF
• overall, AR appears to be associated with worse
  clinical outcomes
• lack of consensus of how to manage aspirin
  resistance, and whether correction of the
  laboratory defect improves outcome

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Aspirin and clopidogrel resistanceOngoing
studies

• ASCET (ASpirin Nonresponsiveness and Clopidogrel
  Endpoint Trial
• does switching to clopidogrel improve outcomes
  in AR patients?
• RESISTOR (Research Evaluation to Study
  Individuals who Show Thromboxane Or P2Y12
  Receptor Resistance
• does modifying antiplatelet therapy prevent
  myonecrosis after PCI in patients with aspirin
  and clopidogrel resistance?

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Questions?
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