Platelet Function TestingPlatelet Function Testing
John Francis Ph.D.John Francis Ph.D.Florida Hospital CenterFlorida Hospital Center
for Hemostasis and Thrombosis,for Hemostasis and Thrombosis,
Orlando, FL, USAOrlando, FL, USA
www.fhitr.comwww.fhitr.com
Typical approach to platelet Typical approach to platelet function testingfunction testing
Typical approach to platelet Typical approach to platelet function testingfunction testing
personal and family bleeding history
CBC and platelet count
bleeding time or PFA-100
Bleeding Time as aBleeding Time as a screening test of screening test of primary hemostasisprimary hemostasis
screening for congenital and acquired
platelet dysfunction
screening for von Willebrand’s Disease
screening for aspirin (and similar) effects
pre-operative risk assessment
The Bleeding Time test lacks The Bleeding Time test lacks clinical benefit*clinical benefit*
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.
*CAP and ASCP position statement 1998
Attempts to simulate the Attempts to simulate the bleeding time bleeding time in vitroin vitro
Platelet retention test
“Machine Bleeding Time”
Platelet-Stat” test
“Hemostatometer”
“Thrombostat 4000”
Platelet Function Analyzer (PFA)-100
Principle of the PFA-100Principle of the PFA-100®®
Collagen/Epinephrine (CEPI) — primary screening cartridge Collagen/ADP (CADP) — differentiates dysfunction due to aspirin
Collagen/Epinephrine (CEPI) — primary screening cartridge Collagen/ADP (CADP) — differentiates dysfunction due to aspirin
Comparison of Bleeding Time Comparison of Bleeding Time and PFA-100and PFA-100®®
BTNormal
BTAbnormal
PFANormal
67(59.3%)
6(5.3%)
PFAAbnormal
23*(20.4%)
17(15.0%)
BTNormal
BTAbnormal
PFANormal
67(59.3%)
6(5.3%)
PFAAbnormal
23*(20.4%)
17(15.0%)
* 20/23 had abnormal platelet aggregation* 20/23 had abnormal platelet aggregation
Francis et al. Platelets 10: 132-136,1999
Comparison of Bleeding Time Comparison of Bleeding Time and PFA-100and 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
platelet count hematocrit platelet function
• drug-induced defects
• other acquired defects
• congenital defects
von Willebrand Factor
Factors that determine closure Factors that determine closure time in the PFA-100time in the PFA-100
Effect of aspirin on the PFA-100Effect of aspirin on the PFA-100
CEPI CADP
True plateletdefect
Abnormal+++
Abnormal+++
Aspirin-likedefect
Abnormal+++
Usuallynormal
PFA-100 Bleeding Time
Type 3 100 100
Type 2 100 100
Type 1 77-100 27-41
All vWD 90-100 65
Relative sensitivity of PFA and Relative sensitivity of PFA and BT to von Willebrand DiseaseBT to von Willebrand Disease
Fressinaud et al 1998 Cattanep et al 1999 Dean et al 2000
Bleeding time vs PFA-100Bleeding time vs PFA-100
Bleeding Time betterBleeding Time better PFA-100 betterPFA-100 better
Von Willebrand’s DiseaseVon Willebrand’s Disease
Aspirin ingestionAspirin ingestion
Platelet secretion defects (CADP)Platelet secretion defects (CADP)
Congenital platelet receptor disordersCongenital platelet receptor disorders
Platelet secretion defects (CEPI)Platelet secretion defects (CEPI)
PFA and BT ‘agree’ in 70-80% cases
PFA correlates more closely with aggregometry
PFA-100 is more useful in clinical practice
Pre-operative hemostatic testingPre-operative hemostatic testing
Koscielny et al Clin Appl Hemost Thromb 10: 195-204, 2004
Positive historyPositive history(n=628)
Positive historyPositive history(n=628)
Abnormal tests (40.8%)Abnormal tests (40.8%)PFA EPI (n=250)PFA ADP (n=2)
PT (n=2)vWF:Ag (n=2)
Abnormal tests (40.8%)Abnormal tests (40.8%)PFA EPI (n=250)PFA ADP (n=2)
PT (n=2)vWF:Ag (n=2)
Negative historyNegative history(n=5021)
Negative historyNegative history(n=5021)
Abnormal tests (0.2%)Abnormal tests (0.2%)APTT (n=9)
Abnormal tests (0.2%)Abnormal tests (0.2%)APTT (n=9)
Pre-op patients Pre-op patients (n=5649)(n=5649)
Pre-op patients Pre-op patients (n=5649)(n=5649)
Hemostatic workupHemostatic workupPlatelets, PT, APTTPFA-100 (EPI, ADP)
Hemostatic workupHemostatic workupPlatelets, PT, APTTPFA-100 (EPI, ADP)
97% detectable by PFA-100
Other tests of platelet Other tests of platelet functionfunction
Platelet aggregation Optical
Impedance
VerifyNow™
Plateletworks™
Flow cytometry
Thromboelastography
Platelet aggregationPlatelet aggregation
Impedance (lumi) aggregometry
Optical aggregometry
Optical aggregometryOptical aggregometry
Light LIGHT
Agonist
Optical aggregometryOptical aggregometry
ADP
Collagen
Arachidonic Acid
Impedance aggregometryImpedance aggregometry
probe inserted in sample
electrical current across
electrodes
platelets form monolayer on
probe
electrical resistance
(impedance) proportional to
increasing platelet recruitment
and aggregation
Lumi-aggregometryLumi-aggregometry
Aggregation
ATP Release
Collagen
Lumi-aggregometry vs optical Lumi-aggregometry vs optical aggregometryaggregometry
faster turnaround time
less processing of blood sample
release easier to assess
requires smaller sample (pediatrics)
technically easier
affected by thrombocytopenia
VerifyNow (Ultegra Rapid PFA)VerifyNow (Ultegra Rapid PFA)
GpIIb/IIIaGpIIb/IIIaFibrinogenFibrinogen
Platelets activated by specific agonist
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
Lightsource
Mixing chamberMixing chamber
%T
Increasing lightIncreasing lighttransmissiontransmission
Plateletworks™Plateletworks™
BaselineBaseline
Single
platelets
Agonist tubeAgonist tube
Single
platelets
Aggregated
platelets
BaselineBaseline
countcount
AgonistAgonist
countcount
BaselineBaseline
countcount
--x x 100 =100 =
PercentPercent
aggregationaggregation
Flow CytometryFlow Cytometry
Applications of flow cytometryApplications of flow cytometry
platelet activation
diagnosis of specific platelet disorders
monitoring antiplatelet agents
reticulated platelets (thrombopoiesis)
platelet-associated antibodies
research applications
Detection of platelet activation by Detection of platelet activation by flow cytometryflow cytometry
RESTINGRESTING
Platelet
Annexin VAnnexin V
Micro-particles
Micro-particles
Fibrinogenbinding toGpIIb/IIIa
Fibrinogenbinding toGpIIb/IIIa
P-Selectin(CD62P)
P-Selectin(CD62P)
DETECTIONDETECTIONACTIVATIONACTIVATION
Activated platelet
ThromboelastographyThromboelastography
ThromboelastographyThromboelastography
Clot TimeFactor levelsAnticoagulants
Clotting RateFibrinogen ++Platelets +
Max AmplitudeFibrinogen +Platelets ++
ThromboelastographyThromboelastographyEffect of platelets on clot formationEffect of platelets on clot formation
Platelets low/defective
Normal plateletsR 100 90
67.5 45.5
MA 64.0 22.5
Anti-platelet effect of aspirinAnti-platelet effect of aspirin
arachidonic acid converted to thromboxane A2 - a potent aggregating agent
aspirin blocks cyclo-oxygenase-1 (COX-1)
Arachidonicacid
PGG2
PGH2
TXATXA22
COX
COX
ASAASA
Assessing the anti-platelet Assessing the anti-platelet effect of aspirineffect 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
Aspirin ResistanceAspirin 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?
Frequency of aspirin resistanceFrequency of aspirin resistance
0 20 40 60 80 100
Percent of patients
Santos 2001
Crowe 2001
von Page 2000
Sambola 2001
Seljeflot 2001
ResistantNormal
Possible causes of aspirin Possible causes of aspirin resistanceresistance
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
How should “Aspirin How should “Aspirin Resistance” be defined?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 ?
How should “Aspirin How should “Aspirin Resistance” be measured?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
Aspirin resistance in normal Aspirin resistance in normal volunteers by PFA-100volunteers by PFA-100
0
5
10
15
20
25
30
35
40
45
Pre 1 2 3 4 5
Aggregation (ohms)
0
50
100
150
200
250
300
350
Pre 1 2 3 4
Closure time (s)
PFA-100® (CEPI)
Days after aspirin (600 mg) ingestion
Arachidonic Acid Aggregation
Aspirin Aspirin ‘‘resistant’resistant’
Francis (unpublished data)
Discordance between PFA-100Discordance between PFA-100 and platelet aggregationand platelet aggregation
325 patients with stable CVD - 325 mg/day platelet aggregation (ADP and AA):
‘resistance’ (ADP+AA) - ~6% ‘semi-responders’ (ADP or AA) - ~24%
PFA-100® (CEPI) ‘resistance’ - ~10%
low concordance between methods for aspirin-resistant subjects - ~22%
Aspirin resistance by PFA-100Aspirin 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
Aspirin resistance by PFA-100Aspirin resistance by PFA-100Real – or due to elevated vWF?Real – 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
Aspirin resistance by PFA-100Aspirin resistance by PFA-100Real – or due to elevated vWF?Real – or due to elevated vWF?
159 + 43Normal<20028Poor
121 + 34>96>30027Good
vWFCADPCEPInResponse
Chakroun et al. Brit J. Haematol 124: 80-85, 2004
Aspirin resistance and outcome
326 patients with stable CAD – on aspirin
aspirin resistance assessed by platelet aggregation >70% (ADP) and >20% (AA)
5.2% - aspirin resistant
hazard ratio of death, MI or CVA = 3.12 (p<0.03)
aspirin resistance associated with more than three-fold increase in major adverse event rate
Platelet Aggregation
Gumm et al JACC 41: 961-965, 2003
VerifyNow Aspirin AssayVerifyNow Aspirin Assay
GpIIb/IIIaGpIIb/IIIaFibrinogenFibrinogen
Platelets activated by Arachidonic Acid
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
Lightsource
Mixing chamberMixing chamber
%T
Increasing lightIncreasing lighttransmissiontransmission
AspirinWorks™ test for AspirinWorks™ test for aspirin resistanceaspirin resistance
11-dehydro-TXB2 is a stable metabolite of TXA2
excreted in urine
normal levels in patients on aspirin indicate ‘resistance’
measured by ELISA
Arachidonicacid
PGG2
PGH2
TXATXA22
COX
COX
ASAASA
11-dehydro-11-dehydro-TXBTXB22
Aspirin resistance and outcomeAspirin resistance and outcome
3.5>2984th quartile
2.5194 – 2983rd quartile
2.0134 – 1932nd quartile
1.0<1341st quartile
Relative Risk of CV death
Range*11-dehydro-TXB2
* pg urinary 11-dehydro-thromboxane B2/mg creatinine (normal value >298)
Eikelboom et al Circulation 105: 1650-1655, 2002
No aspirin
Aspirin
Heparinized bloodActivated FXIII
ReptilaseArachidonic Acid
Thromboelastography for Thromboelastography for measuring aspirin resistancemeasuring aspirin resistance
Prevalence of aspirin resistance Prevalence of aspirin resistance in coronary artery diseasein coronary artery disease
PFA-100: >20%
VerifyNow: >20%
Urinary dehydro-TXB2: >20%
AA-induced aggregation: 5%
TEG Platelet Mapping (AA): <1%
Tests predict clinical outcomeTests predict clinical outcome
Laboratory testLaboratory test
Bleeding time
PFA-100
Aggregation
VerifyNow
Flow cytometry
Urinary 11-dehydro-TxB2
Predictive of MACEPredictive of MACE
No
Yes
Yes
Yes
No
Yes
MACE = Major Adverse Cardiac Events
The ISTH PositionThe 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
Action of clopidogrel (Plavix™)Action of clopidogrel (Plavix™)
ADPreceptors
ADPADP
Release
GpIIb/IIIaGpIIb/IIIa
P2Y12
P2Y1
P2X1
[Ca++]
PLCCa++
cAMP
ClopidogrelClopidogrelClopidogrelClopidogrel
Aggregation blocks GpIIb/IIIa activation
irreversible effect (~7 days)
inhibits aggregation to
exogenous ADP
prevents amplification by
other agonists
no effect on cyclooxygenase
blocks GpIIb/IIIa activation
irreversible effect (~7 days)
inhibits aggregation to
exogenous ADP
prevents amplification by
other agonists
no effect on cyclooxygenase
Testing for clopidogrelTesting 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
Clopidogrel resistanceClopidogrel 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
Possible mechanisms of Possible mechanisms of clopidogrel resistanceclopidogrel resistance
Platelet count
Concomitant medications
Genetic polymorphisms
Cytochrome P450 (CYP344)
P2Y12
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
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
Aspirin and clopidogrel resistanceAspirin and clopidogrel resistanceOngoing studiesOngoing studies
ASCET (ASpirin Nonresponsiveness and Clopidogrel Endpoint Trial
does switching to clopidogrel improve outcomes in AR patients?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?
Questions?Questions?
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