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?

www.fhitr.comwww.fhitr.com