1. Platelet Function Testing
John Francis Ph.D.
Florida Hospital Center
for Hemostasis and Thrombosis,
Orlando, FL, USA

2. Typical approach to platelet function testing
personal and family bleeding history
CBC and platelet count
bleeding time or PFA-100
The investigation of a patients with a suspected platelet function disorder starts with a good clinical and family history. A complete blood count will help to exclude disorders, such as leukemia, that may be associated with bleeding and bruising and platelet count will identify thrombocytopenia.
The bleeding time is currently often used as a first line screening test of platelet function, although platelet aggregation studies are usually required for diagnosis. Von Willebrand’s disease is a common abnormality, but requires a more specific laboratory approach that is outside the scope of this presentation.

3. Bleeding Time as a screening test of primary hemostasis
screening for congenital and acquired platelet dysfunction
screening for von Willebrand’s Disease
screening for aspirin (and similar) effects
pre-operative risk assessment

4. 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.
Recently, the College of American Pathologists and the American Society of Clinical Pathologists issued a position statement clearly identifying the shortcomings of the BT.
Most notably, this recognized the poor performance of the BT as a pre-surgical screening test. Thus, the BT does not identify patients that are likely to bleed excessively, nor does a normal result mean that a patient will not bleed. In addition, the BT does not reliably identify those subjects that have recently ingested anti-platelet drugs such as aspirin.

5. 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

6. Principle of the PFA-100®
Collagen/Epinephrine (CEPI) — primary screening cartridge
Collagen/ADP (CADP) — differentiates dysfunction due to aspirin

7. Comparison of Bleeding Time and PFA-100®
In this study, the BT and PFA agreed in 77/106 (73%) of the patients studied. Twenty three patients had an abnormal PFA-100 despite a normal BT. Platelet aggregation was abnormal in 20 of these. Overall, amongst the 29 discordant subjects, platelet aggregation supported the results of the PFA-100 test in 25 cases (86%).
* 20/23 had abnormal platelet aggregation
Francis et al. Platelets 10: ,1999

8. 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
The PFA-100 detected significantly more abnormalities in our unselected hospital population. The fact that these were ‘true’ abnormalities of platelet function (mainly drug-related) is indicated by the high degree of agreement (86%) between the PFA-100 test and platelet aggregation studies. In the majority of cases, the BT and PFA-100 tests agreed.

9. 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

10. Effect of aspirin on the PFA-100
CEPI
CADP
True platelet
defect
Abnormal
+++
Aspirin-like
defect
Abnormal
+++
Usually
normal

11. Relative sensitivity of PFA and BT to von Willebrand Disease
Fressinaud et al 1998
Cattanep et al 1999
Dean et al 2000

12. Bleeding time vs PFA-100 Bleeding Time better PFA-100 better
Von Willebrand’s 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

13. Pre-operative hemostatic testing
Pre-op patients (n=5649)
Hemostatic workup
Platelets, PT, APTT
PFA-100 (EPI, ADP)
Positive history
(n=628)
Abnormal tests (40.8%)
PFA EPI (n=250)
PFA ADP (n=2)
PT (n=2)
vWF:Ag (n=2)
Negative history
(n=5021)
Abnormal tests (0.2%)
APTT (n=9)
97% detectable by PFA-100
Koscielny et al Clin Appl Hemost Thromb 10: , 2004

14. Other tests of platelet function
Platelet aggregation
Optical
Impedance
VerifyNow™
Plateletworks™
Flow cytometry
Thromboelastography

15. Platelet aggregation Impedance (lumi) aggregometry
A variety of instruments are currently available for the study of platelet function in whole blood. Some of these, like the Sonoclot Analyzer and the Thromboelastograph are also used for monitoring coagulation activity and have found application as point-of-care devices in the operating room or intensive care.
Optical aggregometry

16. Optical aggregometry
Agonist
LIGHT
Light

17. Optical aggregometry
Arachidonic Acid
Collagen
ADP

18. 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

19. Lumi-aggregometry Aggregation Collagen ATP Release
Using the Luciferin-Luciferase (firefly) principle, ATP release from platelets can be assessed at the same time as aggregation. In this illustration, aggregation and ATP release to collagen in a control and a patient are assessed simultaneously in a dual channel lumi-aggregometer.
The patient has delayed aggregation and a very low level of ATP release compared to the control.
ATP Release

20. 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
Although platelet aggregation is considered to be the “gold standard” of routine platelet function tests, it is limited by the slow nature of the method, especially when relatively large amounts of blood have to be processed to obtain platelet-poor plasma. Because of the time and expense involved, it is not well suited to routine screening. In addition, the test does not take into account the role of shear activation or adhesion in platelet function.

21. 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

22. Plateletworks™ Baseline Agonist tube - Single platelets Single
Aggregated
Baseline
count
Agonist -
x 100 =
Percent
aggregation

23. Flow Cytometry

24. Applications of flow cytometry
platelet activation
diagnosis of specific platelet disorders
monitoring antiplatelet agents
reticulated platelets (thrombopoiesis)
platelet-associated antibodies
research applications

25. Detection of platelet activation by flow cytometry
P-Selectin
(CD62P)
Annexin V
Fibrinogen
binding to
GpIIb/IIIa
Platelet
Micro-
particles
Activated platelet
RESTING
ACTIVATION
DETECTION

26. Thromboelastography

27. Thromboelastography Max Amplitude Fibrinogen + Platelets ++
Clotting Rate
Fibrinogen ++
Platelets +
Clot Time
Factor levels
Anticoagulants

28. Thromboelastography Effect of platelets on clot formation
Platelets low/defective
Normal platelets R  MA

29. 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

30. 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

31. 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?

32. Frequency of aspirin resistance

33. 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

34. 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 ?

35. 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

36. Aspirin resistance in normal volunteers by PFA-100
PFA-100® (CEPI)
Days after aspirin (600 mg) ingestion
Arachidonic Acid Aggregation
Aspirin
‘resistant’
Francis (unpublished data)

37. Discordance between PFA-100 and platelet aggregation
325 patients with stable CVD 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%

38. 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

39. Aspirin resistance by PFA-100 Real – or due to elevated vWF?
120 patients on aspirin ( 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

40. Aspirin resistance by PFA-100 Real – or due to elevated vWF?
Normal
<200 28
Poor
>96
>300 27
Good
vWF
CADP
CEPI n
Response
Chakroun et al. Brit J. Haematol 124: 80-85, 2004

41. Aspirin resistance and outcome
Platelet Aggregation
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
Gumm et al JACC 41: , 2003

42. 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

43. AspirinWorks™ test for aspirin resistance
Arachidonic
acid
PGG2
PGH2
TXA2
COX
ASA
11-dehydro-TXB2
11-dehydro-TXB2 is a stable metabolite of TXA2
excreted in urine
normal levels in patients on aspirin indicate ‘resistance’
measured by ELISA

44. Aspirin resistance and outcome
Eikelboom et al Circulation 105: , 2002
11-dehydro-TXB2
Range*
Relative Risk of CV death
1st quartile
<134
1.0
2nd quartile
134 – 193
2.0
3rd quartile
194 – 298
2.5
4th quartile
>298
3.5
* pg urinary 11-dehydro-thromboxane B2/mg creatinine (normal value >298)

45. Thromboelastography for measuring aspirin resistance
Heparinized blood
Activated FXIII
Reptilase
Arachidonic Acid
No aspirin
Aspirin

46. Prevalence of aspirin resistance in coronary artery disease
PFA-100: >20%
VerifyNow: >20%
Urinary dehydro-TXB2: >20%
AA-induced aggregation: 5%
TEG Platelet Mapping (AA): <1%

47. Tests predict clinical outcome
Laboratory test
Bleeding time
PFA-100
Aggregation
VerifyNow
Flow cytometry
Urinary 11-dehydro-TxB2
Predictive of MACE No
Yes
MACE = Major Adverse Cardiac Events

48. 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

49. Action of clopidogrel (Plavix™)
ADP 
Clopidogrel
ADP
receptors
P2Y1
P2X1
P2Y12
PLC
Ca++
cAMP
[Ca++] 
GpIIb/IIIa
blocks GpIIb/IIIa activation
irreversible effect (~7 days)
inhibits aggregation to exogenous ADP
prevents amplification by other agonists
no effect on cyclooxygenase
Aggregation
Release

50. 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

51. Clopidogrel resistance
Study n
Patients
Dose
Time
CR (%)
Jeremo 2002 18
PCI
300/75
24 h 28
Gurbel 2003 92
Mueler 2003
105
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

52. Possible mechanisms of clopidogrel resistance
Platelet count
Concomitant medications
Genetic polymorphisms
Cytochrome P450 (CYP344)
P2Y12

53. Summary Platelet 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

54. Summary Aspirin 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

55. Aspirin and clopidogrel resistance Ongoing 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?

56. Questions?