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

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.

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

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.

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

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

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: 132-136,1999

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.

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

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

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

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

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: 195-204, 2004

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

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

Optical aggregometry Agonist LIGHT Light

Optical aggregometry Arachidonic Acid Collagen ADP

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

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

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.

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

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

Flow Cytometry

Applications 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 flow cytometry P-Selectin (CD62P) Annexin V Fibrinogen binding to GpIIb/IIIa Platelet Micro- particles Activated platelet RESTING ACTIVATION DETECTION

Thromboelastography

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

Thromboelastography Effect of platelets on clot formation Platelets low/defective Normal platelets R 100 90  67.5 45.5 MA 64.0 22.5

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

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

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?

Frequency of aspirin resistance

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

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 ?

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

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)

Discordance between PFA-100 and 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-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-100 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-100 Real – or due to elevated vWF? 159 + 43 Normal <200 28 Poor 121 + 34 >96 >300 27 Good vWF CADP CEPI n Response Chakroun et al. Brit J. Haematol 124: 80-85, 2004

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: 961-965, 2003

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

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

Aspirin resistance and outcome Eikelboom et al Circulation 105: 1650-1655, 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)

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

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%

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

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

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

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

Clopidogrel resistance Study n Patients Dose Time CR (%) Jeremo 2002 18 PCI 300/75 24 h 28 Gurbel 2003 92 31 - 35 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

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

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

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

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?

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