A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease Marie Lordkipanidzé, Chantal Pharand, Erick Schampaert, Jacques Turgeon, Donald A. Palisaitis, Jean G. Diodati

Aspirin is one of the most widely used drugs worldwide. Aspirin: Introduction Aspirin is one of the most widely used drugs worldwide. Aspirin: inhibits platelet aggregation through irreversible acetylation of platelet cyclooxygenase (COX) enzyme blocks the transformation of arachidonic acid (AA) into thromboxane (Tx) A2 reduces the risk of stroke, myocardial infarction, or death by approximately 25% in patients with cardiovascular disease

Concept of aspirin resistance Aspirin may not benefit all patients equally No consensual definition of aspirin resistance exists It is generally accepted that: incomplete suppression of platelet aggregation as assessed by platelet function assays constitutes biochemical unresponsiveness of platelets to the inhibitory action of aspirin

Platelet function testing – The Gold Standard Many tests are available to assess inhibition of platelet function induced by aspirin Light transmission aggregometry (LTA) Current gold standard Evaluates luminosity as aggregation occurs in platelet-rich plasma (PRP) in response to AA Use limited to specialized laboratories because: poorly standardized requires manipulation by a skilled technician

Other platelet function assays available Laboratory testing Whole blood aggregometry (WBA) Preferred agonist: AA Serum measurement of TxB2 Urinary measurement of dTxB2 Flow cytometry Platelet count drop (PCD) Point-of-care assays PFA-100® Using the COLL-EPI cartridge VerifyNow Aspirin® Using AA cartridge Thromboelastograph (TEG®) Cone and Plate(let) Analyzer AspirinWorks® Standardized urinary dTxB2 PlateletWorks® Standardized PCD

Objectives To determine the prevalence of aspirin resistance in stable CAD patients To compare results obtained with widely available platelet function tests This has led us to design this study, which aimed to determine the prevalence of aspirin resistance in patients suffering from stable coronary disease using a platelet function assay specific to the COX pathway and thus sensitive to inhibition by aspirin. We further wished to compare the results obtained with other platelet function assays commonly used in the literature.

Patient selection (n = 201) Inclusion criteria: Adult, stable CAD patients Taking aspirin daily (≥ 80 mg daily) Exclusion criteria: ACS ≤ 6 months Concurrent ingestion of NSAIDs, including COX-2 selective anti-inflammatory drugs, clopidogrel, ticlopidine, dipyridamole, warfarin, or acenocoumarol Self-reported use of non-prescription NSAIDs or aspirin-containing drugs ≤ 10 days Major surgical procedure ≤ 1 month Platelet count outside the 100 to 450 x 109/L range Hb < 100 g/L or Ht < 25% Hemorrhagic diathesis End-stage renal disease requiring dialysis Our population consisted of patients suffering from stable coronary artery disease and taking aspirin daily in prevention of acute ischemic events. We have excluded patients with pathologies or medications known to modulate platelet function.

Definitions of aspirin resistance AA-induced LTA (LTAAA) Platelet aggregation ≥ 20% ADP-induced LTA (LTAADP) Platelet aggregation ≥ 70% AA-induced WBA Impedance ≥ 3 Ω PFA-100® closure time ≤ 193 sec VerifyNow Aspirin® Platelet aggregation ≥ 550 Aspirin Response Units Urinary levels of dTxB2 ≥ 67.9 ng/mmol of creatinine

Results Prevalence of resistance varied according to the platelet function assay used * Definition used by Gum et al

Variability in platelet response to aspirin Point-of-care assays The measure of platelet aggregation by LTAAA segregated patients into two exclusive and distinct groups ( resistant and ￮ sensitive) Much overlap existed above and below the specific cut-off value for other platelet function tests

Correlation between assay results LTA WBA Point-of-care assays Urinary dTxB2 ADP 5 µM ADP 10 µM ADP 20 µM AA 1.6 mM PFA-100® Verify Now Aspirin® LTA AA 1.6 mM ADP 5 µM ADP 10 µM ADP 20 µM 0.063 - - - 0.091 0.827* - - 0.058 0.718* 0.714* - 0.243* 0.290* 0.281* 0.282* -0.120 0.101 0.086 0.094 0.133 -0.030 -0.012 0.023 0.175* -0.064 -0.071 0.014 - 0.061 0.119 -0.025 Point-of-care assays PFA-100® VerifyNow Aspirin® - - 0.189* -0.124 0.151* * p<0.05

Platelet function test Agreement between test to classify subjects as aspirin resistant, when compared with LTAAA Platelet function test κ statistic p-value LTA ADP 5 μM ADP 10 μM ADP 20 μM 0.250 0.168 0.019 < 0.0001 0.002 0.531 WBA, AA 1.6 mM 0.173 0.001 Point-of-care assays PFA-100® VerifyNow Aspirin® 0.247 0.392 Urinary dTxB2 -0.033 0.475

Platelet function tests are NOT equally effective Conclusion Aspirin resistance remains rare in stable CAD, when defined by a COX-specific assay such as LTAAA Platelet function tests are NOT equally effective In measuring aspirin’s antiplatelet effect Correlate poorly with the current gold standard May explain the discrepancies reported in the literature What should you remember from this presentation? A key point is that aspirin resistance is a rare phenomenon in stable coronary artery disease patients, when a platelet function test targeted at the COX pathway is used to quantify platelet response to aspirin. Platelet function tests are not equally effective in measuring aspirin’s antiplatelet effect. They correlate poorly with the gold standard and among themselves. This lack of agreement may explain the discrepancies in prevalence of aspirin resistance reported in the literature.

Results from our study suggest that Conclusion Results from our study suggest that conclusions drawn may be highly dependent on the assay used and results obtained from different assays are not interchangeable. Further research is warranted to better understand the platelet activation pathways involved in platelet response to aspirin in order to allow specific targeting with various platelet function assays to determine the threshold to be used to best predict clinical outcomes.

For full details, please refer to: Lordkipanidzé M, Pharand C, Schampaert E, Turgeon J, Palisaitis DA, Diodati JG. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J 2007; 28:1702-8