1. Measurement of Clopidogrel Resistance by ADP-Inhibition Does Not Reflect the Benefit of Clopidogrel on Overall Thrombotic Status Dr Diana A Gorog Consultant Cardiologist & Honorary Clinical Senior Lecturer E & N Hertfordshire NHS Trust & Imperial College, London, UK Presented at

2. “Clopidogrel resistance”  Definition  clinical ongoing thrombotic events despite medication  laboratory results of platelet function tests  Does it exist?  Clinical resistance infrequent (5-10%)  Laboratory resistance frequent (up to 50%)  Relationship between clinical and lab resistance is unclear, if it exists at all  Is “laboratory resistance” clinically relevant?  unsure, some evidence that it is only a laboratory phenomenon Presented at

3. Limitations of laboratory assessment of clopidogrel resistance  Most tests use citrated blood  Platelet aggregation studies assess response to a specific agonist or combination of agonists, e.g.  PFA 100: Collagen/ADP  VerifyNow: ADP/Epinephrine In addition to physiological doses of ADP used, there is also ADP release from aggregating plts (only in citrated blood) so overall level can be an order of magnitude higher  Clopidogrel effective at inhibiting low conc ADP but not high conc  Overall effect of clopidogrel on global thrombotic status unclear, as the most important contributor to platelet aggregation, thrombin generation, not assessed  Correlation between tests very poor Presented at

4. Effect of ADP in native blood ADP 5-20 μ M aggregation [Ca 2+ ] 1.0-1.3 mM Thrombin generation (only in native blood) Presented at activation stabilization

5. Effect of ADP in citrated blood ADP 5-20 uM aggregation [Ca 2+ ] 10 – 30 uM Thrombin generation Presented at ADP up to mM Platelets in native blood are more sensitive to inhibition of ADP-induced aggregation than platelets in citrated blood (Viigimaa M et. al.,1996) Citrate release of granule contents Only in citrated blood

6. Limitations of platelet function tests in measuring clopidogrel effect  Measuring the effect of clopidogrel on ADP-induced aggregation in citrated blood does not take into account inhibition of ADP-induced thrombin generation  Because of the individually variable release rxn, actual ADP concentration is much higher (mM?) than that used to induce aggregation (5-20uM).  Clopidogrel is less effective at inhibiting high conc of ADP than low concentration Presented at

7. “Ideal” platelet function test for clopidogrel effect  test involves thrombin generation  employs non-citrated blood  assesses response to endogenous ADP concentration

8. Global Thrombosis Test (GTT)  Native (non- anticoagulated) blood  Mechanism involves high shear, thrombin and ADP  “Global” test of platelet function Presented at

9. Normal volunteers before and 12h after 300 mg clopidogrel OT (sec) P=0.001 Effect of clopidogrel on occlusion time (OT)

10. GTT – modified to cause ADP release  Wanted to ↑ actual ADP concentration (to assess specific ADP inhibitory effect of clopidogrel) but still using native blood  Small volume water (0.3 ml) added to cartridge prior to testing at the site where thrombus formation occurs  This caused lysis of red cells and thus ADP release, manifesting in more rapid occlusion (aOT) Presented at

11. P=.0007 Normal vs. Accelerated Occlusion Time (aOT)

13. Acute coronary syndrome study  ACS patients (n=86)  On aspirin and clopidogrel >48 h  Platelet function and “clopidogrel resistance” tested using  GTT and aOT  VerifyNow P2Y12 cartridge

14. Accelerated Thrombosis Test N=60 Criteria OT<150 sec (i.e. <50% of normal OT) Non-responders 4 (6.7%) VerifyNow P2Y12 assay N=86 Criteria <10% inhibition: 19.8% non-responders Criteria <30% inhibition: 32.6% non-responders Criteria <50% inhibition: 46.5% non-responders ACS patients on dual anti-platelet therapy

15. Conclusion  Measurement of clopidogrel resistance by ADP- inhibition in citrated blood does not reflect the benefit of clopidogrel on overall thrombotic status  Detection of clopidogrel “resistance” using citrated blood is likely to be misleading  When assessing efficacy of clopidogrel, should use a global test of platelet function, using native blood, where thrombin makes a significant contribution to the thrombus formation. Presented at