Platelet Function Testing: Is GRAVITAS the Last Word? Michael A. Gaglia, Jr., MD, MSc Center for Platelet Studies Washington Hospital Center/Medstar Research Institute February 27, 2011

Michael A. Gaglia, MD I have no real or apparent conflicts of interest to report.

Overview Platelet agonists and receptors Platelet function tests Light transmission aggregometry (LTA) Vasodilator stimulated phosphoprotein phosphorylation (VASP) VerifyNow P2Y12 High on-treatment platelet reactivity and clinical events Recent and future clinical trials Optimal testing strategy for the cath lab

Major platelet agonists and receptors involved in thrombus formation Major platelet agonists and receptors involved in thrombus formation. Disruption of the endothelium exposes TF and collagen, liberating thrombin and initiating platelet adhesion via the GP Ib/Ix/V and GP VI platelet receptors. Adhesion also initiates the release of platelet agonists, including ADP and TXA2, which thereby activate other platelets. Activated platelets are then cross-linked by fibrinogen, leading to platelet thrombus formation. Gaglia et al. AHJ 2010;160:595

Light Transmission Aggregometry “Gold standard” (misnomer) Various agonists (e.g. ADP, AA) induce platelet aggregation in PRP Strength: most data with clinical outcomes Weakness: variable reproducibility, expensive, time consuming, lots of blood i.e. not practical

Vasodilator Stimulated Phosphoprotein Phosphoryation Flow cytometery-based Measures level of phosphorylated VASP protein; inversely correlated with P2Y12 activity Strength: specific to P2Y12 inhibition; one (small) study proving “tailoring” of therapy Weakness: expensive, time-consuming i.e. not practical

VerifyNow P2Y12 Light transmittance of platelets and fibrinogen-coated beads ADP as agonist (and PGE1 to eliminate P2Y1) Strength: bedside, whole blood, clinical data, FDA approved Weakness: percent inhibition not reliable; can’t test on IIb/IIIa

Verify Now P2Y12 Concept Concept: agonist (ADP or arachidonic acid) used to induce platelet aggregation. Beads coated with fibrinogen. Inhibited platelets (i.e. responders to drug) do not aggregate and produce low level of light transmittance Normal platelets aggregate more rapidly and produce higher level of transmittance (platelets occupy less space because of binding)

Others Dade PFA Collagen/ADP Innovance PFA P2Y Impact-R Thromboelastography (TEG) Clot strength Multiple electrode aggregometry Multiplate analyzer Plateletworks Adaptation of LTA Not useful

High On-Treatment Platelet Reactivity and Adverse Events post-PCI Initial studies examined the difference between pre- and post-clopidogrel platelet reactivity Nearly 30 studies linking high on-treatment platelet reactivity and ischemic events Most data for LTA, VASP, and VerifyNow P2Y12

Thresholds for High On-Treatment Platelet Reactivity Study n Assay Endpoint Sn Sp NPV Threshold Breet 2010 1069 VerifyNow Plateletworks LTA 5 μmol 1 y death, MI, ST, CVA 63% 60% 59% 94% PRU 236 81% 43% Marcucci 2009 683 1 y CV death, MI 61% 70% 96% PRU 240 Price 2008 380 6 mo CV death, MI, ST 78% 99% PRU 235 Frere 2007 195 VASP 30d recurrent ischemia 93% 50% PRI 53% Bonello 2007 144 6 mo CV death, CVA, TVR 100% 25% NR PRU 50% Sibbing 2009 1608 Multiplate analyzer 30d stent thrombosis 84% 468 AUmin Cuisset 2009 598 LTA 10 μmol 68% 67%

JACC “White Paper” Key concept is high on-treatment platelet reactivity, not “responsiveness” to clopidogrel (i.e. the delta). Proposed thresholds for high on-treatment platelet reactivity: LTA 5 μmol ADP: >46% Multiplate: 468 AU/min VASP: PRI >50% VerifyNow P2Y12: PRU >235 to 240 Bonello et al 2010;56:919

GRAVITAS Hypothesis: High-dose clopidogrel for 6 months is superior to standard clopidogrel therapy for the prevention of adverse CV events after PCI in patients with high residual reactivity.

GRAVITAS Study Design R Elective or Urgent PCI with DES* VerifyNow P2Y12 Test 12-24 hours post-PCI PRU ≥ 230 R High-Dose Clopidogrel† clopidogrel 600-mg, then clopidogrel 150-mg daily X 6 months Standard-Dose Clopidogrel† clopidogrel 75-mg daily X 6 months GRAVITAS tested the clinical efficacy of a treatment strategy of prolonged high-dose clopidogrel based upon the results of the VerifyNow P2Y12 test after PCI. The trial enrolled patients undergoing elective or urgent PCI who received a peri-procedural clopidogrel regimen designed to ensure a steady-state level 12 to 24 hours after the procedure. At that time, platelet function was assessed. The VerifyNow P2Y12 test provides a result is in terms of PRU – the higher the PRU, the greater the level of residual reactivity on clopidogrel. In GRAVITAS, high reactivity was defined as a PRU greater than or equal to 230, a cutoff similar to what has been suggested by previous studies to provide the maximal sensitivity and specificity for subsequent cardiac events. Patients with high reactivity were randomized in a double-blind fashion to either 6 months of high dose clopidogrel – that is, an additional loading dose followed by 150-mg a day – or standard dose clopidogrel – that is, 75-mg a day. Platelet reactivity was re-assessed at 30-days and 6 months in a blinded fashion, but no treatment decisions were made based upon the results of these tests. The primary efficacy endpoint was a composite of cardiovascular death, non-fatal MI, or stent thrombosis at 6-months. The Key safety endpoint was GUSTO severe or moderate bleeding. Primary Efficacy Endpoint: CV Death, Non-Fatal MI, Stent Thrombosis at 6 mo Key Safety Endpoint: GUSTO Moderate or Severe Bleeding at 6 mo Pharmacodynamics: Repeat VerifyNow P2Y12 at 1 and 6 months

Primary Endpoint: CV Death, MI, Stent Thrombosis At 6-months, the observed rate of the primary endpoint was 2.3% in the high-dose group compared with 2.3% in the standard dose group, resulting in a hazard ratio of 1.01 and a p value of 0.98. There were a total of 50 events which was lower than the 68 that we had anticipated.

Secondary Comparison: High vs. Not High Reactivity Here are the results of this non-randomized compariion of patients with and without high reactivity treated with clopidogrel 75-mg a day. At 6 months, the observed rate of the composite endpoint was 2.3% in patients with high reactivity compared with 1.4% in patients without high reactivity, representing a hazard ratio of 1.7. The difference in event rates did not reach statistical significance; note that the lower boundary of the 95% CI is less than one. The point estimate of this large hazard ratio appears consistent with previous studies documenting a significant association between residual reactivity and cardiovascular events after PCI.

End of story, right?

Prasugrel vs. Clopidogrel Brandt et al. AHJ 2007;153:66

Ticagrelor vs. Clopidogrel Storey et al. JACC 2007;50:1852

Ongoing Studies ARCTIC TARGET-PCI TRIGGER-PCI PRU ≥230: tailoring of clopidogrel dose TARGET-PCI Clopidogrel naïve: CYP 2C19 Chronic clopidogrel: PRU ≥230 TRIGGER-PCI PRU ≥208: Prasugrel vs. clopidogrel

WHC Testing Protocol Clopidogrel naïve patients: VerifyNow P2Y12 between 4-24 hrs post clopidogrel load and PCI Chronic clopidogrel patients (at least 5 days): VerifyNow P2Y12 just before cath PRU ≥235: recommend change to prasugrel No longer recommending double dose clopidogrel No opinion regarding cilostazol

Practical Advice Conservative: don’t change anything and wait for ticagrelor Less conservative: prasugrel for high-risk patients (e.g. diabetics, Hx stent thrombosis, left main stent) Tailored option: consider prasugrel for patients with PRU ≥235 (or PRI ≥50% if you are from France) Must be mindful of many exclusions (age, weight, CVA, bleeding history)