Platelet Function Testing: Which one Should we Perform and how to Interpret the Data? Dominick J. Angiolillo, MD, PhD, FACC, FESC, FSCAI Director of Cardiovascular Research Assistant Professor of Medicine

Dominick J. Angiolillo, MD, PhD DISCLOSURES Dominick J. Angiolillo, MD, PhD Within the past 12 months, the presenter or their spouse/partner have had a financial interest/arrangement or affiliation with the organization listed below. Honoraria/Lectures: Bristol Myers Squibb, Sanofi-Aventis, Eli Lilly Co, Daiichi Sankyo, Inc., Advisory Board: Bristol Myers Squibb, Sanofi-Aventis, Eli Lilly Co, Daiichi Sankyo, Inc., The Medicines Company, Portola Pharmaceuticals, Novartis, Arena Pharmaceuticals, Accumetrics, Medicure Research Grants: GlaxoSmithKline, Otsuka, Accumetrics, Eli Lilly Co, Daiichi Sankyo, Inc., The Medicines Company, Portola Pharmaceuticals, Schering-Plough, Astra Zeneca, Johnson & Johnson I intend to reference unlabeled/ unapproved uses of drugs or devices in my presentation. I intend to reference Ticagrelor, Elinogrel, Cangrelor, and TRA.

Antiplatelet Drug Resistance / Response Variability: An Emerging Clinical Problem

Platelet Function Tests Platelet Aggregation Light transmittance aggregometry (LTA) Impedance platelet aggregation Flow Cytometry GPIIb/IIIa receptor activation P-selectin expression Monocyte-platelet aggregates Vasodilator-associated stimulated phosphoprotein (VASP) Point-of-care Ultegra rapid platelet function analyzer (VerifyNow) Thromboelastagraph (TEG) PFA-100 Plateletworks Cone and plate(let) analyzer (IMPACT) Genetic testing gold standard adapted from Angiolillo DJ et al. J Am Coll Cardiol. 2007

Definitions of Non/Low – Response using LTA Gurbel PA et al., Circulation 2003 Müller I et al., Thromb Haemostas 2003 Matetzky S et al., Circulation 2004 Serebruany VL et al., J Am Coll Cardiol 2005 Angiolillo DJ et al., Thromb Res 2005 Absolute change in platelet aggregation from baseline < 10% Relative change in platelet aggregation from baseline < 10% Lowest quartile of relative reduction of platelet aggregation Platelet aggregation 2 standard deviations below mean Relative change in platelet aggregation from baseline < 40% (Variable results also depending on the concentration of ADP used)

A B Definitions of Non-Response: Which one should we use? Absolute Change or Relative Change? Absolute Change or Relative Change or Post-treatment platelet reactivity? A B 31% (80 – 55) X 100% 80 = 18% (50 – 41) X 100% 50 Responder Low-Responder + Treatment (Post) 55 Baseline (Pre) 80 + Treatment (Post) 41 Baseline (Pre) 25% 9% Responder Non-Responder %Transmittance 50 %Transmittance 50 100 100 Time (minutes) Time (minutes)

Individual Response Variability to Dual Antiplatelet Therapy in the Steady State Phase of Treatment 20 15 Bleeding risk Ischemic risk Number of Patients 10 5 2.5 12.5 22.5 32.5 42.5 52.5 62.5 72.5 82.5 92.5 7.5 17.5 27.5 37.5 47.5 57.5 67.5 77.5 87.5 97.5 % Platelet Aggregation (LTA-ADP 20mmol/L) Adapted from Angiolillo DJ et al. Am J Cardiol. 2006;97:38-43.

Optimizing Platelet Response in Suboptimal Responders Modifying dosage of currently approved drugs (e.g. higher dose) Adding other agents with antiplatelet properties (e.g. GPIIb/IIIa inhibitors; cilostazol) Using alternative drugs (e.g. ticlopidine or novel antiplatelet agents)

VASP Guided PCI After a 600mg clopidogrel LD, poor responders (PRI ≥ 50%) received additional 600mg bolus (max 2400 mg) until reaching therapeutic target. Mean ±SD Control VASP-guided p VASP after first LD, % 68 ±11 69 ±10 0.4 VASP after adjustment, %  38 ±14* *<0.001 Log rank p =0.007 MACE: CV death, MI, revascularization Bonello et al. J Am Coll Cardiol 2008

VASP and LTA: limitations Not-user friendly Time consuming Require experienced lab personnel Require expensive equipment Not universally available Overall,….expensive

Fibrinogen-coated beads Agglutinated beads aggregate in clusters How does the VerifyNow Assay Work? Whole blood, closed-tube sampling with no pipetting required Assay results in less than 5 minutes (assay time) Good correlation with LTA and VASP Light Source Platelets in whole blood maximally activated by agonist in mixing chamber Fibrinogen-coated beads + Agonist Agglutinated beads aggregate in clusters Mixing Chamber Agonists: Aspirin Assay – AA P2Y12 assay – ADP + PGE1 GpIIbIIIa assay – iso-TRAP

Results are based on the rate and extent of platelet aggregation and are reported in P2Y12 Reaction Units (PRU) and % platelet inhibition Results are reported as PRU Results are also reported as % platelet inhibition

Primary Endpoint Tp >3ULN w/in 48 hs Tailoring Treatment with Tirofiban in patients showing Resistance to aspirin and/or Resistance to clopidogrel Patients with stable, unstable low risk CAD undergoing elective PCI being ASA and/or clopidogrel resistance using Verify Now Primary Endpoint Tp >3ULN w/in 48 hs P=0.009 for superiority 50 RRR: 42% 95%CI: 61-12 45 40 35.1% Placebo 35 Tirofiban 30 25 20.4% 20 15 10 5 Valgimigli M et al Circulation 2009; 119: 3215-22.

Platelet inhibition (%) OPTIMUS (Optimizing Antiplatelet Therapy in Diabetes Mellitus) Impact of high clopidogrel maintenance dosing on platelet function in DM patients with suboptimal clopidogrel response VerifyNow P2Y12 substudy %IPA PRU 20 40 60 80 75 mg 150 mg Platelet inhibition (%) p=0.009 P2Y12 Reactivity Units 50 100 150 200 250 300 p=0.007 Angiolillo DJ et al. Circulation. 2007;115:708-16. Angiolillo DJ et al. Am J Cardiol. 2008;101:440-5.

G R A V T A S Successful PCI with DES without major complication or GPIIb/IIIa use VerifyNow P2Y12 Assay 12-24 hours post-PCI PRU ≥ 230? Yes No Responder Non-Responder Random Selection R ACS A B C N = 1100 N = 1100 N = 583 “Tailored Therapy” clopidogrel 600-mg*, then clopidogrel 150-mg/day “Standard Therapy” placebo loading dose, then clopidogrel 75mg +placebo/day “Standard Therapy” placebo loading dose clopidogrel 75mg +placebo/day Clinical Follow-up And Platelet Function Assessment at 30 days, 6M Primary Endpoint: 6 month CV Death, Non-Fatal MI, ARC definite/prob ST Safety Endpoint: GUSTO Moderate or Severe Bleeding Cost-Effectiveness Analysis Price MJ, Berger PB, Angiolillo DJ, et al. Am Heart J 2009

TRIGGER-PCI Courtesy of F.J. Neumann Successful PCI with DES without major complication and NO GPIIb/IIIa use Post-PCI VerifyNow P2Y12 Assay (PRU) 2 - 4 hours after 1st MD of clopidogrel 75 mg at day 1 post-PCI N ~ 8800 Yes PRU ≥ 208? No Non-Responder Responder PRU ≥ 140? Random Selection A B C D E N = 1075 N = 1075 N = 550 N = 550 “Prasugrel arm” Prasugrel 60 mg LD Prasugrel 10 mg MD + Clopidogrel placebo “Clopidogrel arm” Placebo LD Clopidogrel 75 mg MD + Prasugrel placebo “Prasugrel arm” Prasugrel 60 mg LD Prasugrel 10 mg MD + Clopidogrel placebo “Clopidogrel arm” Placebo LD Clopidogrel 75 mg MD + Prasugrel placebo “Standard Therapy” Clopidogrel 75 mg Neumann Platelet function substudy: VerifyNow Assessment at day 2 (2 – 4 h after 1st MD of study drug) Clinical Follow-up and VerifyNow Assessment at 90 days, 180 days Primary Endpoint: 6 month CV Death and MI 16

Platelet Stimuli Platelet Aggregation AA COX-1 TxA2 TxA2 Collagen Shear rate P2Y12 ADP Thrombin Anti-II (gatrans) Anti X (xabans) Serotonin Thrombin Epinephrine PAR-1 antagonists E5555 SCH 530348 AA COX-1 TxA2 TxA2 TX inhibitors Ridogrel NCX-4016 S18886 GP IIb/IIIa integrin Platelet Aggregation

(oral ingestion of pro-drug) Pharmacogenetics of Cardiovascular Antithrombotic Therapy N S O Cl CH3 C Clopidogrel (oral ingestion of pro-drug) MDR-1 Platelet membrane receptors P2Y12 , GP IIb/IIIa, GP Ia Genetic targets CYP enzyme system Two sequential steps: One step: CYP3A4, CYP3A5, CYP2C9, CYP1A2 Both steps: CYP2B6, CYP2C19 Intestinal absorption Hepatic generation of active metabolite HOOC * HS N O Cl OCH3 Platelet inhibition Marin F & Angiolillo DJ. J Am Coll Cardiol 2009 ;54:1041-57

Intermediate phenotypes: PD/ PK Balancing Safety and Efficacy Inhibition of platelet aggregation High risk of ischemic events bleeding events Risk of any event “Sweet spot” Phenotypes: DM, ACS, CKD Intermediate phenotypes: PD/ PK Genotypes Ischemic risk Bleeding risk Ferreiro & Angiolillo. Thromb Haemost 2010 (in press)

Need for “individualized” treatment! Does one size fit all?? Need for “individualized” treatment!