Clopidogrel Response Variability and Resistance Paul A. Gurbel, M.D. Sinai Center for Thrombosis Research Baltimore, Maryland

P2Y12: A Pivotal Platelet Receptor Thrombin, Collagen, Tx A2 Platelet Activation Degranulation ADP Amplification Tissue Factor Coagulation Cascade Thrombin AA Tx A2 P2Y12 *Platelet* Inflammation WBC Procoagulant Surface Activation P-selectin CD-40L ADP Degranulation Amplification GPIIb/IIIa Activation Aggregation

Laboratory Measurements of Clopidogrel Responsiveness Bisulfate Intestinal Absorption 15% CYP3A4,5,1A2 Conversion Active Thiol Metabolite 85% 1. ADP-Induced Platelet Aggregation - LTA (PRP) - TEG (Whole Blood) 2. Flow Cytometry - ADP- induced P-selectin, PAC-1 P2Y12 Specific 1. P2Y12 Reactivity Ratio- VASP Phosphorylation- Flow cytometry 2. VerifyNow-P2Y12 Assay Point-of-Care Clopidogrel Responsiveness Hydrolysis ADP ADP P2Y1 X P2Y12 Carboxyl Inactive Metabolite G12 Gi Gq Adenylyl Cyclase PLC Rho Kinase PI3-K cAMP Ca++ Mobilization Shape Change Rap-1b Akt VASP-P Granule Secretion ADP Release GPIIb/IIIa Activation, Platelet Aggregation (Gurbel et al. Nat Clin Pract Cardiovasc Med. 2006;3:387-95.)

Definition of Clopidogrel Resistance Failure to Inhibit Target/ Persistent Activity of the Target Antiplatelet Drug Non-responsiveness/Resistance = 1. Absolute change in (ADP) aggregation  10%  Aggregation = Max. baseline aggregation - Max. post-drug aggregation.1 2. Relative platelet inhibition < 10% 2 3 . > 50% PRI (P2Y12 Reactivity- VASP-P assay)3 Clopidogrel 1. Gurbel et al. Circulation. 2003;107:2908-13., 2. Muller et al. Thromb Haemost. 2003;89:783, 3. Barragan et. al. Catheter Cardiovasc Interv. 2003;59:295;

Clopidogrel Resistance/Response Variability: Initial Observations The Plavix Reduction Of New Thrombus Occurrence (PRONTO) Study Wide response variability 20 40 60 80 (%) Inhibition 300 mg Loading (3-24 hours prior to stenting) Prior to Stent 2 hrs 5 hrs 12 hrs 2 day 5 day (Gurbel et al. J Am Coll Cardiol 37: 32 A, 2001; Eur Heart J, 2001; Am Heart J 2003;145:239-47)

Clopidogrel Resistance: Importance of Time and Dose 300 mg 300 mg vs 600 mg 1 2 1Gurbel PA et al. Circulation. 2003;107:2908-2913. 2Gurbel PA et al. J Am Coll Cardiol. 2005;45:1392-1396.

 A (5 M ADP-induced Aggregation) at 24 h Clopidogrel Response Variability (300 vs. 600 mg): Importance of Dose (n = 194) 3 6 9 12 15 18 21 24 27 30 33 300 mg clopidogrel 600 mg clopidogrel Resistance = 28% (300 mg) Resistance = 8% (600 mg) Patients (%) ≤ −30 (−30,−20] (−20,−10] (−10,0] (0,10] (10,20] (20,30] (30,40] (40,50] (50,60] (60,70] > 70  A (5 M ADP-induced Aggregation) at 24 h (Gurbel PA et al. J Am Coll Cardiol. 2005;45:1392)  2005 Dr. Paul Gurbel. All rights reserved. This presentation may not be copied or reproduced without permission.

Variability in Post-Treatment Aggregation 5 M ADP 20 40 60 80 100 5uM ADP-Induced Platelet Aggregation (%) Relative Frequency (%) 21% 32% Cumulative Frequency Distribution: During 75 mg Clopidogrel Maintenance (Adapted from Bliden et al. J Am Coll Cardiol. 2007;49:657-66) Cumulative Frequency Distribution: 24 Hours after a 300mg Clopidogrel Load (Adapted from Gurbel et al.J Am Coll Cardiol. 2005;45:1392-6)

Clopidogrel Resistance: World Experience Investigators n Patients Clopidogrel Dose Resistance (mg Load) 1. Jaremo et al 18 PCI 300 28% (J Invest Med. 2002;252:233) 2. Gurbel et al 92 PCI 300 31-35% (Circulation 2003;107:2908) 3. Muller et al. 105 PCI 600 5-11% (Thromb Haemost. 2003;89:783) 4. Mobley et al 50 PCI 300 30% (Am J Cardiol. 2004;93:456) 5. Lepantalo et al. 50 PCI 300 40% (Eur Heart J. 2004;25-476) 6. Angiolillo et al. 48 PCI 300 44% (Thromb Res. 2005;115:101) 7. Matetzky et al. 60 PCI 300 25% (Circulation 2004;109:3171.) 8. Dzieweierz 31 PCI 300 23% (Kardiol Pol. 2005 ;62:108) 9. Gurbel et al. 192 PCI 300/600 8-32% (J Am Coll Cardiol. 2005;45:1392) 10. Lev Et al. 150 PCI 300 24% (J Am Coll Cardiol. 2006;47:27) Total 616 5-44%

A Common Misconception Lack of Inhibition (Responsiveness) = High Risk Patients with low pre-treatment function and low Inhibition have low post-treatment function and may be at Low Risk (overestimation of risk based on low responsiveness) Patients with high pre-treatment function and inhibition may still have high post-treatment function and may be at High Risk (underestimation of risk based on normal responsiveness) Post-treatment platelet function is a better predictor of ischemic events than platelet inhibition (responsiveness) (Samara et al. Thromb Res. 2005;115:89-94)  2005 Dr. Paul Gurbel. All rights reserved. This presentation may not be copied or reproduced without permission.

? Threshold for Ischemic Event ? Threshold for Ischemic Event What Does High Post-Treatment Platelet Reactivity Mean Clinically? Emergence of a New Quantifiable and Modifiable Risk Factor CREST Study PREPARE Post-Stenting Study (Gurbel et al. J Am Coll Cardiol. 2005;46:1827-32) (Gurbel et al. J Am Coll Cardiol. 2005;46:1820-6) No Ischemic Event (n=154) No ST (n=100) Ischemic Events (n=38) Ischemic Events (n=38) ST (n=20) ? Threshold for ST ? Threshold for Ischemic Event ? Threshold for Ischemic Event Relative Frequency (%) 6 month Follow-up Relative Frequency (%) Relative Frequency (%) p<0.001 for mean values between groups p<0.02 for mean values between groups p<0.02 for mean values between groups 20 uM ADP Aggregation (%) 20 uM ADP Aggregation (%) 20 uM ADP Aggregation (%) 100 No Ischemic Event (n=77) Ischemic Event (n=23) 80 Chronic Clopidogrel Therapy Undergoing PCI -Pretreatment Platelet Function 1 Year Follow-up 60 Relative Frequency (%) 40 (Bliden et al. J Am Coll Cardiol. 2007;49:657-66) 20 p<0.001 for mean values between groups 20 40 60 80 100 5 uM ADP Aggregation (%)

Studies Linking Ex-Vivo Platelet Function to Clinical Events Study Results Clinical Relevance 1. Barragan et al.  P2Y12 reactivity ratio (VASP-P levels) Stent Thrombosis (Catheter Cardiovasc Interv. 2003;59::295) 2. Ajzenberg et al.  Shear- Induced platelet aggregation Stent Thrombosis (J Am Coll Cardiol. 2005;45:1753) 3. Gurbel et al.  P2Y12 reactivity ratio (CREST Study)  ADP- induced aggregation Stent Thrombosis (J Am Coll Cardiol. 2005;46:1827)  Stimulated GPIIb/IIIa expression 4. Matzesky et al.  ADP-Induced platelet aggregation Recurrent Cardiac (Circulation.2005;109:3171) Events (4th quartile) 5. Gurbel et al.  Periprocedural platelet aggregation Myonecrosis and (CLEAR PLATELETS and CLEAR PLATELETS Ib) Inflammation Marker (Circulation. 2005;111:1153, J Am Coll Cardiol;2006 (in press) Release 7. Bliden et al.  Platelet aggregation (pre-PCI) 1 yr Post -PCI Events (J Am Coll Cardiol. 2006; (in press) on chronic clopidogrel 8. Cuisset et al.  Platelet aggregation 30-day Post-PCI events (J Thromb Haemost. 2006;3:542-9) 9. Lev et al. Clopidogrel/Aspirin resistant patients Post-PCI Myonecrosis (J Am Coll Cardiol. 2006;47:27) 10. Cuisset e al.  Platelet aggregation 30-day Post-PCI events (J Am Coll Cardiol. 2006;48:1339-45) 600mg- less events 11. Hochholzer et al.  Platelet aggregation (Upper quartile) 30 day MACE (J Am Coll Cardiol 2006:48:1742-50)

Variable Post-Treatment: P2Y12 Reactivity by VASP- P vs Variable Post-Treatment: P2Y12 Reactivity by VASP- P vs. Reactivity ADP-Induced Platelet Aggregation (n=40) (Patients Loaded with 300 - 600 mg Clopidogrel) 0.0 20.0 40.0 60.0 80.0 100.0 Post-Treatment Platelet Aggregation (20 uM ADP) (%) P2Y12 Reactivity Ratio (%) R=0.703, p<0.001 0.0 20.0 40.0 60.0 80.0 100.0 Post-Treatment Platelet Aggregation (5uM ADP) (%) P2Y12 Reactivity Ratio (%) R= 0.63, p<0.001 (Gurbel PA et al. Thromb Res. 2007 Mar 30; [Epub ahead of print])

Mechanism of Clopidogrel Response Variability: Limited absorption capacity with ceiling effect at 600mg loading dose Esterases 85% Clopidogrel Bisulfate Inactive Carboxylic Acid Metabolite Intestinal Absorption ? P-glycoprotein (MDR1 3435T genotype)1 15% CYP3A4 CYP3A5 CYP2C19 Drug-drug interaction with lipophilic statins, IVS10+12G>a polymorphism Hepatic P450 Cytochromes Genetic polymorphisms Genetic polymorphisms CYP1A2 Smoking Multistep Conversion Active Thiol Metabolite P2Y12 Receptor Inhibition of Platelet Aggregation (Wide Response Variability) ~1.4x 150mg/d vs. 75mg/d for 30days Post-PCI2 ~30% 75mg/day for 30days Post-PCI ~30%-40% 75mg/day for 5-7days volunteers ~30%-40% 300 mg load Post-PCI ~30%-50% 600 mg load Post-PCI (Gurbel PA et al. Thromb Res. 2006 (Epub), . Taubert et al. Clin Pharmacol. 2006, .von Beckerth et al. Eur Heart J. 2007 (epub))

Summary Multiple studies have confirmed marked response variability to clopidogrel therapy and a significant prevalence of resistance/non-responsiveness. Aggregometry remains the gold standard in detection. VASP-P is the most specific available measurement of incomplete P2Y12 blockade. A 600 mg loading dose is associated with less resistance, less response variability, and lower post-treatment aggregation- no conclusive data to support higher doses than 600 mg. Current data suggest that high ex vivo platelet reactivity to ADP/incomplete P2Y12 inhibition are risk factors for post-stenting ischemic events including SAT. EMERGING CARDIOVASCULAR RISK FACTOR

Platelet Receptors Platelet Platelet PAR-1 Thrombin PAR-4 GP IIb/IIIa Fibrinogen GP IIb/IIIa P2Y1 ADP P2Y12 GP IIb/IIIa TBX A2 TBXA2-R Epinephrine EPI-R Serotonin 5HT2A GP VI Collagen Anionic phospholipid surfaces GP Ia

TRA-Background SCH 530348 (himbacine derivative) is an oral, potent, selective thrombin receptor antagonist (TRA) being developed for the prevention and treatment of atherothrombosis. Preclinical and early clinical studies have demonstrated SCH 530348 to have antithrombotic properties, with no increase in bleeding time or clotting times (aPTT, PT, ACT).

Study Design Non-Urgent PCI or Cath possible PCI (All Receive Aspirin) Randomization #1 — 3:1 SCH530348:Placebo (Single Loading Dose) Sequential Groups: 1=10 mg; 2=20 mg; 3=40 mg, or Placebo Cardiac Catheterization Planned PCI (All Receive Clopidogrel and Antithrombin) No PCI** Randomization #2 1:1:1 Maintenance Therapy Once Daily for ~ 60 days SCH 530348 Loading Dose  SCH 530348 Or Placebo Loading Dose  Placebo CABG Medical Management Quantify Postoperative Chest-Tube Drainage, Transfusions, and Re-exploration SCH 530348 0.5 mg n~100 1 mg n~100 2.5 mg n~100 Placebo n~100 Safety: TIMI Major plus Minor Bleeding Efficacy: Death/MACE Safety: TIMI Major plus Minor Bleeding * Primary Evaluable Cohort **Secondary Evaluable Cohort

PCI Cohort Results Placebo SCH 530348 All 10 mg 20 mg 40 mg Number 151 422 129 120 173 TIMI Major/Minor 5 (3.3%) 12 (2.8%) 2 (1.6%) 3 (2.5%) 7 (4.0%) TIMI Major 2 (1.3%) 3 (0.7%) 1 (0.6%) TIMI Minor 3 (2.0%) 9 (2.1%) 6 (3.4%) Non-TIMI bleeding 48 (32%) 170 (40%) 46 (36%) 51 (43%) 73 (42%)

CABG Cohort Bleeding Placebo (n=24) SCH 530348 All (n=51) 10 mg Any Bleed 24 52 10 18 TIMI Major/Minor 19 (79%) 48 (92%) 9 (90%) 17 (94%) 22 (92%) Non-TIMI 8 (33%) 18 (35%) 3 (30%) 6 (33%) 9 (39%) Transfusion PRBC 11 (46%) 32 (62%) 8 (80%) 9 (50%) 15 (63%) >2 Units 5 9 2 Chest Tube Drainage (ml) 996 988 1393 1015 870 Re-exploration 3 1

PCI Cohort 60-Day Death or MACE SCH 530348 8.6% 8.5% 5.9% 5.0% 4.6% 10% p = 0.98 8.6% 8.5% 8% p = 0.26 5.9% p = 0.25 6% 5.0% p = 0.15 4.6% 4% 2% Placebo n=151 All TRA n=422 10 mg n=129 20 mg n=120 40 mg n=173 SCH 530348 p- value relative to placebo

Platelet Aggregation Substudy Subjects with >80% IPA to 15 M TRAP 100% 96 30 minutes 60 minutes 90 minutes 120 minutes 82 80% 68 60% 54 53 46 43 40% 29 21 20% 6 Placebo n=23 10 mg n=15 20 mg n=18 40 mg n=33 SCH 530348

Conclusions TRA- PCI provided rationale for two large scale multinational, randomized, double-blind, placebo-controlled phase 3 studies to evaluate the safety and efficacy of SCH530348 in addition to standard of care therapy: The Thrombin Receptor Antagonist in Secondary Prevention of atherothrombotic events (TRA2P-TIMI 50) trial (n=19,500) -randomized to 2.5 mg qd SCH 530348 or placebo plus standard medical care  (including aspirin and a thienopyridine) for a minimum of one year -Primary endpoint: composite of cardiac death, MI, CVA, UTVR Thrombin Receptor Antagonist in Acute Coronary Syndrome (TRA-ACS) (n=10,000 with non-ST segment elevation ACS) - randomized to placebo vs. 40 mg load/2.5 mg qd SCH 530348 - Primary endpoint: composite of cardiovascular death, MI, rehospitalization for ischemia, urgent coronary revascularization, stroke

PCI Cohort Results Placebo SCH 530348 All 10 mg 20 mg 40 mg Number 151 422 129 120 173 TIMI Major/Minor 5 (3.3%) 12 (2.8%) 2 (1.6%) 3 (2.5%) 7 (4.0%) TIMI Major 2 (1.3%) 3 (0.7%) 1 (0.6%) TIMI Minor 3 (2.0%) 9 (2.1%) 6 (3.4%) Non-TIMI bleeding 48 (32%) 170 (40%) 46 (36%) 51 (43%) 73 (42%)

PCI Cohort Non-TIMI Bleeding Placebo n=151 SCH 530348 All n=422 10 mg n=129 20 mg n=120 40 mg n=173 Overall 48 (32%) 170 (40%) 46 (36%) 51 (43%) 73 (42%) Vascular Puncture 20 (13%) 64 (15%) 16 (12%) 22 (18%) 26 (15%) Arterial Access 19 (13%) 58 (14%) 14 (11%) 20 (17%) 24 (14%) Contusion/Bruise 17 (11%) 13 (10%) 31 (18%) Incidental Cuts 10 (7%) 39 (9%) 5 (4%) 16 (13%) 18 (10%) Epistaxis 12 (8%) 23 (5%) 8 (6%) 6 (5%) 9 (5%) GI 2 (1%) 8 (2%) 2 (2%) 1 (1%) 5 (3%) Gingival 5 (1%) Genitourinary 13 (3%) 4 (3%) 3 (2%) Discontinue for AE 2 (1.3%) 6 (1.4%) 1 (0.8%) 4 (2.3%)

PCI Cohort MACE Results Placebo n= 151 SCH 530348 All n=422 10 mg n= 129 20 mg n=120 40 mg n=173 Death/MACE/Stroke 13 (8.6%) 26 (6.2%) 12 (9.3%) 6 (5.0%) 8 (4.6%) Death/MACE* 25 (5.9%) 11 (8.5%) Death/MI 11 (7.3%) 19 (4.5%) 7 (5.4%) 5 (4.2%) 7 (4.0%) Death 2 (0.5%) 1 (0.8%) 1 (0.6%) MACE 24 (5.7%) MI 18 (4.3%) 6 (3.5%) Recurrent ischemia 1 (0.7%) 1 (0.2%) Revascularization 6 (1.4%) 3 (2.3%) 2 (1.2%) Stroke MACE = Major Adverse Cardiac Event (myocardial infarction, ischemia requiring hospitalization, coronary revascularization) MI = Myocardial Infarction * = primary efficacy endpoint

PCI Cohort MACE Results Placebo n= 151 SCH 530348 All n=422 10 mg n= 129 20 mg n=120 40 mg n=173 Death/MACE/Stroke 13 (8.6%) 26 (6.2%) 12 (9.3%) 6 (5.0%) 8 (4.6%) Death/MACE* 25 (5.9%) 11 (8.5%) Death/MI 11 (7.3%) 19 (4.5%) 7 (5.4%) 5 (4.2%) 7 (4.0%) Death 2 (0.5%) 1 (0.8%) 1 (0.6%) MACE 24 (5.7%) MI 18 (4.3%) 6 (3.5%) Recurrent ischemia 1 (0.7%) 1 (0.2%) Revascularization 6 (1.4%) 3 (2.3%) 2 (1.2%) Stroke MACE = Major Adverse Cardiac Event (myocardial infarction, ischemia requiring hospitalization, coronary revascularization) MI = Myocardial Infarction * = primary efficacy endpoint

TIMI Major/Minor Bleeding PCI Cohort TIMI Major/Minor Bleeding 5% p = 0.73 4.0% 4% 3.3% p = 0.77 p = 0.70 2.8% 3% 2.5% p = 0.35 2% 1.6% 1% Placebo n=151 All TRA n=422 10 mg n=129 20 mg n=120 40 mg n=173 SCH 530348 p- value relative to placebo

PCI Cohort TIMI Bleeding SCH 530348 3.4 2.5 2.1 2.0 1.6 1.3 0.7 0.6 5% TIMI Minor TIMI Major 4% 3.4 3% 2.5 2.1 2.0 2% 1.6 1.3 0.7 1% 0.6 Placebo n=151 All TRA n=422 10 mg n=129 20 mg n=120 40 mg n=173 p = ns for pairwise comparisons SCH 530348

PCI Cohort MACE Results Placebo n= 151 SCH 530348 All n=422 10 mg n= 129 20 mg n=120 40 mg n=173 Death/MACE/Stroke 13 (8.6%) 26 (6.2%) 12 (9.3%) 6 (5.0%) 8 (4.6%) Death/MACE* 25 (5.9%) 11 (8.5%) Death/MI 11 (7.3%) 19 (4.5%) 7 (5.4%) 5 (4.2%) 7 (4.0%) Death 2 (0.5%) 1 (0.8%) 1 (0.6%) MACE 24 (5.7%) MI 18 (4.3%) 6 (3.5%) Recurrent ischemia 1 (0.7%) 1 (0.2%) Revascularization 6 (1.4%) 3 (2.3%) 2 (1.2%) Stroke MACE = Major Adverse Cardiac Event (myocardial infarction, ischemia requiring hospitalization, coronary revascularization) MI = Myocardial Infarction * = primary efficacy endpoint

PCI Cohort 60-Day Death or MI SCH 530348 7.3% 5.4% 4.5% 4.2% 4.0% 10% 8% 7.3% p = 0.53 p = 0.19 6% 5.4% p = 0.28 4.5% p = 0.20 4.2% 4.0% 4% 2% Placebo n=151 All TRA n=422 10 mg n=129 20 mg n=120 40 mg n=173 SCH 530348 p- value relative to placebo

Myocardial Infarction PCI Cohort Myocardial Infarction 8% 4% 2% 6% 10% Placebo 10mg 20mg 40mg p = 0.52 p = 0.28 p = 0.12 1 2 3 4 5 6 7 Days

Conclusions TRA was not associated with an increase in TIMI major, minor, or non-TIMI bleeding Using 15 M TRAP-induced platelet aggregation: 40 mg loading dose of SCH 530348 achieved ≥ 80% IPA in 1-2 hours in 68-96% subjects 1 mg and 2.5 mg maintenance doses sustained ≥ 80% IPA at 30 and 60 days in all subjects While not statistically significant, SCH 530348 was associated with: Death/MACE: 32% overall; 46% with 40 mg MI: 41% overall; 52% with 40 mg

Clopidogrel Resistance: Maximum Aggregation vs. Final Aggregation PCI Patients, n = 100 5 M ADP-Induced Platelet Aggregation 20 M ADP-Induced Platelet Aggregation Final Aggregation Maximum Aggregation Final Aggregation 50 Maximum Aggregation 60 p = 0.001 p = 0.003 p = 0.02 50 p = 0.006 40 40 30 Absolute Change in Aggregation (%) Absolute Change in Aggregation (%) 30 20 20 p = ns p = ns 10 10 Total Patients Non- Responders Responders Total Patients Non-Responders Responders Maximum Final Aggregation Aggregation Aggregation (5uM ADP) Resistance 23% p=NS 17% Correlation R = 0.84, p<0.001 Aggregation (20 uM ADP) Resistance 30% p=NS 27% Correlation R = 0.9, p<0.001 (Gurbel PA et al. Thromb Res. 2007 Mar 30; [Epub ahead of print])

Sinai Center for Thrombosis Research Experience The First Clopidogrel Resistance Study (300 mg): Importance of Time of Post-treatment Measurement n= 92 2 Hours 24 Hours Resistance = 31%  Aggregation (%) % of Patients 10 20 <= -30 (-30,-20] (-20,-10] (-10,0] (0,10] (10,20] (20,30] (30,40] (40,50] (50,60] >60 Resistance  Aggregation (%) % of Patients 12 24 <= -30 (-30,-20] (-20,-10] (-10,0] (0,10] (10,20] (20,30] (30,40] (40,50] (50,60] >60 Resistance = 63% Resistance Sinai Center for Thrombosis Research Experience 5 Days 30 Days Resistance = 15%  Aggregation (%) % of Patients 14 28 <= -30 (-30,-20] (-20,-10] (-10,0] (0,10] (10,20] (20,30] (30,40] (40,50] (50,60] >60 Resistance  Aggregation (%) % of Patients 11 22 <= -10 (-10,0] (0,10] (10,20] (20,30] (30,40] (40,50] (50,60] >60 Resistance = 31% Resistance (Gurbel et al. Circulation. 2003;107: 2908-2913)  2005 Dr. Paul Gurbel. All rights reserved. This presentation may not be copied or reproduced without permission.

5 μM ADP-Induced Aggregation (%) 20 μM ADP-Induced Aggregation (%) Relation of Platelet Reactivity to Long-Term Ischemic Events in PCI Patients (n=352) - 5 μM ADP-Induced Aggregation (%) 70 60 50 40 30 20 10 Without Events With Events (n=250) (n=82) 20 μM ADP-Induced Aggregation (%) 90 80 ? Overtreated ? Bleeding Risk Blockade of the platelet GP IIb/IIIa receptor reduces death and ischemic complications in patients undergoing PCI The role of periprocedural GP IIb/IIIa inhibition in patients undergoing planned PCI was established by seven randomized, placebo-controlled trials that enrolled more than 15,000 patients. Treatment effect was achieved early and maintained over the long-term. Absolute reductions in the 30-day risk of death, MI, or repeat urgent revascularization ranged from 1.5% to 6.5% with some variability among treatment agents used (abciximab, eptifibatide, or tirofiban). Gurbel et al. AHA 2007

Thrombelastograph Platelet Mapping Analysis Parameters Platelet aggregation in response to ADP or AA is calculated with computerized software on the basis of the formula: % Inhibition = [1- (MAADP or AA− MAfibrin)/(MAthrombin − MAfibrin)] × 100 MAThrombin = measure of maximum thrombin-induced platelet-fibrin clot strength; MAFibrin = contribution of fibrin to clot strength; MAADP or AA = represents clot strength after adenosine diphosphate (ADP) or arachidonic acid (AA) stimulation. Gurbel et al. J Am Coll Cardiol. 2005;46:1820-6

Aggregation Curves of Clopidogrel Responders and Non-Responders PRE 24 h POST Responder 300mg 5 uM ADP 5 uM ADP  Agg = 43 and 37% % Aggregation % Aggregation Non-Responder 900mg 5 uM ADP 5 uM ADP % Aggregation % Aggregation  Agg = 5 and -13%

Relation of Platelet Aggregation Measured by Light Transmittance Aggregometry and Thrombelastography Platelet Mapping Assay 100 R=0.821, p<0.001 80 60 LTA (5 M ADP) (%) 40 20 20 40 60 80 100 Thrombelastography (MAADP) (%) (Bliden KP et al. Presented World Congress of Cardiology, Barcelona. 2006)

P2Y12 Reactivity Measured by VASP-P Indicator of Receptor Inhibition by Clopidogrel Biocytex Assay 1. Stimulate Platelets with PGE1  Maximum VASP Phosphorylation: MFI PGE1 2. Stimulate with PGE1 + ADP  MFI PGE1 + ADP If P2Y12 Receptors are unblocked, VASP Phosphorylation Levels Fall after ADP Stimulation Reactivity Ratio = 100 x MFI(PGE1) - MFI(PGE1+ADP) MFI(PGE1) (Gurbel PA et al. J Am Coll Cardiol. 2005:46:1827-32)

Clopidogrel Response Variability As Measured by VASP-Phosphorylation Clopidogrel Response Variability and Impact of P2Y1 on Maximum ADP-Induced Aggregation Platelet Reactivity Index (%) Light Transmission (%) Response Variability P2Y1 Contribution Platelet Aggregation (%) Clopidogrel Response Variability As Measured by VASP-Phosphorylation In vitro Effect of ARC-C69931MX on Platelet Aggregation Induced by 5 uM ADP (Aliel et al. J Thromb Haemost. 2005;3:85-92)

VerifyNow-P2Y12 Assay Assay is based on the ability of activated platelets to bind to fibrinogen. Adenosine diphosphate and prostaglandin E1 are added to activate platelets. Light transmittance increases as activated platelets bind and aggregate fibrinogen-coated beads. The instrument measures this change in optical signal and reports results in P2Y12 reaction units (PRU). Relation of VerifyNow P2Y12 Assay and Aggregometry Assay (van Werkum J.W. et al. Journal Thromb Haemost. 2006; 4:2516-8)

The Future Simple and reproducible point-of-care methods to study ex vivo platelet reactivity and its relation to adverse events: What are the critical receptors/pathways that drive events? Ongoing: On Horizon: Oral reversible P2Y12 antagonists (AZD6140) Blockade of: Parenteral P2Y12 antagonists (cangrelor) P2Y1 Potent irreversible P2Y12 antagonists (prasugrel) GPIb, Increased clopidogrel maintenance dose GPIV Thrombin receptor antagonists Personalized Therapy: Defining the patient’s propensity for thrombosis and adjust antiplatelet therapy to achieve a target response: ? Reduced stent thrombosis ? Reduced post-discharge ischemic events ? Reduced In-hospital ischemic events post-PCI