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

2. 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

3. 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: )

4. 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: , 2. Muller et al. Thromb Haemost. 2003;89:783,
3. Barragan et. al. Catheter Cardiovasc Interv. 2003;59:295;

5. 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 hrs hrs hrs day day
(Gurbel et al. J Am Coll Cardiol 37: 32 A, 2001; Eur Heart J, 2001; Am Heart J 2003;145:239-47)

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

7.  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.

8. 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)

9. Clopidogrel Resistance: World Experience
Investigators n Patients Clopidogrel Dose Resistance (mg Load)
1. Jaremo et al PCI % (J Invest Med. 2002;252:233)
2. Gurbel et al PCI % (Circulation 2003;107:2908)
3. Muller et al PCI %
(Thromb Haemost. 2003;89:783)
4. Mobley et al PCI % (Am J Cardiol. 2004;93:456) 5. Lepantalo et al PCI %
(Eur Heart J. 2004;25-476)
6. Angiolillo et al PCI %
(Thromb Res. 2005;115:101)
7. Matetzky et al PCI %
(Circulation 2004;109:3171.)
8. Dzieweierz PCI %
(Kardiol Pol ;62:108)
9. Gurbel et al PCI / % (J Am Coll Cardiol. 2005;45:1392)
10. Lev Et al PCI %
(J Am Coll Cardiol. 2006;47:27)
Total %

10. 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.

11. ? 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: )
(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 (%)

12. 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 Bliden et al  Platelet aggregation (pre-PCI) yr Post -PCI Events (J Am Coll Cardiol. 2006; (in press) on chronic clopidogrel
8. Cuisset et al  Platelet aggregation day Post-PCI events
(J Thromb Haemost ;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 day Post-PCI events (J Am Coll Cardiol. 2006;48: ) mg- less events
11. Hochholzer et al  Platelet aggregation (Upper quartile) day MACE (J Am Coll Cardiol 2006:48: )

13. 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 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 Mar 30; [Epub ahead of print])

14. 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 (Epub), . Taubert et al. Clin Pharmacol. 2006, .von Beckerth et al. Eur Heart J (epub))

15. 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

16. 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

17. TRA-Background
SCH (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 to have antithrombotic properties, with no increase in bleeding time or clotting times (aPTT, PT, ACT).

18. 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 Loading Dose  SCH
Or Placebo Loading Dose  Placebo
CABG
Medical Management
Quantify Postoperative Chest-Tube Drainage, Transfusions, and
Re-exploration
SCH
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

19. 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%)

20. 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

21. 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
p- value relative to placebo

22. 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

23. 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 SCH 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 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 Primary endpoint: composite of cardiovascular death, MI, rehospitalization for ischemia, urgent coronary revascularization, stroke

24. 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%)

25. PCI Cohort Non-TIMI Bleeding
Placebo
n=151
SCH
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%)

26. PCI Cohort MACE Results
Placebo
n= 151
SCH
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

27. PCI Cohort MACE Results
Placebo
n= 151
SCH
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

28. 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
p- value relative to placebo

29. PCI Cohort TIMI Bleeding SCH 530348 3.4 2.5 2.1 2.0 1.6 1.3 0.7 0.65%
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

30. PCI Cohort MACE Results
Placebo
n= 151
SCH
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

31. 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
p- value relative to placebo

32. 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

33. 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 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 was associated with:
Death/MACE: 32% overall; 46% with 40 mg
MI: 41% overall; 52% with 40 mg

34. 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 % p=NS %
Correlation R = 0.84, p<0.001
Aggregation (20 uM ADP)
Resistance % p=NS % Correlation R = 0.9, p<0.001
(Gurbel PA et al. Thromb Res Mar 30; [Epub ahead of print])

35. Sinai Center for Thrombosis Research Experience
The First Clopidogrel Resistance Study (300 mg): Importance of Time of Post-treatment Measurement
n= 92
2 Hours 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 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: )
 2005 Dr. Paul Gurbel. All rights reserved. This presentation may not be copied or reproduced without permission.

36. 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

37. 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

38. 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%

39. 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)

40. 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: )

41. 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)

42. 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 ; 4:2516-8)

43. 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