Paul Gurbel, Kevin Bliden, Tania Gesheff, Yvonne Kreutz, Udaya Tantry Sinai Center for Thrombosis Research Sinai Hospital of Baltimore Baltimore, Maryland, U.S.A. A Mechanistic Link Between High Platelet Reactivity, Inflammation Marker Release and Myonecrosis in Patients Undergoing Stenting: CLEAR PLATELETS Ib Study

Disclosures Research Grants Honoraria - Schering - Schering - Millennium - Millennium - Astra Zeneca - Astra Zeneca - Bayer - Bayer - NIH - Haemoscope - Medtronic - Boston Scientific This study was supported by Millennium and Schering

Background Thrombosis and inflammation influence the development of ischemic events following PCI. Recent trials established the effectiveness of GPIIb/IIIa inhibitors with clopidogrel and aspirin in reducing thrombotic events following PCI. However, controversy remains regarding the optimal antiplatelet strategy for elective stenting. Response variability to clopidogrel has been demonstrated in patients undergoing elective stenting. 1,2 1. Gurbel et al. J Am Coll Cardiol 2005;45: Gurbel et al. Circulation. 2003;107:

Background In the CLEAR PLATELETS Study a loading dose of clopidogrel with a GPIIb/IIIa inhibitor (eptifibatide) produced: - superior platelet inhibition - lower myocardial necrosis compared to 300 mg or 600 mg clopidogrel loading alone. 1 Potential anti-inflammatory effects of clopidogrel and GPIIb/IIIa inhibitors have been recently reported. 2 The comparative anti-inflammatory effects of clopidogrel ± GPIIb/IIIa blockade during stenting are unknown. 1. Gurbel PA. et al Circulation 2005; 111: Nannizzi-Alaimo L et al. Circulation 2003; 107:

The primary objective of the current investigation (CLEAR PLATELETS -1b): Compare the effects of the antiplatelet regimens employed in the CLEAR PLATELETS study on early inflammation and cardiac marker release after PCI. The secondary objective: Study relation between platelet inhibition and inflammation Objectives

Study Design of CLEAR PLATELETS-1b CLEAR PLATELETS CLEAR PLATELETS 1b N = 60

Study Design of CLEAR PLATELETS-1b 2 X 2 Factorial Elective Stent Study Clopidogrel 300 mg in lab (n = 60) Clopidogrel 600 mg in lab (n = 60) - Eptifibatide (n = 30) + Eptifibatide (n = 30) - Eptifibatide (n = 30) + Eptifibatide (n = 30) Heparin per ESPIRIT dosing, Clopidogrel 75 mg qd, ASA 325 mg qd Laboratory Measurements - Before and hours Post-Stenting Platelet Aggregation Light transmittance ( 5 and 20  M ADP) Platelet Activation Markers ADP-stimulated: - active GPIIb/IIIa - P-selectin by flow cytometry Inflammation Markers - C-reactive protein - Tumor necrosis factor -  by ELISA Necrosis Markers - Creatinine Kinase MB - Troponin I - Myoglobin Biomarker Profile by Luminex®

Results - Patient Demographics Clopidogrel (n=60) Clopidogrel + Eptifibatide(n=60) p-value Age (years)63+/-1465+/-12ns Race (Caucasian) (%)70 ns Gender (Male) (%)6370ns BMI29+/-530+/-6ns Risk Factors (%) Smoking4538ns Family history of CAD3836ns Hypertension6073ns Hyperlipidemia8087ns Diabetes5053ns Prior Myocardial Infarction2535ns Prior CABG2520 Prior PTCA Baseline Medications (%) Beta blockers8797ns ACE Inhibitors6275ns Calcium blockers2027ns Lipid lowering agents CYP3A4 metabolized6257ns Non -CYP 3A4 metabolized20 ns

Results - Procedural Characteristics Clopidogrel (n=60) Clopidogrel + Eptifibatide (n=60) p-value Length of procedure (min.)60+/-2160+/-22ns Ejection Fraction (%)54+/-852+/-9ns Number of vessels treated1.2+/ /-0.6ns Lesion Morphology Denovo (%)9387ns Lesion Location (%) LAD3233ns CX2328ns RCA4032ns SVG57 Stent Types (%) Drug eluting7067ns Bare metal2327ns PTCA only74ns Reference vessel diameter (mm) 3.1+/-0.53+/-0.4ns Total lesion length (mm)20.2+/ /-13.0ns Pre-stenosis (%)85+/-784+/-7ns Post-stenosis (%)3+/-14+/-2ns

Relative Change in Platelet Aggregation, P-Selectin and Active GPIIb/IIIa Expression: Four Treatment Groups Relative Change (%) 300 C600 C300 C+E600 C+E p=0.04 p=0.004 p=nsp<0.01 p=ns p<0.001 LTA LTA Stimulated Stimulated 5 uM ADP 20 uM ADP GPIIb/IIIa P-selectin

Relative Change in Plasma TNF-  and CRP: Four Treatment Groups TNF-  CRP Relative Change (%) 300 C600 C300 C+E600 C+E p<0.001 p = ns p = p = ns

Myocardial Necrosis Markers: Clopidogrel vs. Clopidogrel + Eptifibatide CKMB (>1-3xULN) CKMB (>3xULN) Tn - I (>ULN) Myoglobin (>2xULN)

Clopidogrel Clopidogrel + Eptifibatide Relative Change (%) p<0.001 p=0.095 p<0.001 LTA- LTA- Stimulated Stimulated TNF-Alpha CRP 5uM ADP 20uM ADP GPIIb/IIIa P-Selectin Relative Change in Aggregation, P-Selectin, Active GPIIb/IIIa, TNF- , CRP: Clopidogrel vs. Clopidogrel + Eptifibatide

Relation of Necrosis Marker Release to Plasma CRP and TNF-  Post-Treatment CRP (mg/L) CK-MB (NL) CK-MB (>3X ULN) CK-MB (>1-3X ULN) p=0.01p=0.09 p= Post-Treatment TNF-Alpha (ng/mL) CK-MB (NL) CK-MB (>1-3X ULN) CK-MB (>3X ULN) p<0.001

Absolute Change in Platelet Aggregation and (%) Patients Treated with Eptifibatide in CRP Quartiles < >4.7 Absolute Change in Platelet Aggregation (%) p=0.003 CRP (mg/L) Quartiles < >4.7 CRP (mg/L) Quartiles Frequency of Patients Treated With Eptifibatide (%) p=0.06 p=0.004 p<0.001

Clopidogrel Clopidogrel + Eptifibatide Biomarker Profile by MAP: Clopidogrel vs. Clopidogrel + Eptifibatide MMP-9 < vWF Tissue Factor < Fibrinogen RANTES MIP-Alpha 0.03 TNF-Alpha CRP < VCAM CD40-L Fatty Acid Binding Protein < Myoglobin < Relative Change (%)

Conclusions Clopidogrel + eptifibatide produces a distinctly different periprocedural biomarker profile than clopidogrel alone in elective stenting: significant inhibition of inflammation and myocardial necrosis marker release. Inhibition of platelet aggregation and active GP IIb/IIIa expression but not p-selectin expression was associated with inhibition of inflammation. The mechanistic and clinical implications of attenuated periprocedural inflammation and myocardial necrosis with a strategy of GPIIb/IIIa inhibition warrant further investigation. Necrosis GPIIb/IIIa Inhibition Inflammation