1. Enhancement of thrombolysis in AMI is an unmet clinical need Increase the rate of reperfusion without increasing bleeding Reduce the time to complete reperfusion Reduce the incidence of reocclusion and reinfarction

2. Ultrasound - Facilitated Thrombolysis W. Douglas Weaver, M.D. Henry Ford Heart and Vascular Institute Detroit, MI on Behalf of the PLUS Investigators

3. Ultrasound Mechanism of Action Reversible change to fibrin structure Large diameter fiber bundles (d 1) disaggregate into a greater number of smaller diameter fiber bundles (d 2) Increased number of accessible binding sites Decreased flow resistance increases fluid dispersion into clot (Francis et al, Ultrasound Med Biol 1995;21:419 Braaten et al, Throm Haemost 1997;78:1063) Without Ultrasound With Ultrasound d 1 d 2

4. In vitro studies on thrombolytic agents exposed to ultrasound Reteplase, alteplase, tenecteplase (Centocor, Genentech) No effect on functional activity of the drugs (purified clot lysis assay) No effect on structural integrity of the molecules (SDS polyacrylamide GEL electrophoresis)

5. In vivo rabbit ilio-femoral artery studies of thrombolysis and ultrasound therapy Experimental occlusion using sutures (Suchkova et al, Circulation 2000; 101:2296) or electric current (Luo et al, Circulation 1996; 94:775) Treatment with streptokinase, ultrasound, or combination Enhanced reperfusion using combination by Doppler flow meter (Suchkova) and angiographic patency (Luo)

6. In Vivo Canine Experimental AMI Studies Angiographic Results: t-PA alone vs. t-PA + US (Luo et al, Circulation 2001; 104:II-725) t-PA alone t-PA+US 26% 72% 26% 78% 21% 78% 5 19 4 19 23 32 25 32 29 32 % of Animals p=0.003p=0.0004p=0.0001

7. Thrombolysis Augmented by Ultrasound in a Canine Model of AMI  Baseline left coronary angiogram and ECG  Thrombotically-occluded LAD  Reperfusion after 20 minutes of t-PA and ultrasound (Siegel et al, Circulation 2000; 101:2026)

8. Ultrasound System (Timi3 Systems, Inc.) Components Generator Transducer Ultrasound Energy Low frequency Low intensity Unfocused beam

9. Clinical Feasibility Study Argentine Pilot Study Hospital Italiano Policlinica Bancaria Instituto Cardiovascular de Rosario Hospital Santojanni Health authority (ANMAT) and IEC approvals 25 pts enrolled ST elevation AMI within 6 hrs of symptoms

10. Thrombolytic drug: reteplase or tenectaplase Ultrasound treatment for 60 min Coronary angiography at 90 min to determine TIMI flow grade Serial ECG’s to determine ST-segment resolution In-hospital and 30-day follow-up Clinical Feasibility Study – Protocol

11. HFCC Core Lab, N = 25 Infarct-related artery RCA in 15 pts (60%) LAD in 9 pts (36%) LCx in 1 pt (4%) Residual fixed stenosis of IRA 92% PCI performed in 13 pts after angiography Clinical Feasibility Study – Angiographic Results Clinical Feasibility Study – Angiographic Results

12. Clinical Feasibility Study - TIMI Flow Grade 1 3 5 16 21 % of Patients

13. Clinical Feasibility Study – ECG Results ST-Segment Elevation Recovery >50% At 60 minutes 13/24 pts (54%) At 90 minutes 13/21 pts (62%)

14. No Deaths, Strokes, or Re-infarctions No Unanticipated Major Adverse Events Cardiac Events – enrollment to 30 days PTCA/stent (non-urgent) 3 pts Recurrent ischemia/ urgent PTCA/stent or CABG3 pts Minor Related Adverse Events Skin blisters 3 pts Clinical Feasibility Study – Safety

15. PLUS – Perfusion by ThromboLytic and UltraSound Study design Randomized, double-blinded, controlled trial Standard thrombolysis alone vs. standard thrombolysis with ultrasound therapy 560 patients at up to 75 sites Standard thrombolytic agents: tenecteplase and reteplase

16. PLUS - Primary Objectives To assess whether the Timi3 Ultrasound System improves the proportion of patients achieving TIMI grade 3 flow at 60- minute angiography and To determine whether the Timi3 Ultrasound System improves the proportion of patients with ST-segment resolution at 60 minutes

17. PLUS - Patient Population Inclusion criteria Age between 18 and 75 years Onset of AMI within 6 hours ST-segment elevation greater or equal to 0.1 mV in two contiguous ECG leads Exclusion criteria Contraindications to thrombolytic agents Others

18. PLUS - Study Methods Patients enrolled at hospital presentation Stratified by anterior vs. other infarct Randomization: Thrombolytic and placebo ultrasound transducer Thrombolytic and active ultrasound transducer Simultaneous administration of thrombolytic and ultrasound therapy

19. PLUS - Trial Design