1. TRIP: Thrombotic Risk Progression TRIP: Thrombotic Risk Progression Paul A. Gurbel, MD Director, Sinai Center for Thrombosis Research Sinai Hospital of Baltimore Associate Professor of Medicine Johns Hopkins University School of Medicine Baltimore, MD

2. Transition to an Unstable Coronary Syndrome is Marked by Hypercoagulability, Platelet Activation, Heightened Platelet Reactivity, and Inflammation: Results of the Thrombotic RIsk Progression (TRIP) Study Udaya S. Tantry, Kevin P. Bliden, Rolf P. Kreutz, Joseph DiChiara, Paul A. Gurbel, Sinai Center for Thrombosis Research, Sinai Hospital, Baltimore, MD, USA

3. MI, Stroke, CV Death Pathobiology of Coronary Thrombosis Atherosclerosis Atherothrombosis Plaque Rupture Inflammation (local and systemic)  Platelet Reactivity Hypercoagulability Vulnerable Blood Vulnerable Patient

4. Coronary atherothrombosis is a progressive disease influenced by inflammation, hypercoagulability and heightened platelet function, ultimately leading to catastrophic events after plaque rupture. Independent studies have linked ischemic events to: A) Hypercoagulability (increased fibrinogen and vWF; and high platelet-fibrin clot strength) (1,2). B) Ex vivo measurements of platelet activation and high platelet reactivity (3). C) Elevated inflammation markers, especially C-reactive protein (CRP) (4,5). To date, no single study prospectively evaluated all of these markers of pathophysiological processes together in patients with various stages of acute coronary syndrome. 1. Feinbloom D, Arterioscler Thromb Vasc Biol. 2005;25:2043-2053. 2. Gurbel PA, et al. J Am Coll Cardiol. 2005;46:1820-6. 3. Gurbel PA, et al. Rev Cardiovasc Med. 2006;7 Suppl 4:S20-8. 4. Willerson JT, et al. Circulation 2004;109:II2-II10. 5. Granger DN, et al. Hypertension. 2004;43:924-931. Background

5. To simultaneously study hypercoagulability, platelet function, and inflammation at various clinical stages of CAD. To determine whether changes in these markers indicate a transition in clinical disease state. OBJECTIVE

6. All patients treated with at least 81mg daily aspirin for  1 wk before enrollment. * All patients with MI and 30% of patients with UA treated with heparin prior to blood draw. Patients (n = 188) Patients Undergoing Stenting (n=117) (n = 71) Asymptomatic CAD (AS) CAD documented by prior angiography >6 months prior to enrollment. (n = 84) Stable Angina (SA) Undergoing PCI for at least 1 lesion. All lesions had  75% luminal diameter stenosis. (n = 26) Unstable Angina (UA) Typical symptoms associated with ECG changes, requiring emergent hospitalization and a  75% luminal diameter stenosis. (n = 7) Acute MI (MI) Typical symptoms and elevation of cardiac markers and a  75% luminal diameter stenosis.

7. Methods- Blood Sampling/Processing Symptomatic patients - indwelling femoral vessel sheath in cath. lab. Asymptomatic patients- venipuncture. Samples transferred to vacutainer tubes containing 40 USP lithium heparin for thrombelastography (TEG) and 3.2% citrate for flow cytometry and multianalyte profiling. Flow cytometry and TEG - conducted immediately. Multianalyte profiling plasma samples stored at -70 0 C until analysis.

8. Thrombelastography 1mL heparinized blood transferred to a vial containing kaolin (hydrated aluminum silicate), an intrinsic pathway activator. 500μL activated blood transferred to a heparinase vial to neutralize heparin. 360μL neutralized blood was immediately added to a heparinase-coated cup in the TEG.

9. Whole Blood Flow Cytometry GPIIb/IIIa receptors determined by multicolor analysis before and after stimulating with 5  M ADP: - Fluorescein isothiocyanate-conjugated PAC-1 antibody (recognizes active GPIIb/IIIa) - R-phycoerythrin conjugated CD41a antibody (recognizes total GPIIb/IIIa) Total and activated GPIIb/IIIa receptor levels expressed as mean fluorescence intensity (MFI).

10. MultiAnalyte Profiling (MAP) Fluorokine® MultiAnalyte Profiling (MAP) using a Luminex ® 100™ analyzer, (Rules Based Medicine, Inc., Austin, TX). Plasma samples incubated with fluorokine colored microspheres coated with a specific antibody directed against an analyte then washed and incubated with biotinylated antibodies specific for the analyte and phycoerythrin. Markers: CRP, fibrinogen, IL-1 , IL-3,4,5,7,8,10,and 18; MCP-1, MIP-1 , MIP-1 , RANTES, TNF- , TNF- , VCAM-1, VEGF, and vWF. As microsphere passes through detection chamber, a red laser excites internal dyes allowing the classification of the microsphere specific for each analyte. (Identification). A green laser excites phycoerythrin fluorescence associated with binding of the biotinylated antibody (Quantification).

11. Patient Demographics

12. 0 8 16 24 32 40 48 56 Asymptomatic Patients Stable Angina Unstable Angina Activated GPIIb/IIIa-Unstimulated (MFI) p=0.051 p<0.0001 Platelet Activation

13. Platelet Reactivity 0 50 100 150 200 250 300 ADP-Stimulated GPIIb/IIIa (MFI) p=0.002 p=0.14 Asymptomatic Patients Stable Angina Unstable Angina

14. Plasminogen Activator Inhibitor Type 1 (ng/mL) Fibrinolysis Inhibitor 0 45 90 135 180 p=0.01 p=0.28 p=0.02 Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction

15. Prothrombotic Markers 60 62 64 66 68 70 72 (mm) p=0.002 p=0.053 0 2 4 6 8 (min) p=0.98 p=0.11 Platelet-Fibrin Clot Strength Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction p=0.48 Time to Platelet-Fibrin Clot Formation (Indicator of Thrombin Generation Time) Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction P<0.001

16. Prothrombotic Markers vWF (ug/mL) 0 10 20 30 40 50 60 p=0.34 p=0.17 p=0.67 Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction Fibrinogen 0 1 2 3 4 5 6 (mg/mL) p=0.22 p=0.26 p=0.15 Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction

17. Inflammation Markers (ug/mL) 0 5 10 15 20 25 p=0.006 p=0.2 C-Reactive Protein Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction 0 5 10 15 20 25 (pg/mL) p<0.001 p=0.11 p=0.13 Interleukin- 8 Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction

18. Inflammation Markers (ng/mL) 0 3 6 9 12 15 p<0.001 p=0.8 p=0.64 Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction 0 7 14 21 28 35 (pg/mL) p<0.001 p=0.52 p=0.48 Asymptomatic Patients Stable Angina Unstable Angina Myocardial Infarction Macrophage Inflammatory Protein-1 Alpha

19. Biomarker Profile * P<0.05, **p<0.01; p values are between A-CAD vs. SA

20. Our data suggest that: A) A distinct pathophysiological state of heightened platelet reactivity to ADP, platelet activation, hypercoagulability, and inflammation marks the development of symptomatic cardiovascular disease from chronic stable disease. B) Ex vivo measurements indicating high tensile platelet-fibrin clot strength highlight the patient vulnerable to thrombosis. Our data support the concept that reactive and activated platelets influence inflammation creating a viscious cycle leading to a prothrombotic state culminating in unstable coronary disease. Further studies are required to investigate the primary mechanisms activating the prothrombotic state that destabilizes the disease. Conclusions Reactive Platelets Inflammation ? ? Prothrombotic State (Hypercoagulability) Unstable Coronary Disease

21. Question&Answer