Whole blood RNA signatures accurately classify agonist specific platelet function and highlight common biologic pathways. Deepak Voora, MD, Thomas L. Ortel MD, PhD, Joseph Lucas PhD, Jen-Tsan Chi, MD PhD, Richard C. Becker MD, Geoffrey S. Ginsburg, MD PhD Duke University Divisions of Cardiology and Hematology Institute for Genome Sciences & Policy Durham, NC USA November 15, 2010

Disclosure Information Deepak Voora, MD Whole blood RNA signatures accurately classify agonist specific platelet function and highlight common biologic pathways. FINANCIAL DISCLOSURE: None UNLABELED/UNAPPROVED USES DISCLOSURE: None

Variability in Response to Aspirin Variability in the clinical response to aspirin – 20-30% will experience an event on aspirin Variability in the laboratory response to aspirin Variability in platelet function assays associated with increased risk of events on aspirin Frelinger et al, Circulation 2009 Becker et al, JAMA 2006 ATC, Lancet 2002

COX-1 dependent platelet function Sensitive to COX-1 inhibition by ASA Agonist: Arachidonic acid Minimal variability on ASA Not heritable – no known genetic variants associated with function Gurbel et al, Circulation 2008, Faraday et al, Circulation 2007, Mathias et al, BMC Medical Genomics 2010 AA  Thromboxane Can be robust despite inhibition of COX-1with ASA Agonists: ADP, Collagen, Epi Highly variable on ASA Highly heritable GWAS identified genomic regions associated with function Non COX-1 dependent platelet function vs.

Rationale To use peripheral blood gene expression as a tool to identify novel pathways that underlie Non COX-1 dependent platelet function (NCDPF) on aspirin.

Visit #2 t = 14d Platelet function (post-aspirin) Peripheral blood RNA preserved in PAXgene tubes Visit #1 t = 0 Platelet function (pre-aspirin): Methods – Aspirin challenge study in healthy volunteers 325mg/day aspirin for 14 days Adherence: Medication log Telephone reminder Witnessed dose

Methods – Measuring NCDPF Light transmittance aggregometry – Agonists ADP 10uM Epinephrine 10uM Collagen 5 ug/ul Area under the aggregometry curve (AUC) – Measured in: % min

Baseline characteristics (n = 40) Age Median IQR Female N (%) BMI Median IQR Medications None/OC/other (N) (%) Race White/Black/Other (N) (%) 26 [24,31] 21 (53) 24.6 [26.7, 23.0] 35/4/1 87/10/3 29/6/5 73/15/13 BMI = body mass index; IQR = interquartile range; N = Number; OCP = oral contraceptives

Aspirin reduces NCDPF ADPCOLLAGENEPINEPHRINE Pre-aspirin AUC (% min)

Aspirin reduces NCDPF Pre-aspirin Post-aspirin ADPCOLLAGENEPINEPHRINE AUC (% min)

Methods – RNA analysis overview Affymetrix U133 Plus 2.0 microarray Bayesian factor analysis Linear regression followed by variable selection to identify factors that correlate with each agonist on aspirin – Leave one out cross validation Ingenuity Pathway Analysis of selected factors 54,000 probes 20 factors 6 factors 6 pathways Hypothesis: A whole blood RNA signature can be identified that correlates with NCDPF on aspirin

Factors correlate with NCDPF r = 0.87r = 0.84 P < for all correlations ADP COLLAGENEPINEPHRINE Predicted AUC AUC (% min)

Leave one out cross validation r = 0.87r = 0.84 P < for all correlations Predicted AUC r = 0.58r = 0.40 r = 0.56 ADP COLLAGENEPINEPHRINE AUC (% min)

Top pathways across 3 agonists IFN VEGF IGF-1 ADP Collagen Epinephrine

Top pathways across 2 agonists ADP Collagen Epinephrine N-GLYCAN SYNTHESIS P2YRTLR

Summary NCDPF on 325mg/day aspirin is highly variable in healthy volunteers RNA signatures can be used to develop a model that classifies the response to multiple platelet agonists Analysis of the underlying genes from the derived factors identifies known and novel biology in the platelet response to ADP, Collagen, and Epinephrine

Conclusions Common biological pathways contributing to NCDPF is a consistent finding: – Correlation between assays – Prior GWAS of platelet function demonstrate genomic regions contributing to multiple agonists

Conclusions Pathways analysis suggest that inflammatory pathways contribute to platelet function on aspirin RNA profiling – a testing platform used in commercial labs – may be used to identify those with heightened platelet function on aspirin.

Acknowledgments Collaborators – Geoffrey Ginsburg, MD, PhD (Cardiology, IGSP) – Richard Becker, MD (Cardiology, Hematology) – Thomas Ortel, MD, PhD (Hematology) – Jen-Tsan Chi, MD, PhD (IGSP) – Joseph Lucas PhD (IGSP) Funding: – IGSP, T32HL007101, UL1RR024128