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Supplementary Information Development of Fatty Acid JPET#234781 Supplementary Information Development of fatty acid derivatives of aspirin for inhibition of platelet aggregation Jahnabi Roy c, Reheman Adili e, Richard Kulmacz f, Michael Holinstat e,g, and Aditi Das* a, b, d a Department of Comparative Biosciences, b Department of Biochemistry, Center for Biophysics and Quantitative Biology, Division of Nutritional Sciences, Department of Bioengineering c Department of Chemistry d Beckman Institute for Advanced Science, f The Department of Internal Medicine, Texas Health Science Center at Houston, McGovern Medical School, Houston, TX 77225-0708, e Department of Pharmacology, g Division of Cardiovascular Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, c, a University of Illinois Urbana-Champaign, Urbana IL 61801. Journal: THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS (JPET) CONTENTS Fig S1: Inhibition of platelet aggregation by various inducers S2 Fig S2: Inhibition of platelet aggregation by various inducers -2 S3 Fig S3: TXB2 formed in platelet aggregometry samples S4 Fig S4: Inhibition of platelet aggregation by LA, EPA and DHA S5 Fig S5: Coupled assay schematic and results for parent fatty acids S6 Fig S6: Coupled assay for LA, EPA, DHA and aspirin S7 Fig S7: Representative HPLC trace of DHA anhydride hydrolysis S8 Fig S8: Representative HPLC trace of EPA anhydride hydrolysis S9 Fig S9: Representative HPLC trace of LA anhydride hydrolysis S10 Table 1: NMR and MS characterization of compounds S11 Fig S10: NMR of DHA anhydride S12 Fig S11: NMR of EPA anhydride S13 Fig S12: NMR of LA anhydride S14 S1 JPET#234781 Supplemental figure 1: Inhibition of platelet aggregation in washed platelets by anhydrides (10 μM) in the presence of (A) 1nM Thrombin (B) 50 μM PAR4-AP (C) 1 μM PAR1-AP (D) 1 μM ADP (E) 1 μM U46619 (thromboxane A2 analog). Results show no significant decrease in platelet aggregation using these agonists. The results show data from n=3. S2 JPET#234781 Supplemental Figure 2: Inhibition of platelet aggregation in platelet rich plasma by anhydrides (10 μM) in the presence of (A) 1 μM ADP (B) 2 μg/mL Collagen (C) 1 μM ristocetin (D) 1 μM U46619 (thromboxane A2 analog). Results show no significant decrease in platelet aggregation using alternate agonists. Results show no significant decrease in platelet aggregation. The results show data from n=3 for ADP, collagen and ristocetin and n=2 for U46619. S3 JPET#234781 Supplemental figure 3: Figure shows amount of thromboxane B2 (TXB2) measured in platelet aggregometry samples using AA (5 μM) agonist and 10 μM of C1, C2 or C3, normalized to DMSO control. Compound C1, C2 and C3 all show reduction in TXB2 formation. ** p <0.01. Samples were tried for n=4. S4 JPET#234781 Supplemental figure 4: The effect of LA, EPA and DHA on platelet aggregation. Platelets were incubated with LA, EPA and DHA at concentrations of (A) 2.5 μM, (B) 5 μM and (C) 10 μM and aggregation was induced by 5μM AA. (D) The effect of DHA on AA (5μM) induced platelet aggregation at 0.25 μM, 0.5 μM and 1 μM concentration. S5 JPET#234781 Supplemental figure 5: (A) Schematic of coupled assay. Arachidonic acid (AA) converts to Prostaglandin H2 (PGH2) in the presence of enzyme COX-1 (cyclooxygenase-1). PGH2 converts to Thromboxane A2 (TXA2) in the presence of TXAS (thromboxane synthase) that is stabilized in nanodiscs. (B) Thromboxane formed in the presence of LA, EPA and DHA in a coupled assay with TXAS in nanodisc and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA. (C) Thromboxane formed in the presence of LA, EPA and DHA in a coupled assay with TXAS solubilized in 0.53% β-octyl glucoside and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA. S6 JPET#234781 Supplemental figure 6: Thromboxane formed in the presence of LA, EPA, DHA and aspirin in a coupled assay with TXAS (thromboxane synthase) in nanodisc and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA. However a significant reduction (*p<0.05) is seen for aspirin. S7 JPET#234781 Supplemental figure 7: Representative HPLC trace of DHA anhydride hydrolysis. Peak at 2.57 mins is DHA anhydride (red line) and peak at 32 mins is DHA (blue line). The green line representing hydrolysis shows emergence of DHA as product S8 JPET#234781 Supplemental figure 8: Representative HPLC trace of EPA anhydride hydrolysis. Peak at 2.5 mins is EPA anhydride (red line) and peak at 28 mins is EPA (blue line). The green line representing hydrolysis shows emergence of EPA as product. S9 JPET#234781 Supplemental figure 9: Representative HPLC trace of LA anhydride hydrolysis. Peak at 2.7 mins is LA anhydride (red line) and peak at 34 mins is LA (blue line). The green line representing hydrolysis shows emergence of LA as product. S10 JPET#234781 Supplemental table 1: Characterization of compounds: DHA- aspirin anhydride: HR ESI-MS- 512.03 C31H38O5Na 1H NMR: 0.9 (t, 6H), 1.7 (t, 2H), 2.1 (m, 3H), 2.30 (s, 3H), 2.35 (t, 1H), 2.8 (m, 7H), 5.4 (m, 12 H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d, 1H) EPA- aspirin anhydride HR ESI MS- 487.24 C29 H36O5Na 1H NMR: 0.9 (t, 3H), 1.7 (t, 1H), 2.1 (m, 3H), 2.30 (s, 3H), 2.35 (t, 1H), 2.8 (m, 4H), 5.4 (m, 10 H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d, 1H) LA aspirin anhydride: HR ESI MS- 465.26 C27H38O5Na 1H NMR: 0.9 (t, 3H), 1.3 (m, 14 H), 1.6 (t, 2H), 2.0 (s, 3H), 2.01(m, 2H), 2.3 (m, 4H), 2.75(t, 2H), 5.3 (m, 4H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d , 1H) S11 JPET#234781 1 Supplemental figure 10: H NMR of DHA- aspirin anhydride in CDCl3 S12 JPET#234781 1 Supplemental figure 11: H NMR of EPA- aspirin anhydride in CDCl3 S13 JPET#234781 1 Supplemental figure 12: H NMR of LA- aspirin anhydride in CDCl3 S14 .
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