Myricetin, the Main Flavonoid in <em>Syzygium cumini</em> Leaf, Is a Novel Inhibitor of Platelet Thiol Isomerases PDI and ERp5
Supplementary Figure 1
Chromatographic fingerprint of PESc and flavonoid standards. UV detection of standards for gallic acid, myricetin and quercetin (A) or a sample of PESc (B) were analysed through LC-MS/MS as described in Methods. In addition, each fraction was purified and their identity confirmed by HPLC-MS/MS studies. Peak 1 corresponded to gallic acid, peaks 2 and 3 to myricetin glycoside derivatives, peak 4 to myricetin and peak 5 to quercetin. Structures of the identified compounds are shown in (C).
Supplementary Figure 2
Increased agonist concentration partially overcome anti-platelet effect of PESc. Platelet-rich plasma was pre-treated with PESc (10, 100 or 1000 μg/mL) for 25 minutes and stimulated with ADP (A-D) or thrombin (THB, E-H). Representative traces for 2.5 μM ADP (A) and 5 μM ADP (C). Representative traces for 0.01 U/mL THB (E) and 0.02 U/mL THB (G). Quantified data is shown next to representative curves. a p<0.05 vs first column of graph. b p<0.05 vs second column of graph. c p<0.05 vs third column of graph. Data analysed by paired one-way ANOVA and Tukey as post-test. All bar graphs represent mean ± SEM and individual data points of at least 3 independent experiments. Arrows indicate when agonists were added.
Supplementary Figure 3
Decreased effect of Myricetin in platelet-rich plasma. Platelet-rich plasma (PRP) was pre-treated with myricetin (Myr) or gallic acid (GA) for 10 minutes and stimulated with collagen or TRAP-6. (A) PRP treated with Myr and stimulated with collagen. (C) PRP treated with GA and stimulated with collagen. (E) PRP treated with Myr and stimulated with TRAP-6. (G) PRP treated with GA and stimulated with TRAP-6. Quantified data is shown right next to representative curves. a p<0.05 vs first column of graph. b p<0.05 vs second column of graph. c p<0.05 vs third column of graph. Data analysed by paired one-way ANOVA and Tukey as post-test. All bar graphs represent mean ± SEM and individual data points of at least 3 independent experiments. Arrows indicate when agonists were added.
Supplementary Figure 4
Myricetin does not induce VASP phosphorylation. Resting WP were incubated with myricetin (7.5, 15 and 30 μM) or PAPA-NONOate (100 μM, positive control) for 10 minutes and lysed in laemmli buffer supplemented with reducing agent. Lysed cells were processed as described in Material and Methods and probed for VASPs239 and GAPDH as loading control. Bar graph represent present the mean of four independent experiments run and error bars indicate SEM. Data compared using One-way ANOVA followed by Tukey post-test. There were no statistical differences between groups.
Supplementary Figure 5
Myricetin quenches fluorescence of ERp5, ERp57, ERp72 and PDI. Recombinant proteins were incubated with myricetin (0.01 to 10 μM) in a black 96-wells plate for 10 minutes and fluorescence spectra acquired in a fluorimeter using excitation set at 280 nm. Representative fluorescence spectra shown for ERp5 (A), ERp57 (B), ERp72 (C) and PDI (D). (E) Stern-volmer plot of quenching data is shown as the linear regression between F0/F and log of myricetin concentration in mM where F0 is the fluorescence of vehicle and F is the fluorescence in the presence of increasing concentrations of myricetin. Data represent at least three independent experiments run at least in duplicate and error bars indicate SEM.
Abstract
Background
Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential antiplatelet properties of S. cumini and its constituent flavonoids.
Objective
To evaluate the antiplatelet effects and mechanism of action of a polyphenol-rich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid.
Methods
PESc, myricetin, and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, while molecular docking studies predicted possible binding sites.
Results
PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent antiplatelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting hemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulfide isomerase (PDI) and ERp5 reductase activities. Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5.
Conclusions
PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders.
Acknowledgments
The authors are grateful to the staff of the Laboratory of Immunophysiology (LIF/UFMA) and Laboratory of Experimental Physiology (LeFisio/UFMA), especially to Dr. Ludmila Bezerra and Mr. Victor Vieira for the technical assistance during experimental protocols execution.