A structural basis for the inhibition of collagen-stimulated platelet function by quercetin and structurally related flavonoids

Abstract

Background and purpose: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A(2) receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure-activity relationships with regard to platelet function is also lacking.

Experimental approach: Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3'-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCgamma2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy.

Key results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCgamma2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety.

Conclusions and implications: The structure-activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling.

Figure 2

The extent of inhibition of platelet function by flavonoids and metabolites correlate with their potencies. Washed human platelets (4 × 10⁸ cells·mL⁻¹) loaded with [³H]-5-HT were pretreated with increasing concentrations of flavonoids [quercetin (Q): A.i, A.ii and A.iii, apigenin: B.i, B.ii and B.iii and catechin: C.i, C.ii and C.iii], metabolites (tamarixetin (T) and quercetin-3′-sulphate (Q-3′-S): A.i, A.ii and A.iii) or solvent control [DMSO (0.2% v/v)] for 5 min. Platelets were then stimulated with collagen (5 µg·mL⁻¹) for 90 s and aggregation and 5-HT secretion were measured as a percentage of the DMSO-treated, collagen-stimulated control values (100%). The data points represent the mean (n= 3) aggregation or 5-HT secretion for each treatment (±SEM). *P≤ 0.05, **P≤ 0.01 and ***P≤ 0.001 compared with the control (DMSO-treated, collagen-stimulated platelets).

Figure 1

Flavonoid and metabolite structures are defined by variations in key functional groups. The flavonoid structure comprises 15 carbon atoms arranged into a 3 carbon, oxygenated heterocyclic middle ring (C ring) flanked by two aromatic rings (A and B rings). Subgroups of this family of compounds are defined by variations in functional groups integral to the flavonoid nucleus and substituted to the A, C and B rings. Flavones (apigenin) are characterized by a non-hydroxylated C ring, whereas flavonol (quercetin) C rings contain a C-3 hydroxyl group. Flavan-3-ols (catechin) are defined by a non-planar, C-3 hydroxylated C ring that is not substituted with a C-4 carbonyl group. Metabolites generated by intestinal enterocytes and liver hepatocytes are methylated (tamarixetin), sulphated (quercetin-3′-sulphate) or glucuronidated (quercetin-3-glucuronide) counterparts of parent flavonoid aglyca.

Figure 3

Internalization of quercetin and tamarixetin by MEG-01 cells. MEG-01 cells (4 × 10⁶ cells·mL⁻¹) were incubated with quercetin (40 µM), tamarixetin (20 µM) or solvent control [DMSO (0.2% v/v)] for 30 min. Fluorescence was detected at 480 nm–500 nm after excitation at 430 nm with an argon laser. Images of a single middle layer from z-stacks are shown (DMSO control: A. i–iii, quercetin: B. i–iii tamarixetin: D. i–iii) and higher magnifications of areas of interest are also shown (quercetin: C. i–iii, tamarixetin: E. i–iii). Images represent results from at least three individual experiments.

Figure 6

PLCγ2 tyrosine phosphorylation is inhibited with highest potency by flavonoids with planar, hydroxylated C rings (quercetin) and metabolites with methylated B rings (tamarixetin). Washed human platelets (8 × 10⁸ cells·mL⁻¹) treated with 1 mM EGTA were incubated for 5 min with flavonoids (quercetin: A, B and apigenin: D), metabolites [tamarixetin: B, quercetin-3′-sulphate (Q-3′-S): C and quercetin-3-glucuronide (Q-3-G): C] or solvent control (DMSO 0.2% v/v). Platelets were stimulated with 25 µg·mL⁻¹ collagen for 90 s. PLCγ2 phosphotyrosine residues were detected before equivalent protein loading was verified by reprobing for PLCγ2. Tyrosine phosphorylation was expressed as a percentage of the DMSO-treated, collagen-stimulated control (100%). The bars represent the mean (n= 3) tyrosine phosphorylation for each treatment (±SEM). *P≤ 0.05, **P≤ 0.01 and ***P≤ 0.001 compared with the control (DMSO-treated, collagen-stimulated platelets).

Figure 5

Syk tyrosine phosphorylation in intact platelets is inhibited with greatest potency by flavonoids with planar C rings (quercetin) and metabolites with methylated B rings (tamarixetin). Washed human platelets (8 × 10⁸ cells·mL⁻¹) were treated with 1 mM EGTA prior to incubation for 5 min with flavonoids (quercetin: A, B and apigenin: D), metabolites [tamarixetin: B, quercetin-3′-sulphate (Q-3′-S): C] or solvent control [DMSO (0.2% v/v)], then they were stimulated with 25 µg·mL⁻¹ collagen for 90 s. Syk phosphotyrosine residues were detected and equivalent protein loading was verified by reprobing for Syk. Tyrosine phosphorylation was expressed as a percentage of the DMSO-treated, collagen-stimulated control (100%). The bars represent the mean (n= 3) tyrosine phosphorylation for each treatment (±SEM). *P≤ 0.05, **P≤ 0.01 and ***P≤ 0.001 compared with the control (DMSO-treated, collagen-stimulated platelets).

Figure 4

Flavonoids and metabolites block total protein tyrosine phosphorylation in intact platelets: the C ring C-3 hydroxyl group is necessary for potent inhibition. Washed human platelets (8 × 10⁸ cells·mL⁻¹) treated with 1 mM EGTA were pretreated with increasing concentrations of flavonoids (quercetin: A, B and apigenin: D), metabolites [tamarixetin: B, quercetin-3′-sulphate (Q-3′-S): C and quercetin-3-glucuronide (Q-3-G): C] or solvent control [DMSO (0.2% v/v)] for 5 min. Platelets were stimulated with 25 µg·mL⁻¹ collagen for 90 s before the immunodetection of total protein tyrosine phosphorylation, as a percentage of the DMSO-treated, collagen-stimulated control (100%). The bars represent the mean (n= 3) tyrosine phosphorylation for each treatment (±SEM). *P≤ 0.05, **P≤ 0.01 and ***P≤ 0.001 compared with the control (DMSO-treated, collagen-stimulated platelets).

Figure 7

Flavonoids and metabolites block the kinase activity of Fyn in platelet lysates in a manner influenced by their pattern of hydroxylation. Washed human platelets (8 × 10⁸ cells·mL⁻¹) in the presence of EGTA (1 mM) were stimulated with collagen (25 µg·mL⁻¹) for 90 s. Platelets were lysed with ice-cold 1% (v/v) NP40 and Fyn was precipitated. Platelet lysates were incubated with anti-Fyn antibody and protein G Sepharose (PGS) beads for 1 h at 4°C and Fyn immunoprecipitates were pretreated with flavonoids [quercetin (Q): A–C and catechin: D], metabolites [tamarixetin (T): A,B and quercetin-3′-sulphate (Q-3′-S): C] or solvent control [DMSO (0.2% v/v)] for 5 min. Immunoprecipitates were assayed for kinase activity as described in methods. Equivalent protein loading was verified by reprobing for Fyn. Kinase activity was expressed as a percentage of the DMSO-treated, collagen-stimulated control (100%). The bars represent the mean (n= 3) kinase activity for each treatment (±SEM). *P≤ 0.05, **P≤ 0.01 and ***P≤ 0.001 compared with the control (DMSO-treated, collagen-stimulated platelets).

Figure 8

A working model outlining a structural basis for the actions of flavonoids and their metabolites in inhibition of collagen-stimulated platelet function. Flavonoids (quercetin, apigenin and catechin) and metabolites [tamarixetin, quercetin-3′-sulphate (Q-3′-S) and quercetin-3-glucuronide (Q-3-G)] inhibit platelet aggregation and 5-HT secretion in a differential manner due to variable attenuation of multiple mechanisms. These variations in inhibitory ability are due to distinct structural features including a planar, C-3 hydroxylated and C-4 carbonyl substituted C ring, a B ring catechol moiety and methyl, sulphate and glucuronide groups which are metabolically added. Mechanisms include inhibition of PLCγ2 and Syk tyrosine phosphorylation as well as Fyn kinase activity mediated directly within the cytosol. Although flavonoid and metabolite binding affinities for collagen and fibrinogen did not correlate with their inhibitory potencies, binding to collagen and fibrinogen, disrupting the interactions of these proteins with GPVI and α_IIbβ₃, respectively, may be a potential mode of action. While potent inhibition of platelet function is more dependent on a planar C ring conjugated to a C-4 carbonyl group and a B ring catechol moiety (quercetin and apigenin), elimination of the C ring C-3 hydroxyl group (apigenin) reduces potency for inhibition of signalling. In vivo, methylated and sulphated metabolites of flavonols may be more active inhibitors of platelet function than glucuronidated metabolites. The differential inhibition of signalling by apigenin suggests internalization and preferential inhibition of Syk and PLCγ2 rather than Fyn by this flavone indicates a degree of selectivity.