The ex vivo antiplatelet activation potential of fruit phenolic metabolite hippuric acid

Abstract

Polyphenol-rich fruit and vegetable intake has been associated with reduction in platelet hyperactivity, a significant contributor to thrombus formation. This study was undertaken to investigate the possible role of hippuric acid, a predominant metabolite of plant cyclic polyols, phenolic acids and polyphenols, in reduction of platelet activation-related thrombogenesis. Fasting blood samples were collected from 13 healthy subjects to analyse the effect of varying concentrations of hippuric acid (100 μM, 200 μM, 500 μM, 1 mM and 2 mM) on activation-dependant platelet surface-marker expression. Procaspase activating compound-1 (PAC-1) and P-selectin/CD62P monoclonal antibodies were used to evaluate platelet activation-related conformational changes and α-granule release respectively using flow cytometry. Platelets were stimulated ex vivo via the P2Y1/P2Y12- adenosine diphosphate (ADP) pathway of platelet activation. Hippuric acid at a concentration of 1 mM and 2 mM significantly reduced P-selectin/CD62P expression (p = 0.03 and p < 0.001 respectively) induced by ADP. Hippuric acid at 2 mM concentration also inhibited PAC-1 activation-dependant antibody expression (p = 0.03). High ex vivo concentrations of hippuric acid can therefore significantly reduce P-selectin and PAC-1 expression thus reducing platelet activation and clotting potential. However, although up to 11 mM of hippuric acid can be excreted in the urine per day following consumption of fruit, hippuric acid is actively excreted with a recorded Cmax for hippuric acid in human plasma at 250-300 μM. This is lower than the blood concentration of 1-2 mM shown to be bioactive in this research. The contribution of hippuric acid to the protective effects of fruit and vegetable intake against vascular disorders by the pathways measured is therefore low but could be synergistic with lowered doses of antiplatelet drugs and help reduce risk of thrombosis in current antiplatelet drug sensitive populations.

Bioactive polyphenols and cardiovascular disease: chemical antagonists, pharmacological agents or xenobiotics that drive an adaptive response?

“…Similar findings were obtained for hippuric acid, the predominant metabolite of some phenolic acids and polyphenols, which reduced platelet aggregation and P‐selectin/CD62P expression at 1–2 mM. Hippuric acid, however, is actively excreted from the body with maximal plasma concentrations of ~200–300 μM (Santhakumar et al , ). Meanwhile, pelargonidin aglycone, but not the glucoside adduct, inhibited thrombin‐induced fibrin polymerization and platelet aggregation and elicited anticoagulant effects in mice.…”

Oxidative stress biomarkers in type 2 diabetes mellitus for assessment of cardiovascular disease risk

“…Numerous studies have already shown the ability of free radicals (O2 -, H2O2, NADPH Oxidase; NOX) to increase cellular adhesion molecules such as P-selectin, PCAM, E-selectin and VCAM-1 on platelet and endothelial cell surfaces. P-Selectin is a cellular adhesion molecule released from activated platelet and endothelial cells; it is known to play a key role in leukocyte recruitment and the formation of large thrombus [19,20]. Similarly E-Selectin, an endothelial-leukocyte adhesion molecule, is overexpressed under elevated oxidative stress and inflammatory states.…”

“…Endothelial expression of these adhesion molecules plays a major role in platelet aggregation and the atherogenesis. Studies have shown that with the increase in expression of P-Selectin and E-Selectin, elevated platelet-fibrin and plateletleukocyte aggregation ensues thrombogenic activity [19]. The elevated ICAM-1 and VCAM-1 at the endothelial cell also contribute to the increased state of platelet aggregation and adhesion.…”

Polyphenols: Modulators of Platelet Function and Platelet Microparticle Generation?

“…Moreover, a review of 97 bioavailability studies showed that plasma concentrations and urinary excretion differ depending on the type and not the proportion of polyphenols in the diet. Due to such important parameters associated with bioavailability, it has been suggested that the metabolites of polyphenols should rather be used to study antiplatelet effects [52,58,102].…”