Inhibition by theobromine, a cocoa methylxanthine, of platelet aggregation and adhesion stimulated by various agonists

Zetty Nadia Mohd Zain, Tim Warner. Department of Translational Medicine and Therapeutics,William Harvey Research Institute,Barts and The London,Queen Mary School of Medicine and Dentistry, London, United Kingdom.

Previous studies have suggested cardioprotective effects of cocoa, such as an inhibition of platelet activation and aggregation (Engler et al, 2004). As well as its high content of flavonoids, cocoa has abundant levels of methylxanthine theobromine (3,7-dimethylxanthine) which is a caffeine metabolite (Smit et al, 2004). Platelet aggregatory responses are routinely measured by light transmission techniques (Born & Cross, 1962) which can be developed for use in 96-well plates with platelet adhesion determination (Armstrong et al, 2008). We have evaluated the effects of theobromine on aggregation and adhesion stimulated by various agonists in platelet-rich plasma (PRP) using a 96-well plate method.

Human blood was collected by venepuncture into tri-sodium citrate (3.2% w/v final) and centrifuged to obtain PRP. The PRP was then incubated (37°C, 30min) with theobromine

(2-50μg/ml) or vehicle before being transferred to the wells of a 96-well plate in the presence or absence of various agonists. Platelet agonists used in this study were arachidonic acid (0.03-1mM), ADP, TRAP-6, U46619 (all 0.1-30μM), collagen (0.1-30μg/ml), adrenaline (0.001-100μM) and ristocetin (0.2-3mg/ml). Changes in absorbance, as a measure of platelet aggregation, were then determined at 595nm every 15s for 16min. Final platelet adhesion was determined by the conversion of p-nitrophenol phosphate to p-nitrophenol dependent upon platelet acid phosphatase.

In this study, theobromine displayed concentration dependent inhibitory effects upon platelet function. For instance, platelet aggregation in response to ADP 3μM was decreased from 45±9% to 35±9% and 32±7% by 5 and 10μg/ml theobromine, respectively (n = 5 for each). Platelet aggregation in response to 3μg/ml collagen, 69±13% was also inhibited by 5μg/ml theobromine from 69±13% to 33±14%, respectively (n = 4 for each). Incubation with theobromine also inhibited platelet aggregation and adhesion in response to arachidonic acid, adrenaline, and TRAP-6 but not to U46619 or ristocetin.

Our data demonstrate that theobromine inhibits platelet activation in response to some but not all agonists. These effects may due to the inhibition of platelet phosphodiesterase, increasing platelet levels of cyclic AMP and cyclic GMP and so reducing platelet reactivity.

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