Discussion
Cocoa (T. cacao L.) is derived from the cacao bean and has a storied history of diverse therapeutic benefits, including vascular effects based on its flavanol, procyanidin and methylxanthine content.18–20 It has substantially contributed to the socioeconomic development of Trinidad and Tobago for over 200 years, at which one point it was responsible for ‘producing 20% of the world’s cocoa’. 21 In fact, as of 1930, the Cocoa Research Centre had pioneered and innovated this sector, making it the oldest cocoa research institution globally.22
Cocoa products contain catechins ((+)-catechin) and epicatechins ((−)-epicatechin), classified as flavanols (flavan-3-ols), which display accentuated vascular benefits. They possess a much higher flavanol concentration than wine, tea or berries.6 Several nutraceutical studies have been inconsistent in demonstrating a definitive CV benefit, largely attributed to the ambiguity of flavanol concentration.11 The recently published ‘Effect of cocoa flavanol supplementation for the prevention of CVD events: the COcoa Supplement and Multivitamin Outcomes Study’ randomised clinical trial revealed no significant effect on the primary outcome of total CV events; however, CV mortality was significantly reduced by 27%.10 Numerous mechanistic studies evaluating cocoa have also alluded to enhanced cardiometabolic effects with respect to endothelial function, blood pressure, inflammation, insulin resistance and platelet reactivity.23–27
When activated, platelets adhere to sites of vascular injury within a complex milieu of factors promoting aggregation and stabilisation of the haemostatic plug.28 These factors include ADP, thromboxane A2 (TXA2), serotonin, collagen (COL) and thrombin.29 The release of ADP and TXA2 leads to several mechanistic, morphological and proinflammatory effects, including change in shape, increased expression of P-selectin, soluble CD40 ligand and conversion of the glycoprotein (GP) IIb/IIIa receptor into its active form.30
ASA is an irreversible cyclooxygenase-1 (COX-1) inhibitor that blocks TXA2 production. TXA2 is produced from arachidonic acid (AA) through enzymatic conversion by COX-1 and thromboxane synthase. TXA2 binds to the thromboxane receptors, resulting in platelet shape and aggregation of platelets to the primary platelet plug.31 By preventing the formation of TXA2, ASA decreases platelet activation and aggregation promoted by TXA2 but not by other agonists.32 In this study, there was a non-significant reduction in ARU of 17.65. The VN–ARU assay uses AA as an agonist (sensitive to ASA therapy) and expresses results in ARUs. Despite this study not demonstrating any significant effect of cocoa on ARU with the VN system, Rein et al demonstrated that consumption of cocoa caused an ‘ASA-like’ effect on platelet function, as measured in terms of platelet-related primary haemostasis via the platelet function analyser (PFA-100; Siemens Healthineers AG, Erlangen, Germany).33 This alternative analyser measures COL–ADP-stimulated or COL–epinephrine (EPI)-stimulated platelet function under shear conditions.34 The COL–EPI system detected qualitative platelet abnormalities induced by ASA and was prolonged 6 hours after consuming the cocoa beverage, suggesting an antiplatelet effect.33 In that study, the 30 participants consumed 300 mL of a beverage containing 18.75 g cocoa powder, whereas our study included 20 patients consuming 30 g/day of 65% cocoa for 1 week and evaluated with a different platelet function assay.
The consumption of the cocoa intervention significantly decreased PRU by 26.85, with a relative reduction of 11.9%. The VN-P2Y12 assay reports results as P2Y12 reaction units (PRUs). This assay mimics turbidimetric aggregation and uses disposable cartridges containing 20 mM ADP and 22 nM prostaglandin E1 (PGE1). Aggregation testing using ADP as a sole agonist activates P2Y1 and P2Y12 purinergic signalling, while adding PGE1 increases the test’s specificity for P2Y12 signalling. A baseline value for platelet function is obtained in a separate channel of the cartridge in which isothrombin receptor activating peptide (TRAP) is used as an agonist.35 36 The VN system exhibits moderate concordance with other platelet function tests and has reliably stratified high-risk patients for MACE.37
Platelet morphology and transient aggregation are mediated by P2Y1. Binding of ADP to the P2Y12 receptor results in cascade amplification that culminate in platelet aggregation and stabilisation.38 In Ostertag et al, cocoa significantly decreased ADP-induced platelet aggregation, TRAP-induced platelet aggregation and P-selectin expression, and increased COL/EPI-induced ex vivo bleeding times.39 Their methodology included a 60 g cocoa (dark chocolate) bar assessed 6 hours after consumption with platelet function via a PFA-100 analyser and flow cytometry. Additionally, Pearson et al demonstrated that cocoa inhibited several measures of platelet activity, including EPI-induced and ADP-induced GP IIb/IIIa and P-selectin expression, platelet microparticle formation, and EPI/COL and ADP-COL induced primary haemostasis.40 Montagnana et al revealed a significant increase of COL/ADP-induced PFA-100 closure time, but not COL/EPI, 4 hours after ingestion of dark chocolate.5 Platelet aggregation induced by COL was unchanged after low flavanol or high flavanol dark chocolate, whereas both attenuated responses to ADP and TRAP relative to baseline.41 Shear stress-dependent platelet adhesion was also attenuated in a study by Flammer et al using another modality of platelet function testing.42 Platelet hyper-reactivity is critical in acute coronary syndrome pathophysiology; thus, mitigation of shear stress-dependent platelet adhesion may beneficially affect atherothrombosis.42
Our study displayed a potentiated effect with respect to ADP-induced platelet aggregation in patients on DAPT with ASA and clopidogrel. These comparative studies were performed in patients without established CVD, whereas our study involved patients with a medical history of prior MI, type 2 diabetes mellitus, and who received PCI and CABG.
The P2Y12 receptor and COX-1 pathway are complementary with respect to platelet inhibition, and thus it is surprising that the ARU and PRU signals were not significantly concordant in this study. This could result from an unknown confounder, an interaction effect, or reduced intrinsic power of the study with respect to the number of participants enrolled and the duration of the cocoa intervention. Of the 20 subjects, 5 (25%) had a baseline ARU of >550 which remained unchanged post cocoa intervention, while 12 (60%) had a baseline PRU of >208 compared with 8 (40%) subjects post cocoa intervention, alluding to a non-significant reduction in HPR.
Study limitations
Despite this study being sufficiently powered for prospective pharmacodynamic outcomes with respect to PRUs and ARUs, it was not designed for prespecified clinical outcomes, and thus no definitive conclusions on clinical efficacy and safety can be ascertained. As with previous studies conducted by this group in Trinidad, there was a preponderance of South Asian patients, alluding to a selection bias during study enrolment.14 43 A double-blind, randomised controlled trial would have been the gold standard for ascertaining the antiplatelet effect of this cocoa intervention; however, there are logistical challenges in executing such methodology in our limited resource setting without a dedicated clinical research organisation.44
Additionally, this study did not evaluate the composition of the cocoa (dark chocolate) bars with respect to caloric, glucose, protein and lipid content, as any beneficial antiplatelet effect may be potentially offset by paradoxical glycaemic or lipidaemic effects. However, Hamed et al reported improved lipid profiles (low-density lipoprotein reduction of 6%, high-density lipoprotein increase of 9%) with decreased platelet reactivity.45 Also, this study did not quantify flavanol concentrations with relatively low bioavailability, and their downstream plasma metabolite concentrations are temporally variable and may not correlate with other nutraceutical mechanistic studies.5 Our study also involved the cocoa intervention being distributed throughout the day (three 10 g bars consumed at breakfast, lunch and dinner), which may affect the pharmacodynamic effects of the flavanol metabolites on platelet function as compared with the form and timing in other studies, for example, one-time beverage consumption. This cocoa intervention was also relatively short, with a time frame of 1 week, and each cocoa bar contained 65% cocoa solids and 35% sugar. As a result, thus, there can be no long-term extrapolation of clinical and biochemical outcomes such as glycaemic control in patients with diabetes, dyslipidaemic effects or net weight gain, which can have negative implications.
To our knowledge, this prospective study is novel in determining the effect of a 1-week trial of 30 g/day of 65% cocoa consumption intervention on platelet reactivity using the VN system on patients with CAD on DAPT. As such, it may not be clinically pertinent to patients on more potent antithrombotic therapies such as prasugrel, ticagrelor or direct oral anticoagulants. Additionally, many of these therapies and their generic counterparts are not readily available due to regulatory or financial issues. A more inclusive and detailed array of platelet function testing using PFA, flow cytometry and thromboelastography may be revelatory; however, these are unavailable in Trinidad due to technical and personnel logistical issues.