Epicatechin Impact on Primary Hemostasis, Coagulation and Fibrinolysis
Authors: Sinegre Thomas , Mazur André , Berger Marc G , Milenkovic Dragan , Sapin Anne-Françoise , Morand Christine , Lebreton Aurelien 
Published: October 2019

Abstract
Introduction
Cardiovascular diseases are the leading causes of death worldwide. Among cardiovascular risk factors, some are linked to the stages of hemostasis : primary haemostasis, coagulation and fibrinolysis. There is a growing interest on the potential beneficial effects of some nutritional components on cardiovascular health, in particular in the field of thrombosis. Dark chocolate is associated with a one-third reduction in the risk of cardiovascular disease. Cocoa is particularly rich in a flavonoid subclass called flavan-3-ol which includes epicatechin. Epicatechin is a micronutrient found mainly in chocolate, green tea and grape. It has already been proven that epicatechin impacts platelet functions but there are only poor data on the role of epicatechin on coagulation and fibrinolysis. The aim of this study was to evaluate the impact of epicatechin in vitro on the different stages of hemostasis.
Patients and methods
The study was performed on 10 major healthy subjects. Exclusion criteria were antiplatelet or anticoagulant therapy intake, clinical history of thrombosis or bleeding, thrombocytopenia (platelet count < 150 G/L) and abnormal coagulation profile (fibrinogen < 1.8 g/L, Prothrombin time > 13 s, and activated partial thromboplastin time > 39 s).
Platelets agregometry were performed according the ISTH recommendations with (final concentrations) : ADP (2 µmol/L), epinephrin (5 mmol/L), collagen (2 µg/ml), arachidonic acid (1 mmol/L) (Helena Laboratories, Beaumont, USA), thromboxan A2 analog, U46619 (1 mmol/L) (Merck, Darmstadt, Germany), Thrombin Receptor Activating peptide (TRAP, 10 µmol/L) (Roche Rotkreuz, Switzerland). Area under curve at 4 minutes and maximal agregation were the main parametres analysed.
Thrombin generation assays (TGA) were performed with PPP reagent low (phospholipids (4 µmol/L) and tissue factor (TF, 1 pmol/L); Stago, Asnières sur Seine, France) on platelet free plasma (PFP). The platelets procoagulant activity was evaluated with PRP reagent (TF 1 pmol/L) (Stago) on platelets rich plasma (PRP) and microparticles procoagulant activity with PRP reagent on plasma rich in microparticles (without platelets). After activation by Ca ionophore ??
Fibrinolysis was evaluated by a clot lysis assay determined by measurements of optical densitometry at 405 nm overtime. Coagulation was initiated by PPP reagent (Stago) sensibilised by an activator of plasminogen (t-PA, actylise®, Boehringer, France ; 200 ng/mL, final concentration). The plasma fibrinolytic potential was assessed by the clot lysis time (CTL), defined as the time from the midpoint of the baseline to maximum turbid transition, to the final plateau phase.
In each of the experiments plasmas were used pure or supplemented with a range of (-)-epicatechin (Extrasynthese; Lyon, France) concentration (final concentrations of 0, 1, 10 and 100 mM).
All ethical considerations were obtained.
Results and discussion
5 women and 5 men were included in this study, with a median age of 33 years (Q1 - Q3) (28-38).
Epicatechin 100 µmol/L inhibits platelet aggregation induced by ADP and collagen with maximal agregation of 45 % versus 73 % in control (p< 0.001) and 67 % versus 79 % in control (p < 0.05) respectively. Epicatechin 10 µmol/L impact coagulation and decrease the TGA parameters such as peak or endogenous thrombin potential with 1345 (1161 - 1563) nM.min versus 1503 (1262 - 1728) nM.min (p<0.05) in control. Epicatechin did not seem to interact significantly on the platelet procoagulant activity. No impact of epicatechin was found in microparticles procoagulant activity assessed by TGA.
Epicatechin play a profibrinolytic role as evaluated by a clot lysis assay : the CLT with epicatechin 100 µmol/L was significantly lower than without epicatechin with 11,0 (8,0 - 16,0) min versus 15,0 (9,5 - 20,8) min in control (p < 0.01).
Conclusion
Epicatechin seems to play a role in each stage of haemostasis. The impact on platelets functions is highlighted by lower maximal agregation induced by ADP and collagen. The coagulation is slightly decreased as shown by TGA on PFP. Epicatechin also impairs fibrinolysis evaluated with a clot lysis assay. Further in vivo studies are needed to confirm these results.

Disclosures
No relevant conflicts of interest to declare.