Impact of Epicatechin on the Procoagulant Activities of Microparticles

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 μM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.


Introduction
Microparticles are small, anucleate vesicles ranging from 100 to 1000 nm in size. They are released by many cell types, including platelets, monocytes, red and endothelial cells by exocytosis from the cell membranes upon cell activation, stress or apoptosis [1]. Microparticles are delimited by a phospholipid bilayer and express proteins from the cell of origin [2]. Microparticles are detected in healthy subjects, and their release increases in various pathological conditions, such as cancer, diabetes, sepsis and cardiovascular diseases (CVD) [3][4][5].
Platelet microparticles (PMPs) represent the main fraction of circulating microparticles and are formed upon platelet activation, glycoprotein (Gp) IIb-IIIa signaling or after exposure to shear stress [6,7]. PMPs express Gp IIb-IIIa (CD41) and Gp Ib (CD42b), phospholipids (e.g., phosphatidylserine (PS) from PPP centrifugation at 14,000 g at room temperature for 1 h. The microparticle-containing pellet was resuspended in 100 µL of the supernatant after centrifugation (and thus microparticle-poor) to concentrate microparticles by 5.0-fold.
(-)-Epicatechin stock solution (Extrasynthèse, Lyon, France; 12.5 mM in DMSO) was diluted with phosphate buffered saline (PBS) to 0.1, 1 and 10 mM working solutions that were then added to the plasma samples to reach the target final concentrations of 1, 10 and 100 µM with a constant 1/100 dilution. An equivalent volume of vehicle was added to samples without epicatechin. Plasma samples were incubated with epicatechin at 37 • C for 10 min.

Cytometry Analysis of Microparticles
All cytometry analyses were performed with fresh samples. The impact of epicatechin on microparticles was determined in three conditions (all samples were pre-incubated with epicatechin): after incubation of PRP samples with PBS, to assess the direct role of epicatechin on microparticle production and their phospholipid and protein membrane composition (condition 1); after PRP incubation with platelet activators to simulate microparticle production (calcium ionophore A23187 (Sigma-Aldrich, Saint-Louis, MO, USA)) (condition 2) and with thrombin receptor activating peptide (TRAP; Roche, Mannheim, Germany) (condition 3), and to study epicatechin role in microparticle production and composition ( Figure A1). Afterwards, samples were incubated with FITC-conjugated annexin-V (PS labeling), anti-CD41a-PE (glycoprotein IIb, clone HIP8), anti-CD42b-APC (glycoprotein Ib, clone HIP1) and anti-CD62P-BV421 (P-selectin, clone AK-4) antibodies (all from BD Biosciences) at room temperature in the dark for 20 min. Isotype controls (at the same concentration as the primary antibodies) were used as negative controls to differentiate non-specific background and specific antibody signals. Immediately after labeling, samples were resuspended in 250 µL of 0.20 µm-filtered annexin-V buffer and were analyzed on a BD FACS Canto II (BD Biosciences, Le Pont de Claix, France), equipped with three lasers (407, 488 and 633 nm wavelengths) and the BDFACS Diva software (v.8.0.1).
Flow cytometer performance tracking was performed daily using the BD cytometer setup and tracking beads (BD Biosciences). To ensure a limited background noise, filtered PBS (0.20 µm filter) was analyzed before each run at least for 10 min.
For each analysis, 100 µL of fresh PPP diluted at 1/100 or 1/200 was transferred to a TruCount tube (BD Biosciences) containing a lyophilized pellet that releases a known number of fluorescent beads to allow microparticle quantification.
Before each series of sample analysis, fluorescent Megamix-Plus SSC Beads (Biocytex, Marseille, France), a mix of fluorescent beads ranging from 0.1 to 1 µm, were used to define the gate consistent with the microparticle size, according to the manufacturer's instructions. The Megamix-Plus by its standardized acquisition defines microparticles between 0.17 and 0.5 µm equivalent-SSC and allows discriminating between small and large microparticles (i.e., smaller and bigger than 0.22 µm-eq SSC). PMPs were characterized on the basis of the side scatter threshold defined using Megamix-Plus SSC Beads and labeling with CD41a, a constitutive platelet receptor.

Phospholipid-Induced Procoagulant Activity of Microparticles
Microparticle procoagulant activity was determined using Zymuphen TM MP-ACTIVITY (Hyphen-biomed, Neuville, France), a functional immunological assay, according to the manufacturer's protocol. Briefly, after pre-incubation with epicatechin, 5 µL of each PPP sample was incubated in a well of a microplate coated with annexin V that can bind to electronegative phospholipids at the microparticle surface. In the presence of calcium, factors (F) Xa and FVa, prothrombin are activated into thrombin in relation with microparticle exposure to phospholipids. Thrombin activity was measured by absorbance at 405 nm on a spectrophotometer (Spark, Tecan, Switzerland) following cleavage of a specific substrate. Plasma microparticle concentration was expressed in nM of PS equivalent.

Tissue factor (TF)-Induced Procoagulant Activity of Microparticles
Microparticle procoagulant activity was determined using Zymuphen TM MP-TF (Hyphen-biomed), a functional immunological assay, according to the manufacturer's protocol. Briefly, after pre-incubation with epicatechin, 20 µL of PPP was incubated in wells coated with an anti-TF monoclonal antibody. In the presence of calcium, FVIIa and FX, TF-positive microparticles form the TF-FVIIa complex and activate FX into FXa. TF-induced microparticle procoagulant activity was correlated with FXa activity on a specific substrate measured by absorbance at 405 nm on a spectrophotometer (Tecan) and was expressed in pg/mL.

Thrombin Generation Assays
Thrombin generation assays (TGA) were used in two experimental conditions to measure epicatechin effect on microparticle procoagulant activity by following the thrombin formation kinetics. First, MRP samples were incubated with increasing concentrations of epicatechin, as before, to evaluate the anticoagulant impact of epicatechin. Second, PRP samples were incubated with epicatechin before platelet stimulation by addition of 20 µM of the calcium ionophore A23187 (Sigma-Aldrich) that promotes platelet activation and apoptosis [40]. Then, MRPs were prepared as described above, and TGA were performed to evaluate the impact of epicatechin on the release of procoagulant microparticles from platelets.
TGA were performed using a modified Calibrated Automated Thrombogram method developed by Hemker [41], with a fluorometer (Fluoroscan Ascent, ThermoLab Systems, Franklin, TN, USA) equipped with a dispenser. Briefly, in 96-well plates (Immulon 2HB, Waltham, MA, USA), 30 µL of MRP samples was used as the source of phospholipids and TF. Then, 70 µL of a pool of normal plasma from 10 healthy donors, 20 µL of fluorogenic substrate and CaCl 2 (FluCa-Kit ® , Thrombinoscope BV) were added to the wells. In parallel, each sample was calibrated with Thrombin Calibrator ® (Stago, Asnières, France) and the same pool of normal plasma. The main parameter was the endogenous thrombin potential (ETP, area under the curve). All tests were performed in duplicate with a maximum difference <10% for ETP (nM.min) between curves. Raw data were analyzed using Thrombinoscope TM V5 (Thrombinoscope BV, Maastricht, The Netherlands).

Statistical Analysis
Statistical analyses were performed with the Prism software, version 6 (GraphPad software, Inc., La Jolla, CA, USA). Tests were two-sided, with a type I error set at α = 0.05. Continuous data were presented as the mean ± standard deviation (SD). The statistical significance of differences between classes was determined with ANOVA, or the Friedman test when the ANOVA conditions were not met (normality and homoscedasticity verified with the Bartlett test), followed by the appropriate multiple comparison post-hoc tests (Tukey-Kramer or Dunn test, respectively). conditions. Of note, the inter-individual variability of PMP concentration was high, as indicated by the coefficient of variation of 70%.
These data showed that 100 µM epicatechin can inhibit microparticle-mediated coagulation without affecting the lag time of thrombin generation.

Discussion
There is strong evidence of the antiplatelet effects of dietary polyphenols, and it is suggested that polyphenols may have an impact on microparticles [42]. The present study investigated epicatechin role in microparticle formation and procoagulant potential that plays a key role in CVD ( Figure 6) [43,44]. Our findings show that overall, epicatechin does not influence the concentration of PMPs, which were identified by labeling with the constitutive platelet receptor CD41a, even after platelet activation by agonists, such as TRAP or a calcium ionophore. However, some PMP sub-populations seem to be affected by epicatechin. Indeed, the percentage of total (small and large) P-selectin-expressing PMPs

Discussion
There is strong evidence of the antiplatelet effects of dietary polyphenols, and it is suggested that polyphenols may have an impact on microparticles [42]. The present study investigated epicatechin role in microparticle formation and procoagulant potential that plays a key role in CVD ( Figure 6) [43,44]. These data showed that 100 μM epicatechin can inhibit microparticle-mediated coagulation without affecting the lag time of thrombin generation

Discussion
There is strong evidence of the antiplatelet effects of dietary polyphenols, and it is suggested that polyphenols may have an impact on microparticles [42]. The present study investigated epicatechin role in microparticle formation and procoagulant potential that plays a key role in CVD ( Figure 6) [43,44]. Our findings show that overall, epicatechin does not influence the concentration of PMPs, which were identified by labeling with the constitutive platelet receptor CD41a, even after platelet activation by agonists, such as TRAP or a calcium ionophore. However, some PMP sub-populations seem to be affected by epicatechin. Indeed, the percentage of total (small and large) P-selectin-expressing PMPs Our findings show that overall, epicatechin does not influence the concentration of PMPs, which were identified by labeling with the constitutive platelet receptor CD41a, even after platelet activation by agonists, such as TRAP or a calcium ionophore. However, some PMP sub-populations seem to be affected by epicatechin. Indeed, the percentage of total (small and large) P-selectin-expressing PMPs decreased, while PS expression was unchanged. The expression of GpIb at the PMP surface, particularly in small PMPs, was reduced already by incubation with 1 µM of epicatechin. Epicatechin Nutrients 2020, 12, 2935 9 of 13 also generated an imbalance between large and small GpIb-positive PMPs, following stimulation with the calcium ionophore, by increasing the large microparticle fraction.
There is evidence that microparticles smaller than 0.5 µm have specific properties, such as inhibition of the collagen/adenosine-diphosphate-mediated formation of platelet thrombi [45]. The epicatechin-induced decrease in GpIb-positive microparticles could impair their reactivity to thrombin and von Willebrand factor [46], and might have a direct impact on myocardial infarction pathogenesis [47]. Furthermore, epicatechin effect from 1 µM on P-selectin-expressing PMPs could have a beneficial effect on the risk of major adverse cardiovascular events after myocardial infarction [48,49], probably because of P-selectin role in thrombosis and in the recruitment of leukocytes in inflammation [50,51].
Microparticles are procoagulant factors due to their membrane that supports the coagulation enzymatic cascade. This property is reinforced by anionic phospholipids (e.g., PS) and TF, the main coagulation activator [52]. Here, we observed a functional impact of epicatechin on microparticle procoagulant role. Specifically, incubation with epicatechin reduced their phospholipid-mediated procoagulant activity (from 10 µM of epicatechin), but not the activity mediated by TF. Interestingly, TGA, which uses microparticles as phospholipid source and TF to trigger coagulation, is decreased by epicatechin. These data seem to demonstrate that epicatechin can inhibit microparticle-mediated coagulation by affecting the number and phenotype of the released microparticles, and/or the enzymatic reaction of coagulation.
Several studies have shown that initiation of thrombin generation is mainly supported by microparticles derived from monocytes, and not by PMPs [53,54]. However, PMPs contribute to clot propagation and to prothrombotic activities after initiation [55], Surprisingly, we found that PS-expressing PMPs were not modified by epicatechin. It was previously reported that epicatechin can inhibit thrombin activity [56,57]. This could explain the effect on the procoagulant activity mediated by phospholipids and on thrombin generation, and the absence of effect on the procoagulant action mediated by TF. Taken together, these data support epicatechin interest in CVD through its action on microparticles (PMP generation and size/properties) and on thrombin generation. Its capacity to modulate microparticles could contribute to its health protective effects [58].
This study has some limitations. Ottaviani et al. showed that among the stereoisomers of flavan-3-ol, (-)-epicatechin is the one with the highest bioavailability [59]. It would also be interesting to explore the effect of long-term in vivo exposure to lower concentrations of epicatechin, instead of short in vitro exposure to higher concentrations [60]. In vivo, epicatechin is present in the plasma as conjugated derivatives, resulting from phase II metabolism occurring after its intestinal absorption [61]. More studies are needed to thoroughly assess the bioactivity of epicatechin metabolites at physiologically relevant concentrations that we could not perform because they are not available yet. This is an in vitro study that must be completed by mechanistic investigations.
In conclusion, we demonstrated that epicatechin positively affects microparticle generation, phenotype and procoagulant properties, particularly PMPs. Given microparticle importance in CVD and the major complications of CVD, these data open new perspectives on how epicatechin can affect coagulation that deserve to be confirmed in in vivo studies.