The influence of Rubus idaeus and Rubus caesius leaf extracts on platelet aggregation in whole blood. Cross-talk of platelets and neutrophils

Recently, polyphenols have gained attention as potential natural cardioprotective therapeutics, due to their antiplatelet, anti-inflammatory and anticoagulant activity. Species belonging to the genus Rubus sp. have been reported to be a source of polyphenolic compounds with antioxidative proprieties and beneficial biological activities. This study investigates the effects of leaf extracts obtained from red raspberry (Rubus idaeus L.) and European dewberry (Rubus caesius L.) on the reactivity of blood platelets. In ADP-stimulated blood, raspberry and dewberry extracts (15 µg/ml) markedly decreased platelet surface membrane expression of activated GPIIbIIIa receptor by 16% and 21%, respectively (P < 0.01) and significantly inhibited platelet aggregation (by 31-41% for raspberry and by 38-55% for dewberry, P < 0.01). In platelet-rich plasma (PRP), the extracts had no effect on ADP-induced platelet aggregation. The effectiveness of the extracts in whole blood and the lack of their activity in PRP indicate that leukocytes are likely to participate in the platelet response to the extracts. Our experiments show that the extracts significantly reduced the amount of free radicals released by activated neutrophils in whole blood (P < 0.001), as well as in suspensions of isolated neutrophils (P < 0.05). Moreover, the reduced number of neutrophils leads to the decreased efficiency of the extracts in the inhibition of platelet aggregation. In summary, our findings show that the raspberry and dewberry leaf extracts considerably modulated blood platelet reactivity in whole blood: they influenced blood platelet aggregation, possibly via the modulation of the redox status dependent on the oxidative activity of neutrophils.

shown that DMSO at the concentrations of 0.01% or 0.02% had no impact on platelet aggregation and neutrophil functions (expression of receptors and ROS production).
The concentrations of the extracts corresponded to those used in our earlier study of endothelial function (1-15 µg/ml) [1].

Blood samples and donors
The study was performed according to the guidelines of the Helsinki Declaration for

Isolation of neutrophils
Neutrophils were isolated as described previously form heparinized whole blood layered onto Polymorphprep (1:1) and centrifuged (500 x g, 30 min). Gradient centrifugation allows the separation of mononuclear and polymorphonuclear cells by density. The neutrophils were harvested and washed with PBS without calcium and magnesium (400 x g, 10 min). Residual erythrocytes were removed by lysis with lysing buffer (BD Pharm Lyse). The isolated neutrophils were suspended in an appropriate amount of a buffer or platelet-poor plasma (PPP). In some experiments, neutrophil granulocytes were isolated from blood by the sedimentation method with the use of 3% dextran D-500 in PBS. The cells were counted in a haemocytometer.
The viability of cells was assessed with the use of the trypan-blue method.

Whole blood aggregometry (WBA)
Blood was incubated with extracts for 15 min (37°C), either alone or in combination with purinergic receptor inhibitors, A3P5 (35 µM) or cangrelor (6.5 nM), added to the blood for the final 3 min or 5 min of incubation (37°C) . The concentration of platelet inhibitors, A3P5 or cangrelor, and the time of blood incubation with platelet inhibitors were established experimentally [2,3,4]. Blood incubated with 0.02% DMSO was The effect of the extract on platelet aggregation was measured also in the blood samples with a reduced neutrophil number. In the neutrophil depletion experiments, 1 ml aliquots of whole blood were preincubated with magnetic microbeads conjugated with anti-CD15 antibodies for 15 min at an ambient temperature. The mixture of blood and beads was placed on a magnet (MACS system, Miltenyi Biotec, Bergisch Gladbach, Germany) and neutrophils coated with beads were removed from the blood. The blood samples were carefully collected and measurements of aggregation were performed as described above. The efficiency of neutrophil depletion was assessed by counting the cells before and after the exposure of blood samples to the magnetic field. This technique allowed the number of neutrophils to be reduced by approximately 90%. The results were calculated as the inhibition vs.
the relevant paired control as follows: for the samples, treated with either extract or cangrelor, the inhibition was calculated in relation to the untreated sample (control), for the samples depleted with neutrophils ("beads +"), treated with the extracts or cangrelor, the inhibition was calculated in relation to the untreated samples depleted with neutrophils (control "beads +").

Light transmission aggregometry (LTA)
Measurement of platelet aggregation by LTA was performed in platelet-rich plasma (PRP). After withdrawal of blood, whole blood samples were immediately centrifuged for 12 min at 190 x g to obtain PRP. The PRP was then removed and the remaining sample was subsequently centrifuged (12 min, 2000 x g) to get PPP to adjust the platelet count in PRP to 2-3 x 10 8 /ml. Platelet aggregation was monitored for 10 min in Chrono-Log 490-2D (Chrono-Log, Havertown, PA, USA) after stimulation with 6.4 µM ADP. In some experiments, platelets were isolated by gel filtration, as described previously [5].

Measurements of platelet viability
Platelet viability in whole blood preincubated with the examined plant extracts was determined by flow cytometry as described in detail elsewhere [6].

Determination of VASP Phosphorylation
The phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) has been widely used in a literature to determine the blood platelet response to stimulation by ADP via the P 2 Y 12 receptor [7,8]. In our study, the effect of plant extracts on the VASP phosphorylation in blood platelets was determined in whole blood by the use of PLT VASP/P 2 Y 12 assay. Prior to the activation with ADP, blood

Oxidative burst of neutrophils
The production of ROS by neutrophils in the response to the exogenous stimulation (oxidative burst) was measured in whole blood samples and in isolated neutrophils suspensions, using the luminol-enhanced chemiluminescence (L-CL) method described elsewhere [9,10]

Statistical analysis
The Shapiro-Wilk test was used to verify whether the data was normally distributed.
Levene's test was used to verify the homogeneity of variances. Depending on data distribution, mean ± SE or median and interquartile ranges (IQR): from lower (LQ, 25%) to upper quartile (UQ, 75%), are given for all parameters. In the case of normally-distributed data, the significance of the differences between samples and controls was determined with one-way or two-way ANOVA for repeated measures, followed by either the analysis of contrasts (two way analyses) or the post-hoc multiple comparison test (Scheffe's test, Dunnett's test or the paired Student t-test with the Bonferroni's correction for multiple comparisons). For the comparing a few extract concentrations the dose-dependent effect was verified with the linear trend testing in the analysis of contrasts. In the case of non-normally-distributed data, the significance of differences between samples and controls was determined with the rank Friedmann test followed by the Wilcoxon signed-rank test with the Bonferroni's correction for multiple comparisons. The Pearson's linear correlation or Spearman rank correlation was used to assess associations between the measured parameters (maximal platelet aggregation vs. neutrophil-platelet aggregate fraction in whole blood; ROS production vs. CD11b expression).