The study protocol was approved by the University of Pécs Clinical Centre Regional and Institutional Research Ethics Committee (Ref. number: 6735, Clinical Trial No: NTC03679858). All procedures were performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from both patients and healthy volunteers. A total of 18 patients (median age at 66 years, 12 males) on antiplatelet therapy (75 mg clopidogrel once daily) due to secondary stroke prevention were prospectively recruited into this study. The selected patients with previous anterior circulation large artery atherothrombosis were on regular medical check-ups at the Outpatient Clinic of the Department of Neurology at the University of Pécs.

Venous blood samples were drawn with 21G needle after short strangulation from an antecubital vein into closed blood collection system tubes with 3.2% (0.109 M) Na${}_3$-citrate, K${}_3$-EDTA (Becton Dickinson, Diagon LTD Hungary) and hirudin (Sarstedt S-Monovette® 1.6 mL Hirudin) as anticoagulant. Upon blood collection the first 3 mL blood was discarded. The blood samples for measurements were transported immediately to laboratory and were processed within 1 hour.

The modified whole blood gravity sedimentation technique was developed for studying platelet and neutrophil sedimentation properties [11]. After a 1-hour gravity sedimentation, the upper and lower half of the venous blood column were separately removed from the EDTA and hirudin containing tubes and transferred into another EDTA and hirudin tube for further analysis (Fig. 1). The total blood cell count and the neutrophil (%) count were measured from the whole blood and after 1-hour gravity sedimentation from the upper and lower part of the blood on a Sysmex XN 9000 integrated automated haematology analyser (Sysmex Co., Kobe, Japan, 2017).

Fig. 1.

Chart of sample preparation, separation of the upper and lower blood sample after 1-hour gravity sedimentation and further analytical process.

Platelet function test was performed in the whole blood and after 1 hour of sedimentation from the upper and lower part of the hirudin anticoagulated blood with a Multiplate® Analyzer (Roche Diagnostics, Mannheim, Germany). Platelet aggregometry was uniformly carried out 60 minutes after blood sampling using adenosine diphosphate (ADP; 6.5 M) as agonist [12]. Aggregation level was expressed as the area under the curve (AUC). AUC was calculated by a Multiplate® Analyzer using the product of aggregation unit (AU) $\times$ time (minutes). After ADP stimulation the normal aggregation range was expected as AUC: 53-220 — according to the manufacturer. Based on the whole blood AUC, patients on clopidogrel were categorized as responders with AUC 53 and non-responders with AUC $\ge$53.

After a one-hour sedimentation the upper and lower part of the citrated blood were centrifuged at 2500 $\times$ g for 20 minutes at room temperature. The supernatant was transferred into a new test tube and centrifuged at 2500 $\times$ g for further 20 minutes to obtain cell-free plasma. The top of the cell-free plasma was transferred into an Eppendorf tube and was immediately frozen on liquid nitrogen and stored at –80 °C until further use. MVs measurement was described in our previous article [13]. Briefly, the samples for measurement were thawed on melting ice and pelleted at 18000 $\times$ g for 10 minutes. The supernatant was carefully removed, leaving 25 $\mu$L of MV rich plasma at the bottom of the Eppendorf tube. MVs were suspended with gentle vortexing for 20 seconds in 1.0 mL Apo-binding buffer (10 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl${}_2$, 136 mmol/L NaCl, pH = 7.4 HEPES was obtained from Sigma-Aldrich Ltd., Budapest, Hungary, other analytical grade reagents were obtained from Reanal Ltd., Budapest, Hungary) without CaCl${}_2$. The selected CD markers, their cellular origin, the fluorescent dye used for labelling and the manufacturer specification for our MV measurements are summarized in Table 1. For sample labelling 10 $\mu$L MV in Ca${}^{2+}$ free buffer was incubated in 100 $\mu$L Apo-binding buffer supplemented with 2.5 mmol/L CaCl${}_2$ with a total of 10 $\mu$L antibody, previously diluted to optimal labelling concentration. Staining was incubated for 30 minutes at room temperature in a dark chamber. All buffers were filtered through 0.2 $\mu$m membrane filters.

Flow cytometric measurements and data analysis were performed on a Beckman-Coulter FC-500 cytometer with CXP software (Version 2.3. Beckman Coulter Life Sciences, Indianapolis). The MV’s reference gate was defined with Megamix beads (Biocytex). Side scatter, forward scatter and fluorescence channels were set in a logarithmic scale. MV size gate was determined between 0.5 $\mu$m and 1.0 $\mu$m size range. Events in the MV gate were further discriminated by labelling with Annexin [14]. MVs were defined as Annexin V positive events in the MV gate with fluorescence intensity above the isotype control. For determination of the MV number, known concentration (1 $\times$ 10${}^6$/mL) of 3 $\mu$m diameter microbeads (Becton Dickinson) were used. To determine the optimal labelling concentrations all antibodies and Annexin V were titrated. Labelling concentrations were defined by antibody staining of samples and sample-free buffers in the presence or absence of CaCl${}_2$. Labelling was considered optimal if CaCl${}_2$ labelled sample measurement events were clearly distinguishable from background, CaCl${}_2$ free staining, as well as from isotype controls.

Statistical analysis was performed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Summary statistics of the participants were constructed using frequencies and proportions for categorical data and as mean and standard deviation (SD) for continuous variables. Conformity of data to normal distribution was determined by histogram and Kolmogorov–Smirnov test. The between-group difference was calculated with $\chi$${}^2$, Fisher’s exact and Mann–Whitney U tests in line with suitability. Data with nonparametric distribution were presented as median and interquartile range (IQR). Correlations of microvesicle counts with aggregometry data (area under the curve, AUC; velocity of the slope of aggregation) were tested by linear regression using Spearman correlation coefficient (Rho). The significance level was considered as p 0.05.

A total of 18 convalescent ischemic stroke patients on clopidogrel (all patients suffered from large vessel occlusion) and 20 age-matched healthy subjects were recruited into this study prospectively. Demography of patients and healthy controls and baseline laboratory parameters of the study population are summarized in Table 2. There was no significant difference in regard to age and gender. The total number of circulating microvesicles (p 0.001), and particularly the endothelial-derived CD31${}^{+}$ (p = 0.016) and platelet derived CD42a${}^{+}$ (p 0.001) MVs measured in the whole blood were significantly higher in post-stroke patients compared to healthy subjects. Interestingly, CD62P${}^{+}$ (P-selectin) positive MVs showed no significant difference between groups. All significant MV data from Table 2 are presented as Fig. 2.

Fig. 2.

Comparison of the total count of MVs (2A), the CD31${}^{\mathrm{+}}$ MVs (2B) and the CD42a${}^{\mathrm{+}}$ MVs (2C) in the whole blood of post-stroke patients and healthy subjects (Mann-Whitney U test).

We analysed the correlation between MVs and aggregometry data. The platelet aggregation in the whole blood (area under the curve, AUC) measured by Multiplate® in patients taking clopidogrel, but not in age-matched healthy controls, showed a significant negative correlation with the total number of MVs ($\times$10${}^5$/mL) in the lower blood sample after 1-hour gravity sedimentation (r = –0.650, p = 0.005; Fig. 3). Nevertheless, we did not observe any correlation between the AUC and the total number of MVs in the whole blood.

Fig. 3.

Correlation between the area under the curve (AUC) in whole blood measured by Multiplate® and total number of microvesicles ($\mathrm{\times }$10${}^{\mathrm{5}}$/mL) in the lower blood sample after 1-hour gravity sedimentation in patients (Spearman correlation, p = 0.005).

Next, we explored the potential associations between MVs and aggregometry data (area under the curve, AUC and velocity respectively) obtained from clopidogrel responders and non-responders based on the previously defined cut-off value (AUC: 53). Both the AUC and the velocity in the whole blood showed negative correlation with the total number of MVs ($\times$10${}^5$/mL) in the lower blood sample after 1-hour gravity sedimentation. Importantly, a significant negative correlation was observed for the velocity (r = –0.801, p = 0.005), but not for the AUC in responders (n = 11) (Fig. 4).

Fig. 4.

Correlation between velocity measured by Multiplate® aggregometry and total number of microvesicles ($\mathrm{\times }$10${}^{\mathrm{5}}$/mL) in the lower blood sample in clopidogrel responders and non-responders respectively. Blue line indicates a negative correlation in responders (Spearman correlation, r = –0.801, p = 0.005).

Activation-induced conformational epitope on CD41/CD61 complex positive (PAC-1${}^{+}$) MVs in the lower blood sample showed a significant positive correlation with the percentage of neutrophil granulocytes in the lower blood sample after 1-hour gravity sedimentation (r = 0.634, p = 0.008; Fig. 5). In contrast, this positive correlation disappeared when whole blood indices were analysed. Furthermore, the constitutive platelet marker (CD42a${}^{+}$) positive MVs measured in the upper blood fraction showed a significant correlation with the percentage of the neutrophils in the lower blood sample (r = 0.652, p = 0.006; Fig. 6). Nevertheless, no significant correlation was found in the whole blood samples.

Fig. 5.

Correlation between platelet derived PAC1${}^{\mathrm{+}}$ MVs ($\mathrm{\times }$10${}^{\mathrm{5}}$/mL) and neutrophils (%) in the lower blood sample (Spearman correlation, p = 0.008).

Fig. 6.

Correlation between platelet derived CD42a${}^{\mathrm{+}}$ MV number ($\mathrm{\times }$10${}^{\mathrm{5}}$/mL) measured in the upper blood sample and neutrophils (%) in the lower blood sample (Spearman correlation, p = 0.006).