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Clinical Trial
. 2017 Jun;430(1-2):69-80.
doi: 10.1007/s11010-017-2955-7. Epub 2017 Feb 16.

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients

Agnieszka Morel  1 , Joanna Rywaniak  2 , Michał Bijak  2 , Elżbieta Miller  3   4 , Marta Niwald  3   4 , Joanna Saluk  2
Affiliations
Clinical Trial

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients

Agnieszka Morel et al. Mol Cell Biochem. 2017 Jun.

Abstract

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Keywords: Blood platelets; Flow cytometry; Multiple sclerosis; Prothrombotic risk.

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Figures

Fig. 1
Fig. 1
Expression of P-selectin on resting (a) or agonist-stimulated platelets: ADP 20 µM (b) and collagen 20 µg/ml (c) in whole blood samples obtained from SP MS patients and healthy controls. Analyses were performed in whole blood of healthy volunteers (control) and patients with SP MS. The blood platelets were distinguished based on the expression of CD61/PerCP. For each sample, 10 000 CD61-positive objects (platelets) were acquired. For the assessment of P-selectin expression, samples were labeled with fluorescently conjugated monoclonal antibody CD62P/PE. Results are shown as the percentage of platelets expressing CD62P. Statistical analysis was performed using Mann–Whitney U test
Fig. 2
Fig. 2
Expression of the active form of GPIIb/IIIa on resting (a) or agonist-stimulated platelets: ADP 20 µM (b) and collagen 20 µg/ml (c) in whole blood samples obtained from SP MS patients and healthy controls. Analyses were performed in whole blood of healthy volunteers (control) and patients with SP MS. The blood platelets were distinguished based on the expression of CD61. For each sample, 10,000 CD61-positive objects (platelets) were acquired. For the assessment of P-selectin expression, samples were labeled with fluorescently conjugated monoclonal antibody PAC-1-FITC. Results are shown as the percentage of platelets binding PAC-1/FITC. Statistical analysis was performed using Mann–Whitney U test
Fig. 3
Fig. 3
Formation of platelet aggregates in control (a) and agonist-stimulated platelets: ADP 20 µM (b) and collagen 20 µg/ml (c) in whole blood samples obtained from SP MS patients and healthy controls. The fraction of platelet aggregates was distinguished from platelets (CD61-positive objects) based on their size and granularity on the forward light scatter (FSC) vs. side light scatter (SSC) plots. CD61-positive objects with FSC higher than 104 were characterized as platelet aggregates. In each sample, 10,000 CD61-positive objects (platelets) were measured. Statistical analysis was performed using Mann–Whitney U test
Fig. 4
Fig. 4
Formation of platelet-derived microparticles in control (a) and agonist-stimulated platelets: ADP 20 µM (b) and collagen 20 µg/ml (c) in whole blood samples obtained from SP MS patients and healthy controls. The fraction of platelet-derived microparticles was distinguished from platelets (CD61-positive objects) based on their size and granularity on the forward light scatter (FSC) vs. side light scatter (SSC) plots. CD61-positive objects with FSC lower than 102.3 were characterized as PMPs. In each sample, 10 000 CD61-positive objects (platelets) were measured. Statistical analysis was performed using Mann–Whitney U test
Fig. 5
Fig. 5
Representative histograms of the expression level of P-selectin on non-stimulated platelets (shown as control) (a, d) and on stimulated platelets with ADP (b, e) or collagen (c, f) in whole blood samples obtained from SP MS patients (d–f) and healthy controls (a–c). The blood platelets in whole blood were distinguished based on the expression of CD61 conjugated with PerCP. For each sample, 10 000 CD61-positive objects (platelets) were acquired. The blood platelets were labeled with monoclonal antibody anti-CD62P (PE)
Fig. 6
Fig. 6
Representative histograms of the expression level of active form of GPIIb/IIIa on non-stimulated platelets (shown as control) (a, d) and on stimulated platelets with ADP (b, e) or collagen (c, f) in whole blood samples obtained from SP MS patients (d–f) and healthy controls (a–c). The blood platelets in whole blood were distinguished based on the expression of CD61 conjugated with PerCP. For each sample, 10 000 CD61-positive objects (platelets) were acquired. The blood platelets were labeled with monoclonal antibody PAC-1 (conjugated with FITC) against the active form of GPIIb/IIIa
Fig. 7
Fig. 7
Representative dot plots of the percentage of platelet-derived microparticles and platelet aggregates in a population of resting platelets (shown as a control) (a, d) and in a population of stimulated platelets with ADP (b, e) or collagen (c, f) in whole blood samples obtained from SP MS patients (d–f) and healthy controls (a–c). The blood platelets in whole blood were distinguished based on the expression of CD61 conjugated with PerCP. For each sample, 10,000 CD61-positive objects (platelets) were acquired
Fig. 8
Fig. 8
Level of sP-selectin in plasma obtained from SP MS patients and healthy controls. sP-selectin was determined by ELISA method. Statistical analysis was performed using unpaired Student’s t test (p < 0.0001)
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