Aleksandra Gasecka1,2, Rienk Nieuwland3, Edwin van der Pol4, Najat Hajji3, Agata Ćwiek5, Kinga Pluta5, Michał Konwerski5, Krzysztof J Filipiak5. 1. First Chair and Department of Cardiology, Medical University of Warsaw, Poland. a.agasecka@wum.edu.pl. 2. Vesicle Observation Centre, and Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands. a.agasecka@wum.edu.pl. 3. Vesicle Observation Centre, and Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands. 4. Biomedical Engineering & Physics, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands. 5. First Chair and Department of Cardiology, Medical University of Warsaw, Poland.
Abstract
BACKGROUND: Activated platelets release platelet extracellular vesicles (PEVs). Adenosine diphosphate (ADP) receptors P2Y1 and P2Y12 both play a role in platelet activation, The present hypothesis herein is that the inhibition of these receptors may affect the release of PEVs. METHODS: Platelet-rich plasma from 10 healthy subjects was incubated with saline, P2Y1 antagonist MRS2179 (100 μM), P2Y12 antagonist ticagrelor (1 μM), and a combination of both antagonists. Platelets were activated by ADP (10 μM) under stirring conditions at 37°C. Platelet reactivity was assessed by impedance aggregometry. Concentrations of PEVs- (positive for CD61 but negative for P-selectin and phosphatidylserine) and PEVs+ (positive for all) were determined by a state-of-the-art flow cytometer. Procoagulant activity of PEVs was measured by a fibrin generation test. RESULTS: ADP-induced aggregation (57 ± 13 area under curve {AUC] units) was inhibited 73% by the P2Y1 antagonist, 86% by the P2Y12 antagonist, and 95% when combined (p < 0.001 for all). The release of PEVs- (2.9 E ± 0.8 × 10⁸/mL) was inhibited 48% in the presence of both antagonists (p = 0.015), whereas antagonists alone were ineffective. The release of PEVs+ (2.4 ± 1.6 × 10⁷/mL) was unaffected by the P2Y1 antagonist, but was 62% inhibited by the P2Y12 antagonist (p = 0.035), and 72% by both antagonists (p = 0.022). PEVs promoted coagulation in presence of tissue factor. CONCLUSIONS: Inhibition of P2Y1 and P2Y12 receptors reduces platelet aggregation and affects the release of distinct subpopulations of PEVs. Ticagrelor decreases the release of procoagulant PEVs from activated platelets, which may contribute to the observed clinical benefits in patients treated with ticagrelor.
BACKGROUND: Activated platelets release platelet extracellular vesicles (PEVs). Adenosine diphosphate (ADP) receptors P2Y1 and P2Y12 both play a role in platelet activation, The present hypothesis herein is that the inhibition of these receptors may affect the release of PEVs. METHODS: Platelet-rich plasma from 10 healthy subjects was incubated with saline, P2Y1 antagonist MRS2179 (100 μM), P2Y12 antagonist ticagrelor (1 μM), and a combination of both antagonists. Platelets were activated by ADP (10 μM) under stirring conditions at 37°C. Platelet reactivity was assessed by impedance aggregometry. Concentrations of PEVs- (positive for CD61 but negative for P-selectin and phosphatidylserine) and PEVs+ (positive for all) were determined by a state-of-the-art flow cytometer. Procoagulant activity of PEVs was measured by a fibrin generation test. RESULTS:ADP-induced aggregation (57 ± 13 area under curve {AUC] units) was inhibited 73% by the P2Y1 antagonist, 86% by the P2Y12 antagonist, and 95% when combined (p < 0.001 for all). The release of PEVs- (2.9 E ± 0.8 × 10⁸/mL) was inhibited 48% in the presence of both antagonists (p = 0.015), whereas antagonists alone were ineffective. The release of PEVs+ (2.4 ± 1.6 × 10⁷/mL) was unaffected by the P2Y1 antagonist, but was 62% inhibited by the P2Y12 antagonist (p = 0.035), and 72% by both antagonists (p = 0.022). PEVs promoted coagulation in presence of tissue factor. CONCLUSIONS: Inhibition of P2Y1 and P2Y12 receptors reduces platelet aggregation and affects the release of distinct subpopulations of PEVs. Ticagrelor decreases the release of procoagulant PEVs from activated platelets, which may contribute to the observed clinical benefits in patients treated with ticagrelor.
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