P2Y12 antagonist ticagrelor inhibits the release of procoagulant extracellular vesicles from activated platelets

Figures

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  Figure 1. A transmission electron microscope image of platelet extracellular vesicles from human plasma. The vesicles were labelled with gold nanoparticles conjugated to CD61 antibodies. Image courtesy of Chi M. Hau, Vesicle Observation Center, Academic Medical Center, University of Amsterdam, the Netherlands.

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  Figure 2. The design of the experiments on the role of the P2Y1 and P2Y12 receptors in the release of platelet extracellular vesicles. CaCl2 — calcium chloride; ADP — adenosine diphosphate; CD — cluster of differentiation; PEVs — platelet extracellular vesicles.

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  Figure 3. Representative images of platelet extracellular vesicles (PEVs) from adenosine diphosphate-activated platelets in plasma of a healthy donor measured by flow cytometry (A60-Micro, Apogee Flow Systems, Hertfordshire, UK); A. PEVs exposing CD61 but not CD62P and phosphatidylserine (PS) and labelled with anti-CD61-APC; B. PEVs exposing CD61, CD62P and PS and labelled with anti-CD61-APC, anti-CD62P-Violet and lactadherin-FITC, respectively. In the first step all plasma extracellular vesicles exposing PS (binding to lactadherin) were recorded (data not shown). Out of all PEVs exposing PS, PEVs exposing CD61 and CD62P were recorded (Q2); a.u. — arbitraty units.

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  Figure 4. The effect of platelet pre-incubation with a P2Y1 antagonist, a P2Y12 antagonist and a combination of both antagonists on platelet aggregation (A) and the release of platelet extracellular vesicles (PEVs) exposing CD61 but not CD62p and phosphatidylserine (PS) (B) and PEVs exposing CD61, CD62p and PS (C) in response to adenosine diphosphate (ADP). Whereas platelet aggregation correlated weakly with the concentration of CD61+/CD62P−/PS− PEVs, platelet aggregation correlated strongly with the concentration of CD61+/CD62P+/PS+ PEVs (D).

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  Figure 5. Proposed procoagulant activity of platelet extracellular vesicles (PEVs) in plasma. Fibrin generation test was performed in platelet-depleted, but PEVs-containing plasma stimulated with adenosine diphosphate (ADP). A part of PEVs expose phosphatidylserine (CD61+/PS+) (A). When the plasma clots, the optical density increases (B). ADP-stimulated plasma containing phosphatidylserine (PS)-exposing extracellular vesicles (EVs) did not clot (red). Upon the addition of recombinant human tissue factor (TF) to this plasma, clotting was triggered as measured by fibrin generation (blue). The extent of clotting was not affected by the presence of the P2Y1 and P2Y12 receptor antagonists (data not shown). Covering PS-exposing EVs with an excess of lactadherin, or inhibiting the TF-initiated extrinsic coagulation with an anti-factor VIIa antibody, both inhibited plasma clotting (black and green, respectively). Hence, both PS-containing EVs and TF are indispensable for propagation of coagulation.

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  Figure 6. The potential proinflammatory and procoagulant effects of platelet extracellular vesicles (PEVs). PEVs contribute to coagulation both directly by providing procoagulant surface (phosphatidylserine) for the assembly of coagulation factors, and indirectly by binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) on monocytes, leading to an exposure of tissue factor on monocytes. In addition, interaction between P-selectin and PSGL-1 contributes to inflammation by stimulating the release of pro-inflammatory cytokines from monocytes.