Background and aims: Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional alpha(IIb)beta(3) integrins, indicating that alpha(IIb)beta(3) integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin alpha(IIb)beta(3), phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release.
Methods: EVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.
Results: Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of alpha(IIb)beta(3) integrin activation and subsequent closure paralleled by PS exposure. Prevention of alpha(IIb)beta(3) activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using a alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects.
Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.
|Number of pages||9|
|Publication status||Published - Oct 2020|
- Actin cytoskeleton
- Blood platelets
- Extracellular vesicles
- Platelet activation
- MICROPARTICLE FORMATION
- AMINOPHOSPHOLIPID EXPOSURE