Abstract
Background and aimsPlatelets 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 αIIbβ3 integrins, indicating that αIIbβ3 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 αIIbβ3, phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release. MethodsEVs 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. ResultsPlatelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of αIIbβ3 integrin activation and subsequent closure paralleled by PS exposure. Prevention of αIIbβ3 activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using αIIbβ3-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. ConclusionPlatelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.
Highlights
Besides platelets role in haemostasis and thrombosis, there is a growing body of literature recognizing that they contribute to several other processes like vascular integrity, wound healing, inflammation and immune processes [1,2,3,4,5,6,7]
The importance of integrin αIIbβ3 in extracellular vesicles (EVs) release was seen in Glanzmann thrombasthenia [27]
To investigate how integrin activation and inactivation followed by phosphatidyl serine (PS) exposure are related to EV release, washed platelets were stimulated with CVX or thrombin and integrin activation and PS exposure were followed in time by flow cytometry
Summary
Besides platelets role in haemostasis and thrombosis, there is a growing body of literature recognizing that they contribute to several other processes like vascular integrity, wound healing, inflammation and immune processes [1,2,3,4,5,6,7]. The EV outer membrane is similar in composition as its cell of origin, e.g. EVs derived from platelets can possess procoagulant properties (phosphatidylserine) and display of various integrins (e.g. αIIbβ3) on their outer surface. Platelets from patients with Glanzmann thrombasthenia have reduced EV release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. We investigated the role of integrin αIIbβ, phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release. Kinetic measurements indicated that EV release followed the pattern of αIIbβ integrin activation and subsequent closure paralleled by PS exposure. Similar results were obtained using αIIbβ3-deficient platelets from patients with Glanzmann thrombasthenia. Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement
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