Complementary roles of platelet αIIbβ3 integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles
Alexandra C.A. Heinzmann, Mieke F.A. Karel, Daniëlle M. Coenen, Tanja Vajen, Nicole Meulendijks, Magdolna Nagy, Dennis Suylen, Judith M.E.M. Cosemans, Johan W. M. Heemskerk, Tilman M. Hackeng, Rory R. Koenen
Abstract
BACKGROUND AND AIMS: , 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: -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.