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 M M Meulendijks; Magdolna Nagy; Dennis P L Suylen; Judith M E M Cosemans; Johan W M Heemskerk; Tilman M Hackeng; Rory R Koenen

doi:10.1016/j.atherosclerosis.2020.07.015

Complementary roles of platelet α_IIbβ₃ integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles

Atherosclerosis. 2020 Oct:310:17-25. doi: 10.1016/j.atherosclerosis.2020.07.015. Epub 2020 Aug 1.

Affiliations

¹ Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands.
² Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology and Vascular Medicine, Medical Faculty, Heinrich-Heine-University, Düsseldorf, the Netherlands.
³ The Netherlands Organisation for Applied Scientific Research (TNO), Material Solutions, Eindhoven, the Netherlands.
⁴ Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands. Electronic address: r.koenen@maastrichtuniversity.nl.

PMID: 32877806
DOI: 10.1016/j.atherosclerosis.2020.07.015

Abstract

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 α_IIbβ₃ integrins, indicating that α_IIbβ₃ 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 α_IIbβ₃, 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 α_IIbβ₃ integrin activation and subsequent closure paralleled by PS exposure. Prevention of α_IIbβ₃ activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using α_IIbβ₃-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.

Keywords: Actin cytoskeleton; Blood platelets; Extracellular vesicles; Integrins; Platelet activation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blood Platelets
Extracellular Vesicles*
Humans
Integrin beta3
Phosphatidylserines*
Platelet Activation
Platelet Glycoprotein GPIIb-IIIa Complex

Substances

Integrin beta3
Phosphatidylserines
Platelet Glycoprotein GPIIb-IIIa Complex