Formation of tight junction-like structures of zonula occludens 2 in platelet-platelet interaction

Magdolna Nagy; Markus Bender; Natalie S Poulter; Jeremy A Pike; Albert Sickmann; Sonja Vondenhoff; Natalia Bielicka; Marc A M J van Zandvoort; Rory R Koenen; Hugo Ten Cate; Xavier Stéphenne; Johan W M Heemskerk; Constance C F M J Baaten

doi:10.1016/j.rpth.2025.102845

Formation of tight junction-like structures of zonula occludens 2 in platelet-platelet interaction

Res Pract Thromb Haemost. 2025 Apr 8;9(3):102845. doi: 10.1016/j.rpth.2025.102845. eCollection 2025 Mar.

Authors

Magdolna Nagy¹, Markus Bender², Natalie S Poulter^{3

4}, Jeremy A Pike^{4

5}, Albert Sickmann⁶, Sonja Vondenhoff⁷, Natalia Bielicka⁸, Marc A M J van Zandvoort^{7

9}, Rory R Koenen¹, Hugo Ten Cate^{1

10

11

12}, Xavier Stéphenne¹³, Johan W M Heemskerk^{1

14}, Constance C F M J Baaten^{1

7}

Affiliations

¹ Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
² Institute of Experimental Biomedicine-Chair I, University Hospital Würzburg, Würzburg, Germany.
³ Department of Cardiovascular Sciences, School of Medical Sciences, College of Medicine and Health, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
⁴ Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, United Kingdom.
⁵ The Research Software and Analytics Group, University of Exeter, Exeter, United Kingdom.
⁶ Department of Protein dynamics, Leibniz Institute for Analytical Sciences-ISAS-e.V, Dortmund, Germany.
⁷ Institute for Molecular Cardiovascular Research (IMCAR), University Hospital RWTH Aachen, Aachen, Germany.
⁸ Department of Biopharmacy and Radiopharmacy, Medical University of Bialystok, Bialystok, Poland.
⁹ Department of Molecular Cell Biology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
¹⁰ Department of Internal Medicine, Maastricht University Medical Centre+, Maastricht, the Netherlands.
¹¹ Thrombosis Expertise Centre, Maastricht University Medical Centre+, Maastricht, the Netherlands.
¹² Center for Thrombosis and Haemostasis (CTH), Gutenberg University Medical Center, Mainz, Germany.
¹³ Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium.
¹⁴ Synapse Research Institute, Maastricht, the Netherlands.

Abstract

Background: In tissues, cell-cell adhesion, barrier formation, and communication are regulated by gap, adherens, and tight junction (TJ) proteins. Platelets express several of these proteins. Platelets express key building blocks of gap junctions, the connexins, with known functions in integrin αIIbβ₃ regulation. While, for some expressed TJ proteins like junctional adhesion molecule A and endothelial cell-specific adhesion molecule, a role in platelets has been uncovered, for other TJ proteins, like zonula occludens (ZO)-2 a contribution to platelet function is still unknown.

Objectives: This study aimed to elucidate the role of ZO-2 in the stabilization of tight interplatelet contacts.

Methods: Isolated human platelets from healthy volunteers and a patient deficient in ZO-2 were spread on fibrinogen and laminin surfaces in the presence of platelet agonists and inhibitors. Samples were fixed and prepared for microscopy.

Results: Confocal and superresolution fluorescence microscopy indicated a redistribution of ZO-2 molecules forming clusters at sites of stable interplatelet contacts that was dependent on the platelet activation status. In the tight contacts, ZO-2 colocalized with endothelial cell-specific adhesion molecule and junctional adhesion molecule A. Furthermore, platinum replica electron microscopy revealed that interplatelet contacts resulted in compaction, detected as interwoven circumferential actin filaments, of interacting platelets. These changes were antagonized by cyclic adenosine monophosphate elevation and inhibition of αIIbβ₃ integrins. In a blood sample from a patient deficient in ZO-2, we observed an increased thrombus stability, suggesting a potential regulation of thrombus stability by these TJ-like structures.

Conclusion: Jointly, these data point to the assembly of TJ-like structures of interacting platelets, which enforce platelet adhesion contacts but lower 3-dimensional thrombus stability.

Keywords: actin cytoskeleton; integrin alpha IIb beta 3; platelets; tight junctions; zonula occludens 2.