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

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 aIIbß3 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 aIIbß3 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.