Abstract/Summary

Background: Cell junctions form multiprotein complexes that facilitate contact or adhesion between neighbouring cells or between a cell and the extracellular matrix. Among these, gap junction-mediated intercellular communication has been found to regulate platelet function in an αIIbβ3-dependent manner. Notably, previous studies on JAM-A, a component of tight junctions, have reported clusters of JAM-A on thrombin-activated platelet contacts. JAM-A associates with integrin αIIbβ3 and suppresses its activation; however, this interaction is disrupted during platelet activation. Zonula occludens 2 (ZO-2) is another essential component of tight junctions, connecting the actin cytoskeleton with transmembrane components. Interestingly, approximately 3,900 copies of ZO-2 were detected in the platelet proteome. Additionally, significant levels of transcripts for zonula occludens proteins (ZO-1, ZO-2, and ZO-3) were found in megakaryocytes. Despite this, the protein expression or function of these ZO-proteins in platelets remains unknown. Therefore, our study aimed to explore potential ZO-containing complexes that mediate platelet functions. Result: Immunoprecipitation and western blotting experiments confirmed the expression of ZO-1, ZO-2, and ZO-3 in human platelets. Both ZO-2 and JAM-A were found to form physical complexes in resting and thrombin-stimulated platelets. Confocal and super-resolution microscopy further confirmed complex formation, showing increased levels upon platelet stimulation as ZO-1 and ZO-2 translocated to the plasma membrane vicinity of both platelets and MEG-01 cells. Notably, the reduction of either ZO-1 or ZO-2 expression in MEG-01 cells using ZO-1 siRNA or ZO-2 siRNA hindered the disengagement of JAM-A from integrin αIIbβ3, which was associated with reduced fibrinogen binding. The role of ZO-1 in integrin αIIbβ3 regulation was further validated using the peptide inhibitor αCT1. Targeting ZO-1 with aCT1 in MEG-01 cells prevented JAM-A dissociation from integrin αIIbβ3, leading to reduced fibrinogen binding. The αCT1 peptide also diminished P-selectin exposure, platelet spreading, adhesion, and aggregation, highlighting its regulatory role in platelet function. Conclusion: These findings suggest that ZO-1 and ZO-2 form a complex with JAM-A to mediate platelet activation. This new understanding of the molecular processes involved in platelet function suggests a promising new target for developing anti-thrombotic therapies.