Plasmin, the product of tissue plasminogen activator (tPA) treatment for ischemic stroke, impairs human brain endothelial barrier integrity

Abstract

BackgroundtPA is used for the acute treatment of ischaemic stroke because it converts plasminogen to active plasmin, which breaks down clots. Previous studies show that tPA-activated plasminogen impairs brain endothelial barrier function. However, it is unclear whether the plasmin product of this reaction directly contributes to brain endothelial barrier deterioration. ObjectiveDetermine whether plasmin directly influences the human brain endothelial barrier. MethodsWe developed a new serum-free hCMEC/D3 culture model with ECIS real-time monitoring to establish how plasmin in isolation influences the brain endothelial barrier. ResultsECIS monitoring demonstrated that plasmin caused a concentration-dependent decline in hCMEC/D3 barrier integrity, which was primarily mediated by a reduction in endothelial cell-to-cell interactions. Whilst a decrease in membrane capacitance and increase in basolateral adhesion were also observed, these changes were less marked. The inclusion of 2-antiplasmin ameliorated the changes in hCMEC/D3 barrier properties, suggesting this response is mediated by plasmins proteolytic activity. Quantitative immunocytochemistry confirmed that plasmin stimulated a decline in the key junctional molecules, Claudin-5, VE-Cadherin (CD144), {beta}-Catenin, ZO-1 and PECAM-1 (CD31), which likely contributed to the deterioration of paracellular cell-to-cell interactions. Interestingly, using this serum-free model, tPA alone didnt influence hCMEC/D3 barrier properties, whilst tPA with plasminogen did, implicating plasmins involvement. ConclusionPlasmin directly impaired the barrier function of hCMEC/D3 brain endothelial cell monolayers by stimulating a decline in key junctional molecules. This plasmin-mediated brain endothelial barrier deterioration has important implications for tPA use and should be considered whilst designing safer thrombolytic treatment options for patients experiencing acute ischemic stroke.