Tissue plasminogen Activator (tPA) is currently the only drug approved by the United States Food and Drug Administration for the treatment of ischemic stroke. However, besides its thrombolytic activity, tPA can also act as a signaling molecule via protein-protein interactions, stimulating the production of matrix degrading enzymes. These enzymes may compromise the integrity of the blood-brain barrier (BBB), leading to intracerebral hemorrhage. Furthermore, tPA is capable of interacting with other cell surface components of the neurovascular unit, which may contribute to a worsening of the condition. Therefore, a tPA construct that can selectively interact with other proteins may be desired so that any undesirable protein-protein interactions can be reduced. These interactions include receptor-mediated productions of degrading enzymes at the brain capillary endothelial cells as well as a receptor-mediated excitotoxicity in a neuronal cell model. The PI and coworkers have observed that the secretion of a matrix degrading enzyme (matrix metalloproteinase (MMPs), MMP-9 in particular) from the cultured human brain microvascular endothelial cells (HBMEC) was attenuated when the cells were exposed to low molecular weight heparin (LMWH)-tPA conjugate compared to the native tPA. The purpose of this study is to examine whether or not the conjugation of LMWH to tPA reduces the MMP-9- associated vascular leakiness and excitotoxic neuronal cell death in vitro.
The LMWH-tPA will be synthesized by site-specific modification of tPA on its protease domain followed by conjugation with LMWH, according to the method established by the PI's laboratory. The conjugate will be isolated by ion-exchange chromatography will be characterized for its enzymatic activity. An in vitro culture of HBMEC will be used as a model constituent of human BBB to evaluate the effect of treatments, which are native tPA and the modified tPA, on the production of MMP-9. Gelatin zymography assay will be used to evaluate the production of MMP-9 by HBMEC. Permeability through HBMEC monolayers will be studied using a transwell system using fluorescein-labeled human serum albumin (FITC-HSA) upon exposure to the different tPAs. Also, a receptor-mediated excitotoxic neuronal cell death will be studied in vitro. Student t-test and one-way analysis of variance (ANOVA) will be used to assess whether the difference exist between the different tPAs.
It is expected that the LMWH-tPA conjugate will significantly address, compared to the native tPA, the MMP-9-induced compromise in the cultured endothelial cells as well as a receptor-mediated excitotoxic cell deaths in a neuronal cell model in vitro.