ATTENUATION OF THE RELEASE OF MATRIX DEGRADING ENZYMES FROM IN VITRO CULTURE OF HBMEC BY LMWH-TISSUE PLASMINOGEN ACTIVATOR CONJUGATE

POSTER PRESENTATIONS

Objective. The purpose of this study is to examine whether or not the conjugation of Low-Molecular Weight Heparin (LMWH) to t-PA reduces the production of matrix degrading enzyme (MDE) (matrix metalloproteases; presumably MMP-9) from Human Brain Microvascular Endothelial Cells (HBMEC) in vitro. Background. Tissue plasminogen Activator (t-PA) is only FDA-approved drug for ischemic stroke treatment. However, t-PA can act as a cytokine that stimulates the production of MDE that may compromise the integrity of the blood-brain barrier (BBB), leading to intracranial hemorrhage (ICH). Therefore, a sterically hindered t-PA construct may be desired to control t-PA activity and diminish the production of MDEs at the brain capillary endothelial cells. Methods. A camouflaged-tPA construct that enables triggered plasminogen activation was used, wherein LMWH-tPA is a constituent. LMWH-tPA was synthesized, and then isolated by ion-exchange chromatography and centrifugal filtration. The construct activity was evaluated by an indirect chromogenic assay. An in vitro culture of HBMEC was used as a model constituent of human BBB to evaluate the effect of treatments on the production of MDEs. Gelatin zymography assay was used to evaluate the production of MMPs. Student t-test and one-way ANOVA were used. Results. The modified-tPA retained ~95% of enzyme activity compared to the native t-PA. The LMWH-tPA conjugate significantly reduced the production of MDEs compared to the native t-PA, from an in vitro culture of HBMEC. Student ttests and ANOVA test showed statistically significant different when comparing the fold increasing in MDE levels among treatments (p<0.05). Conclusion. The generation of MDEs by the HBMEC was attenuated in the presence of LMWH-tPA 55 conjugate compared to the native t-PA. Grants. This study was supported by President’s Faculty Research & Department Grant (PFRDG), NSU, and Saudi Arabian Cultural Mission (SACM).