Abstract
Abstract: :
Purpose: Plasmin enzyme has been proposed as an aid to create posterior vitreous separation based on its ability to cleave laminin and fibronectin, both known as structural components of the vitreoretinal interface. Its precursor, plasminogen, can be activated to plasmin either by direct proteolytic cleavage by urokinase or tissue plasminogen activator, or by rapid formation of a 1:1 complex with streptokinase. This complex is then converted to a streptokinase-plasmin complex (SK-PL), which displays similar fibrinolytic activity to free plasmin and can catalytically generate free plasmin by proteolytic cleavage of plasminogen. The purpose of this study is to compare the efficacy of SK-PL, SK-PL activated free plasmin and urokinase activated plasmin in cleaving laminin and fibronectin. Methods: Plasmin activity was determined with a chromogenic assay. Plasminogen was isolated by lysine affinity chromatography from human plasma and eluted with aminocaproic acid, which was subsequently removed by G-25 sephadex chromatography. Plasminogen yields were determined with a BCA-assay. Human streptokinase was added to plasminogen in molar ratios between 0.1:1 to 2:1, generating SK-PL at ratios ≥ 1:1, and mixtures of SK-PL and free plasmin (SK-PL/plasmin) at lower ratios. Various doses of SK-PL, SK-PL/plasmin and urokinase activated human plasmin (Sigma) were added to purified laminin and fibronectin (Sigma). The mixtures were incubated at 37°C for 30min-22h, then processed for SDS-PAGE, with enzymes and untreated substrates as controls. Results: Streptokinase activated plasminogen rapidly and completely at all molar ratios. All enzyme preparations cleaved laminin and fibronectin. SK-PL/plasmin was more effective in cleaving both laminin and fibronectin with increasing free plasmin content (decreasing SK-PL to free plasmin ratio). Urokinase activated plasmin displayed similar cleavage as SK-PL/plasmin at the 0.1:1 ratios. Degradation of laminin by all enzymes was slower that that of fibronectin. Conclusions: The different modes of plasminogen activation determine its efficacy in cleaving fibronectin and laminin. Lower streptokinase to plasminogen ratios result in generation of increasing amounts of free plasmin, which is more effective in laminin and fibronectin degradation than the SK-PL complex, and may therefore be more useful than SK-PL as a surgical tool to facilitate posterior vitreous separation.
Keywords: vitreoretinal surgery • enzymes/enzyme inhibitors • vitreous