Differential vitreous dye diffusion following microplasmin or plasmin pre-treatment.

Purpose: Plasmin and microplasmin are related enzymes that differ mainly in size. The differential effect of plasmin and microplasmin on vitreous structure, protein degradation, and dye diffusion through porcine vitreous was evaluated. Methods: The enzymatic effect was examined using a number of approaches on fresh porcine eyes: (1) structural integrity of vitreous after a 2-hr incubation using the electron microscope (EM); (2) effect on soluble proteins within the vitreous using gel electrophoresis after incubation at various time points over a 24-hr period; (3) fluorescein dye diffusion within the vitreous cavity measured over a 1-hr period following a 2-hr incubation. The chosen enzymatic activities for plasmin 0.5 IU and microplasmin 125 mu g were within the clinical range, and were chosen for equipotence. A saline control was also used in all experiments. Results: Significant structural changes were seen with both microplasmin and plasmin when examined by EM. Gel electrophoresis showed that microplasmin and plasmin digested the same proteins, mainly molecular weights above 50 kDa. The enzymatic effect was noticeable earlier in microplasmin-treated eyes and was more significant by the end of the incubation period. Differential fluorescein diffusion rates were seen between normal saline, plasmin, and microplasmin within the vitreous cavity. The greatest diffusion rate was seen with microplasmin and was statistically significantly higher than plasmin. Conclusion: Microplasmin and plasmin have a similar enzymatic effect on vitreous. However, an equipotent amount of microplasmin appears to have a more extended effect on vitreous gel. This may, in part, be related to its smaller size allowing it to diffuse more readily through the vitreous matri

Microplasmin - Ex vivo characterization of its activity in porcine vitreous.

PURPOSE: Microplasmin is a recombinant protein limited to the enzymatic moiety of plasmin without any of its cringle domains. Its enzymatic activity is similar to that of plasmin enzyme. The present study characterizes in a porcine eye model the vitreolytic ability of microplasmin. METHOD: Freshly harvested porcine eyes were used in these trials. Eyes were injected with escalating doses of microplasmin (62.5, 125, 250, 400 microg) for 1 hour or with 125 microg microplasmin with increasing time exposures (15, 30, 60, 120 minutes). Eyes were fixed by a very slow dehydration process to preserve the integrity of the vitreous retinal interface. They were examined by light microscopy to determine the degree of posterior vitreous detachment and by scanning electron microscopy (SEM) to study structural changes. RESULTS: Effective separation of the posterior hyaloid appeared to be dose dependent. After 1 hour, the posterior pole was detached in 100% of porcine eyes exposed to 125 microg microplasmin and in the midperiphery to 250 microg microplasmin. Vitreous at the ora did not detach. At 120 minutes of exposure, midperipheral detachment was observed with 125 microg microplasmin. A smooth retinal surface was seen where the enzyme caused posterior vitreous detachment. There was also significant change to the integrity of the vitreous without any obvious structural alterations to the retina by histology or scanning electron microscopy. CONCLUSIONS: Microplasmin caused vitreolysis and posterior vitreous separation in an ex vivo porcine eye model in an apparent dose- and time-dependent fashion. In this model system, the minimal effective dose appeared to be 125 microg