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M.K. Hartzer, W.A. Dailey, M.T. Trese, G.A. Williams, M.A. Hermel, D. Trese; Rapid Purification of Autologous Plasmin for Use in Vitreoretinal Surgery . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3046.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Development of a device for the rapid purification of autologous plasmin for use as an adjunct to vitreoretinal surgery. Plasmin aids in the creation of a posterior vitreous detachment by cleavage of fibronectin and laminin at the vitreoretinal interface. Methods: Blood is collected in six tubes containing ACD solution A. The tubes are centrifuged and 22 ml of plasma are collected using a 30 ml syringe with a directional flow valve. The plasma is injected through a previously activated lysine affinity cartridge. The affinity cartridge is washed with 60 ml of 100 mM potassium phosphate buffer, pH 7.5 to remove any unbound plasma proteins. The bound plasminogen is eluted from the cartridge with 2.5 mM epsilon aminocaproic acid (Amicar). The Amicar is subsequently removed by a gel filtration cartridge. The eluted plasminogen (2.6 ml) is then concentrated to a volume of 0.5-0.6 ml using a G-50 Sephadex concentrator. The concentrated plasminogen is passed through a 0.22 micron filter into a sterile vial. Plasminogen is converted to plasmin by addition of 6250 units of streptokinase. After five minutes, the presence of plasmin activity is demonstrated by injecting 0.02 ml of enzyme solution into an indicator device containing a chromogenic substrate (S-2251) Results: The resulting plasmin preparation has an activity of approximately 12 units. This provides for a dose of approximately 1.8 units in a volume of 0.15 ml. The preparation can be completed in 30 minutes. Conclusions: Autologous plasmin can be rapidly prepared for use in vitreoretinal surgery.
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