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T. Sakuma, J. Inoue, M. Kiyokawa, N. Hatano, Y. Tamiya, K. Shiina, A. Mizota, M. Tanaka; Purification of Autologous Plasmin for Vitreoretinal Surgery . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4684.
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© ARVO (1962-2015); The Authors (2016-present)
Plasmin purified from autologous blood is a useful enzyme for vitreoretinal surgery, but it requires a long and difficult time to isolate. The purpose of this study was to develop an improved method to obtain safe and highly–purified autologous plasmin in a shorter time for use as an adjunct to vitreoretinal surgery.
To isolate plasmin, the proteinase inhibitors, aprotinin and benzamidine, were used to suppress protein denaturation. The collected blood (30 ml) was centrifuged and 10 ml of plasma was used. The plasma was injected into a previously activated lysine affinity cartridge. The affinity cartridge was washed with 10 ml of 50 mM potassium phosphate buffer at pH 7.5 to remove any unbound plasma proteins. The bound plasminogen was eluted from the cartridge with 2.5 mM epsilon aminocaproic acid, and the epsilon aminocapric was subsequently removed by a concentration kit. The concentrated plasminogen was passed through a 0.22 micron filter into a sterile vial. The samples were incubated for 48 hours to check for contamination. Plasminogen was converted to plasmin by adding 500 units of urokinase. The activity of the activated plasmin was checked before use.
By adding the suppressors of protein denaturation in the purification process, high–purity plasmin can be prepared from autologous blood in a shorter time. The 10 ml of plasma provided a dose of approximately 1 unit in a volume of 0.1 ml. No bacterial contamination was detected.
Our new method allows us to isolate autologous plasmin safely, easily, and in a shorter time. This method should expand the use of plasmin in the clinic
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