Parameters affecting optimum activity and stability of urokinase-bound fibrocollagenous tubes

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Abstract
Urokinase was immobilized by entrapment to fibrocollagenous tubes in order to develop a small-caliber fibrinolytic vascular prosthesis. Several parameters associated with the immobilization process were studied in order to optimize bound urokinase activity and stability. A total of 37% of the absorbing enzyme was attached to the collagen tube and 38% of the attached enzyme retained esterolytic activity, under optimal conditions. In the crosslink step of the entrapment process, the glutaraldehyde concentration was varied from 0.01 to 5.00% (i.e., 0.01, 0.1, 1.0, 3.0, and 5.0%). Urokinase activity was optimized at a 1.0% glutaraldehyde crosslink concentration. Urokinase-bound fibrocollagenous tubes (UK-FCT) prepared at the above glutaraldehyde concentrations were tested for their activity with time. The UK-FCT's with 0.1, 1.0, and 3.0% glutaraldehyde retained constant activity for at least 75 h operation time. The UK-FCT's with 0.1, 1.0, and 3.0% glutaraldehyde retained constant activity for at least 75 h operation time. The UK-FCT's with 5.0 and 0.01% glutaraldehyde remained stable for the first 50 h operation time, but begandeactivating beyond 50 h. UK-FCT'S Crosslinked with 0.01, 0.1, 1.0, and 5.0% glutaraldehyde were recrosslinked with 0.02% glutaraldehyde for 24 h, after they have been operating for 50 h, and the effect of reexposing the crosslink agent on the stability of the UK-FCT's was studied. The results showed that 0.02% glutaraldehyde reexposure had no effect on 0.1, 1.0, and 5.0% glutaraldehyde crosslinked UK-FCT's but exerted an inhibitory effect on a 0.01% crosslink density UK-FCT. Several fibrocollagenous tubes were exposed to various glutaraldehyde concentrations prior to immobilizing urokinase. The subsequent immobilization process occurred under optimal conditions. The effect of the precrosslink step on the activity of the UK-FCT was studied. Results indicated that UK-FCT activity decreases as the precrosslink density increases. The UK-FCT's made under optimal conditions remained stable for at least 75 h operation time, corresponding to ca.1 year of storage time. Ex vivo exposure of UK-FCT's to whole canine blood did not affect catalytic activity. Implantation of a UK-FCT by carotid arterial interposition via an end-to-end anastomosis and subsequent excision after 60 days resulted in an enhanced esterolytic activity which decreased with time to a level close to preoperative levels.

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