Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein-LKs effect to protect rats against thrombosis. Thus, in this study, CTB-LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and G(M1)-ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB-LK had a more significant anti-thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant-derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB-LK was designed for oral administration, thus the expression of CTB-LK in edible sunflower seeds eliminated the need for downstream processing of proteins. (C) 2014 American Institute of Chemical Engineers