Recombinant Plasmin from Lemna: Purification, Characterization and In Vitro Comparison with Human Plasma-Derived Plasmin.

Background: The fibrinolytic enzyme plasmin has gained renewed interest as a direct thrombolytic agent. Local delivery of plasmin via catheter into thrombi, combined with the efficient neutralization of plasmin that may escape from the clot by its inhibitors α₂-antiplasmin (α₂-AP) and α₂-macroglobulin may provide safe and effective removal of pathologic blood clots.

Objective: To overcome the potential for pathogen contamination associated with plasmin prepared from pooled human plasma, we have generated recombinant plasmin (r-PLM) from transgenic Lemna plants and compared its biological properties with those of plasma-derived plasmin (pd-PLM).

Results: Lemna minor, an aquatic plant, was transfected with the c-DNA encoding human plasminogen, and grown in media under controlled conditions. Lemna-derived plasminogen (r-PLG) was extracted from plant-tissue, separated on lysine-Sepharose and subsequently converted into plasmin (r-PLM), using urokinase-type plasminogen activator (u-PA). r-PLM was further purified to homogeneity using affinity chromatography on benzamidine-Sepharose. r-PLG and r-PLM co-migrate with pd-PLG and pd-PLM on non-reducing SDS-PAGE with the respective molecular weights of about 90,000 kDa and 82,000 kDa. When analyzed on reducing SDS-PAGE, r-PLM, just like pd-PLM, separates into a 56,000 kDa heavy chain and a 26,000 light chain. On Western blots r-PLG and r-PLM cross-react with polyclonal antibodies that were raised against pd-PLG, indicating immunologic identity of r-PLG/PLM and pd-PLG/PLM. All three plasminogen activators (PA), tissue-type PA, u-PA and streptokinase, activate r-PLG. Activity assays using the PLM-specific chromogenic substrate S-2403 show that catalytic rates of r-PLM are indistinguishable from pd-PLM, indicating that in this assay r-PLM and pd-PLM have comparable activity. Purified r-PLM and pd-PLM have similar specific activities. Like pd-PLM, r-PLM activity is inhibited by its physiological inhibitor α₂-AP and Kunitz-type inhibitors SBTI and aprotinin. Inhibition of r-PLM by α₂-AP results in the formation of a high-molecular weight SDS-stable complex, which migrates on SDS-PAGE with a molecular weight of 150,000 kDa, the cumulative molecular weight of r-PLM (82,000 kDa) and α₂-AP (65,000 kDa). In microtiter-based fibrinolysis assays, both pd- and r-plasmin dissolve fibrin clots at the same rate and in a concentration-dependent manner.

Conclusion: This work demonstrates that proteins of the fibrinolytic system can be expressed in Lemna. Lemna-derived r-PLM and pd-PLM have comparable biochemical properties, activity, and are equally sensitive to known plasmin inhibitors. Plant-derived r-PLM may be a valuable alternative to PLM preparations from pooled human plasma.