Antithrombotic activity of a lumbrokinase immobilized polyurethane surface

Six fractions of strong and novel fibrinolytic enzymes (lumbrokinase, LK) were extracted from the earthworm Lumbricus rubellus. The enzymes in these fractions appeared to be very stable and showed greater antithrombotic activity than other currently used antithrombotics. The authors immobilized an LK fraction that shows the most potent fibrinolytic activity on a polyurethane (PU) surface to investigate its enzymatic and antithrombotic activity. The methanol extracted PU surface was treated with a 3% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC) solution and finally incubated in an LK solution in PBS (pH 7.4). The immobilized LK activity was estimated by the fibrin plate method and caseinolytic activity assay. The antithrombotic activity was evaluated by in vitro 125I-fibrinogen adsorption in fresh whole blood and 99mTc platelet adhesion tests. In addition, the occlusion time was determined through ex vivo rabbit A-A shunt experiments. The content and unit activity of immobilized LK were found to be 24 micrograms/cm2 and 18 IU/cm2, respectively. The relative activity ratio of immobilized LK to soluble LK was found to be approximately 34%. Immobilized LK was stable within a various pH range and resistant to inhibitors and thermal inactivation. Less fibrinogen was adsorbed and fewer platelets adhered on an LK-immobilized surface than on PU and PU-MAMEC controls. The ex vivo occlusion time of untreated PU and PU-MAMEC surfaces were only 32 and 42 minutes, respectively. But that of LK-immobilized PU was extended to 140 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)