SURFACE CHARACTERISTICS AND PROPERTIES OF LUMBROKINASE-IMMOBILIZED POLYURETHANE

Potent and never fibrinolytic enzymes (lumbrokinase [LK]) were extract ed from the earthworm, Lumbricus rubellus. These enzymes were very sta ble and showed greater antithrombotic activity than other currently us ed fibrinolytic proteins. An LK fraction showing the most potent fibri nolytic activity was immobilized onto a polyurethane (PU) surface to i nvestigate its enzymatic activity and antithrombotic activity. A metha nol-extracted PU surface was coated with 3% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC)/tetrahydrofuran (THF) solution, an d the surface was incubated in an LK solution/phosphate-buffered salin e (PBS, pH 7.4). The surface properties were characterized by attenuat ed total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR ), electron spectroscopy for chemical analysis (ESCA), and dynamic con tact angle. The stability of immobilized LK was determined by caseinol ytic activity assay and the specificity of immobilized LK on fibrinoge n/fibrin was observed by sodium dodecyl sulphate-polyacrylamide gel el ectrophoresis (SDS-PAGE). The antithrombotic activity of immobilized L K was evaluated using an ex vivo rabbit A-A shunt experiment. LK immob ilization was confirmed by ATR-FTIR and ESCA. Immobilized LK demonstra ted stable proteolytic activity during various incubation periods. Imm obilized LK proteolyzed fibrinogen and fibrin almost specifically, whi le it hardly hydrolyzed other plasma proteins including plasminogen an d albumin. In the ex vivo A-A shunt experiment, the LK-immobilized sur face significantly prolonged occlusion time over control surfaces. Thi s is primarily due to the high thrombolytic activity of immobilized LK . In this work, a highly efficient surface modification method on the PU surface was developed, and this LK immobilization technique will be very useful in improving the blood compatibility of blood-contacting devices. (C) 1995 John Wiley & Sons, Inc.