Surface characteristics and properties of lumbrokinase-immobilized polyurethane.
Potent and novel fibrinolytic enzymes (lumbrokinase [LK]) were extracted from the earthworm, Lumbricus rubellus. These enzymes were very stable and showed greater antithrombotic activity than other currently used fibrinolytic proteins. An LK fraction showing the most potent fibrinolytic activity was immobilized onto a polyurethane (PU) surface to investigate its enzymatic activity and antithrombotic activity. A methanol-extracted PU surface was coated with 3% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC)/tetrahydrofuran (THF) solution, and the surface was incubated in an LK solution/phosphate-buffered saline (PBS, pH 7.4). The surface properties were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), electron spectroscopy for chemical analysis (ESCA), and dynamic contact angle. The stability of immobilized LK was determined by caseinolytic activity assay and the specificity of immobilized LK on fibrinogen/fibrin was observed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antithrombotic activity of immobilized LK was evaluated using an ex vivo rabbit A-A shunt experiment. LK immobilization was confirmed by ATR-FTIR and ESCA. Immobilized LK demonstrated stable proteolytic activity during various incubation periods. Immobilized LK proteolyzed fibrinogen and fibrin almost specifically, while it hardly hydrolyzed other plasma proteins including plasminogen and albumin. In the ex vivo A-A shunt experiment, the LK-immobilized surface significantly prolonged occlusion time over control surfaces. This 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.
Ryu
G H
GH
Department of Biomedical Engineering, College of Medicine, Seoul National University, Korea.
Han
D K
DK
Park
S
S
Kim
M
M
Kim
Y H
YH
Min
B
B
D000818:Animals; D004577:Electron Probe Microanalysis; D010450:Endopeptidases; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005343:Fibrinolytic Agents; D008297:Male; D011140:Polyurethanes; D011817:Rabbits; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013499:Surface Properties
D004800:Enzymes, Immobilized; D005343:Fibrinolytic Agents; D011140:Polyurethanes; D010450:Endopeptidases; C083421:lumbrokinase