Urokinase (UK) has been immobilized on nylon monofilaments, tubes and sheets, by the method of Edelman et al. (1971). The quantity of immobilized UK by this method was estimated to be 0.05-0.06U/cm2.
The fibrinolytic activities were observed on the standard fibrin plates after incubation at 37°C, for 12-24 hours.
The immobilized UK showed fibrinolytic activity even after preservation at 4°C, for long term and even after heat-treatment at 70°C for 15min. These results have been published previously.
The fibrinolytic activity of immobilized UK was prevented by “antiplasmin” which exists in normal human plasma. So, we have devised a modified technique to immobilize UK on nylon for the purpose of increasing the quantity of binding UK on nylon and of suppressing the activity of antiplasmin. In our technique, Gantrez (G) (copolymer of maleic anhydride and methylvinyl ether) was used to increase quantity of UK (immobilized) on nylon and Nitrophthalic acid (NP) as antiplasmin inhibitor.
The samples we had produced by the devised method revealed remarkable fibrinolytic activities in the existence of human plasma.
The fibrinolytic activities of immobilized UK decreased gradually by repeated incubations. After several repetitions, they settled to a constant value, which was measured at 10-25% of the initial activity. The constant value is considered to represent the quantity of UK which had coupled covalently on the nylon surface.
Antithrombogenicity of immobilized UK was observed employing the method of chandler, by taking measure of thrombus formation time (TFT). Two kinds of anticoagulants were used to test the antithrombogenicity.
The anticoagulants were FP-522 and EG-626, which had been tested as anti-platelet-aggregation medicaments.
The results of these studies were summarized like follows:
1) TFT of non-treated nylon tubes were within ten min.
2) TFT of UK-immobilized nylon tubes with G and NP, were over 45min.
3) TFT of non-treated nylon tubes with clinical dosage of FP-522 solution were over 45min.
Antithrombogenecity of immobilized UK and clinical dosage of FP-522 were found to be effective.
Possibilities of immobilization of another fibrinolytic or thrombolytic agents must be examined. We are carrying out the next studies in vivo, employing the immobilized UK.
