Studies on the immobilized urokinase

Abstract

Plasminogen activators are serine proteases with trypsin-like specificity which convert the inactive zymogen plasminogen to enzymatically active plasmin and thereby initiate fibrinolysis $\mbox{\underline{in}}$ $\mbox{\underline{vitro}}$ and $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$. Among them, human urinary type plasminogen activator, known as urokinase (EC.3.4.99.26) has been used clinically for treatment of thrombosis and/or vascular obstructions. The half-life of urokinase $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$, however, is very short and hence large amount of urokinase and repeated injections are required to maintain enough fibrinolytic potential $\mbox{\underline{in}}$ $\mbox{\underline{vivo}}$, when urokinase is used for the clinical treatment. In addition, many prosthetic materials such as artificial organs are widely used. Because of development of thrombus on the surfaces of body prosthetic material contact to blood, continuous use in body has severe limitations and application of urokinase to artificial organs is gradually required. To improve these disadvantages and to develope clinical application of urokinase requirements to these fundamental scientific knowledge on urokinase, immobilization/conjugation were attempted in studying 1) model study of immobilization of urokinase on Sepharose matrix, 2) development of conjugated form of urokinase on soluble dextran, 3) development of immobilized urokinase on cellulose hollow fiber membrane used in artificial kidney set. Immobilization of urokinase on insoluble Sepharose gel was succeeded as follows ; To the CNBr-activated Sepharose gel, urokinase was directly coupled. The resulting urokinase showed 37\% caseinolytic activity compared with initial loading amount, and 14.4 CTA units of specific activity. And Sep-$C_n$-COOH-UK was prepared by coupling to NOS-activated Sep-($CH_2$)n_COOH which was previously synthetized from CNBr-activated Sepharose gel and $NH_2$-($CH_2$)n_CO...