Urokinase, immobilization

Fig. 8 Immobilization of urokinase on the surfaces of islet cells, (a) Surface modification (/) chemical structure of ssDNA-PEG-lipid, and (2) ssDNA-PEG-lipid anchoring to the cell membrane. (b) Introduction of a complementary ssDNA onto urokinase, which was first modified with a madeimide group by a cross-linker, EMCS. (c) Urokinase-immobilization through DNA...

S. Watanabe, Y. Shimizu, T. Teramatsu, T Murachi, T Hino, The in vitro and in vivo behavior of urokinase immobilized onto collagen-synthetic polymer composite material. Journal of Biomedical Materials Research 15 (4) (1981) 553-563. 

Takeda K, Harada A, Kubo M, Inenaga T, Tsuruya K, Mitsuiki K, et al. Successful use of single-lumen, urokinase immobilized femoral catheters as a temporary access for haemodialysis. Nephrol Dial Transplant 1998 13(1) 130-3. 

Immobilization of fibrinolysis enzyme Urokinase immobilized polymer ... 

Fig. 9 Surface modification of cells with ssDNA-PEG-lipid. (a) Real-time monitoring of PEG-lipid incorporation into a supported lipid membrane by SPR. (r) A suspension of small unilamellar vesicles (SUV) of egg yolk lecithin (70 pg/mL) was applied to a CH3-SAM surface. A PEG-lipid solution (100 pg/mL) was then applied, (ii) Three types of PEG-lipids were compared PEG-DMPE (C14), PEG-DPPE (C16), and PEG-DSPE (C18) with acyl chains of 14, 16, and 18 carbons, respectively, (b) Confocal laser scanning microscopic image of an CCRF-CEM cell displays immobilized FITC-oligo(dA)2o hybridized to membrane-incorporated oligo(dT)20-PEG-lipid. (c) SPR sensorigrams of interaction between oligo(dA)2o-urokinase and the oligo (dT)2o-PEG-lipid incorporated into the cell surface, (i) BSA solution was applied to block nonspecific sites on the oligo(dT)20-incorporated substrate, (ii) Oligo(dA)20-urokinase (solid line) or oligo(dT)20-urokinase (dotted line) was applied...

Fig. 10 Confocal laser scanning microscope images of islets with urokinase (UK) immobilized on the membrane. The green fluorescence indicates positive immunostaining for UK. (a) Islets were modified with oligo(dT)2o-PEG-lipid (C16) or (b) oligo(dT)2o-PEG-lipid (C18) then, oligo (dA)2o-UK was added to the media, (c) Unmodified islets with (left) and without (right) oligo (dT)20-PEG-lipids added to the solution. Insets. Bright field images. Scale bars 100 pm...

Fig. 11 Islets with immobilized urokinase (UK-islets) were tested for the ability to dissolve fibrin, (a) Fibrin in the plate gel medium was dissolved by UK-islets (clear areas). Fifty islets were applied to each spot, and the plate was observed after incubation at 37 °C for 14 h. (1) untreated islets (2) UK-islets treated with oligo(dT)2o-PEG-lipid (C16), just after preparation (3) UK-islets treated with oligo(dT)2o-PEG-lipid (C16) lost activity after 2 days in culture (4) UK-islets treated with oligo(dT)20-PEG-lipid (C18), just after preparation and (5) UK-islets treated with oligo (dT)20-PEG-lipid (C16) lost activity after 2 days in culture, (b) Morphology of UK-islets after 1 and 7 days of culture...

Chen H, Teramura Y, Iwata H (2011) Co-immobilization of urokinase and thrombomodulin on islet surfaces by poly(ethylene glycol)-conjugated phospholipid. J Control Release 150 229-234... 

Totani T, Teramura Y, Iwata H (2008) Immobilization of urokinase on the islet surface by amphiphilic poly(vinyl alcohol) that carries alkyl side chains. Biomaterials 29 2878-2883... 

At present, the binary water-soluble preparation of heparin and proteolytic enzymes is being applied for the treatment of thromboses. For instance, injection into the bloodstream of heparin-plasmin complex or a heparin-plasmin-streptokinase preparation leads to the total dissolution of the thrombus, while if introduced separately, heparin and streptokinase do not display the lytic action at all, and plasmin, alone or together with streptokinase, dissolves the thrombus only partially 132>. The treatment of acute thrombophlebitis with trypsin resulted in a full dissolution of the thrombus and in an increase of antithrombin III in the blood 133). Administration of trypsin together with heparin has an effect similar in efficiency to the action of the heparin-plasmin complex 134>. The use of a mix of heparin and urokinase for improving tbrom-boresistance of polymeric materials was also described 13S). These substances were immobilized by preliminary coating of the surface of a polymer with a graphite layer and subsequent adsorption of heparin and the enzyme. 

Kumar A, Bansal V, Andersson J, Roychoudhury PK, Mattiasson B (2006), Super-macroporous cryogel matrix for integrated protein isolation - immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line, J. Chromatogr. A 1103 35-42. 

Blood-compatible polymer materials are required to inhibit both platelet adhesion and coagulation just as the endothelial on the polymer surface. It is known that there are many investigations in the design and the synthesis of socalled antithrombogenic materials. The immobilization of biologically active substances such as heparin [74, 75], urokinase [76], and prostaglandins [77-81] is one of the practical approaches. 

Another approach for studying a cell s capacity to degrade a specific substrate lies midway between zymography and the immobilized component method described above. Here, the cells are seeded on the bottom of a dish, which is then overlaid with soft agar containing a substrate for proteolytic activity. After incubation under appropriate conditions, lithic plaques appear within the agar, indicative of enzymatic activity. The assay is described below to evidence urokinase-plasminogen activator (uPA) activity. 

Ultimately, the cost of immunosorbent isolation will depend on the entire process and must be evaluated against alternative processes. Consider, as an example, the costs and decisions involved in the purification of urokinase. One course of drug therapy consists of 33 mg of urokinase (4,000,000 CTA units). At the hospital pharmacy the drug costs for one course of treatment are currently 3,000 (9), or 91,000/gram. There are approximately 76,000 patients in the U.S. that could be treated with urokinase therapy each year requiring an annual production of approximately 2,500 g. We have selected a monoclonal antibody that has allowed the purification of urokinase from urine, tissue culture media, and bacterial culture media in a single step with 85% retention of urokinase activity (6). This monoclonal antibody was immobilized at 2 gL l with an immobilization yield of 0.8 and a cycle half number of 300 cycles. The urokinase capacity for the first cycle would be 1.2 gL l of immunosorbent. 

Allowing for the decrease in activity with cycle number the total amount of immunosorbent required is 21.4 L. At 2 gL-l loading and 200 g l for the monoclonal antibody and 200 IT for the matrix and immobilization, the total cost of the immunosorbent is 12,840. This amounts to 4.90 g l of urokinase. In this example the low costs are obviously attractive. 

Oshiro,T. 1983. Thrombosis, antithrombogenic characteristics of immobilized urokinase on synthetic polymers. In Biocompatible Polymers, Metals, and Composites, M. Szycher, Ed. pp. 275-299. Technomic, Lancaster, PA. 

Urokinase has been widely used for the clinical treatment of thrombogenetic disease and hemorrhoidal disease. Artificial organ materials, on which urokinase was immobilized for its fibrinolytic activity, have been developed for blood-compatible materials. For example, Liu et al. immobilized urokinase by encapsulation in poly(2-hydroxyethyl methacrylate) and Kbnig et al. introduced urokinase on the surface of the polytetrafluoroethylene using plasma modification technique by covalent bond. Another example of immobilized urokinase application was reported by Kato and coworkers, who had used mokinase immobilized in a Teflon catheter for treatment of thrombosis. 

Kbnig, U. et al.. Plasma modification of polytetrafluoroethylene for immobilization of the fibrinolytic protein urokinase. Surf. Coat. TechnoL, 119, 1011, 1999. 

Bhaigava, K., and Ando H., 1992, Immobilization of active urokinase on albumin micro-spheres Use of chemical dehydrant and process monitoring, Pharm. Res. 9 776-781. 

Affinity chromatography of mammalian phospho-fructokinase on immobilized adenine nucleotides Studies of the effect of the chain-length on the adsorption of albumin removal of albumin from sera Purification of commercial preparations of urokinase Purification of Solanum tuberosum agglutinin (sta-lectin)... 

Immobilization of the enzyme urokinase has also been utilized [35-37], Urokinase is used chnically as a thrombolytic agent in the treatment of severe or deep venous thrombosis and occluded intravenous cannulas made with SPUs. 

Urokinase AT-III PGEiiMethyl Dopa Complex Immobilized Albumin-Blended Chitosan Membranes - Antithrombotic and Permeability Properties 297... 

Here we report an attempt to immobilize an urokinase antithrombin III PGEirmethyl dopa conjugate on albumin blended chitosan membranes and the evaluation of their antithrombotic and permeability properties. Further studies have also been undertaken to improve the mechanical properties of the albumin blended chitosan membranes through optimum cross-linking with carbodiimide. A novel approach of producing other protein blends of chitosan, as non-thrombogenic membranes, for improved permeability had been demonstrated and compared to that of the standard cellulose membranes. 

The results of the platelet adhesion to the urokinase complex modified albumin blended chitosan membranes is shown in Table 3. The number of adhering platelets seen on the urokinase derivative immobilized surface has been dramatically reduced when compared with the albumin blended membrane. The permeability of various molecules through the albumin blended chitosan and the urokinase derivative immobilized mem-branes, as a function of time, is demonstrated in Figures 1 and 2, respectively. It is evident from these studies that the urokinase complex modified membranes had similar permeability properties to those of the... 

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