Urokinase activator

Czekay RP, Kuemmel TA, Orlando RA, Farquhar MG. Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity. Mol Biol Cell 2001 12(5) 1467-1479. 

Resnati M, Pallavicini I, Wang JM, Oppenheim J, Serhan CN, Romano M, et al. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci USA 2002 99(3) 1359-1364. 

Figure 8. Renaturation of low molecular weight urokinase observed in samples collected from continuous flow refolding. UK was injected in 9.3 M urea into 2.0 M urea, 20 mM Bis-Tris, pH 7.8 buffer with a gradient from 2.5 mM reduced glut-athione (GSH) to 2.5 mM oxidized glutathione (GSSG). Upper panel Data for GSSG concen-tration are from direct absorbance measurements at the secondary UV detector data for GSH were determined by DTNB titration of collected fractions. Lower panel urokinase activity in collected fractions measured by spectrophotometric assay using S-2444 (12) in a Molecular Devices titerplate reader.

GLA has been shown in experimental model of cancer to inhibit metastasis of cancer. Urokinase concentration is increased in malignant cancer cells, and it is reported to play a role in invasiveness and metastasis of cancer, du Toit et al. (97) studied the effect of GLA on urokinase activity. They observed that GLA is a competitive inhibitor of urokinase activity with a Ki value of 120 pM. In a... 

Converted to fibrinolysin Precursor of urokinase Activates plasminogen Inhibits thrombin, IXa, Xa, XIa, Xlla Inhibits factors Va, Villa Co-factor for protein C Inhibits binding of fibrinolysin... 

K2. Katoh, K., and Miyazaki, H., Assay method for urokinase activity by capillary-tube isotachophoresis using a synthetic substrate. J. Chromatogr. 188, 383-390 (1980). 

The concentration of urinary glycoproteins may play an important role in stone formation. Therefore, the level and activity of urinary proteases, for example, urokinase and plasmin, might be of great significance. According to this hypothesis, low activities or decreased production of urinary urokinase or plasmin may increase the urinary uromucoid concentrations, as well as stone formation. Increased levels of a urokinase inhibitor, namely urinary trypsin inhibitor (67,000 Da) and decreased urokinase activity in the urine of stone formers are well established (T2). Urinary urokinase activity was therefore studied, as well as the desialylation of urinary glycoproteins. (Fig. 4). 

Spectrophotometric analysis of urinary inhibition on the active forms (HMW-UK and LMW-UK) revealed a significant difference between subjects with and without renal calculi (VI). A positive correlation exists between the percentage of inhibition and the urinary urate concentration. Urate inhibits both the LMW-UK and the HMW-UK, but not plasmin. These results are in perfect agreement with reports that stone patients have higher urinary urate concentrations (C2). These observations may explain why allopurinol is administered to kidney stone patients. Allopurinol causes a decrease in urinary urate excretion by inhibiting the xanthine oxidase, which could cause a higher urinary urokinase activity. 

Because age and sex are risk factors of urolithiasis (Fig. 1), radioimmunoassays on males of different ages show that the total urinary testosterone concentrations in stone patients are significantly lower than those in their healthy counterparts (V2). A positive correlation also exists between the total urinary testosterone concentrations and the activity of urokinase (LMW-UK). Steroids such as estradiol, catecholestrogens, and dihydrotestosterone inhibit the urokinase activity in vitro at concentrations higher than 10-5 M (C. H. van Aswegan and D. J. du Plessis, unpublished data). It therefore seems that the activity of urokinase plays a role in the complex process of stone formation by regulating the concentration of glycoproteins present in urine. 

T2. Toki, N., and Sumi, H, Urinary trypsin inhibitor and urokinase activities in renal diseases. Acta Haematol. 67, 109-113. 

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. 

Tapiovaara H, Matikainen S, Hurme M, Vaheri A (1994) Induction of differentiation of promyelocytic NB4 cells by retinoic acid is associated with rapid increase in urokinase activity subsequently downregulated by production of inhibitors. Blood 83 1883-1891... 

Fig. 3. Overview of puriftcation sequence for the nonrecombinant tissue plasminogen activator (t-PA) which also contains urokinase plasminogen activator (u-PA). Serum-free culture conditional media is from normal human ceU line. The temperature for aU. steps, except for size-exclusion chromatography...

Fig. 4. Fibrinolytic system where SCUPA is single-chain urokinase plasminogen activator rTPA is recombinant tissue plasminogen activator APSAC is acylated plasminogen streptokinase activator complex SK is streptokinase and UK is urokinase.

Tissue Plasminogen Aetivator (tPA). While streptokinase and urokinase can effectively induce clot dissolution in the majority of patients if given early, they lack clot specificity. Treatment with these enzymes results in a systemic lytic state attributable to their degradative action on circulating fibrinogen. Tissue plasminogen activator (tPA) was developed to achieve rapid and specific thrombolysis. 

Urokinase (from human urine) [9039-53-6] Mr 53,000, [EC 3.4.21.31]. Crystn of this enzyme is induced at pH 5.0 to 5.3 (4") by careful addition of NaCl with gentle stirring until the soln becomes turbid (silky sheen). The NaCl concentration is increased gradually (over several days) until 98% of saturation is achieved whereby the urokinase crystallises as colourless thin brittle plates. It can be similarly recrystd to maximum specific activity [104K CTA units/mg of protein (Sherry et al. J Lab Clin Med 64 145 1964)]. [Lesuk et al. Science 147 880 1965 NMR Bogusky et al. Biochemistry 28 6728 1989.] It is a plasminogen activator [Gold et al. Biochem J 262 1989 ]. 

Fibrinolytics. Figure 3 Plasminogen activation (a) Kinetics of plasminogen activation by uPA (urokinase-type) and tPA (tissue-type) plasminogen activators. Effect of fibrin (b) Ternary complex formation between enzyme (tPA), substrate (Pg) and cofactor (F) Abbreviations plasmin (P), fibrin (F), plasminogen (Pg). Plasmin, formed in time, is expressed in arbitrary units. 

In a different approach, a TNF fusion protein designated TNF-Selectokine has been described [5]. This TNF prodrug is comprised of a trimeric scFv-TNF fusion protein to which a TNFR fragment has been fused at the C-terminal. A flexible peptide linker between TNF and the blocking receptor domain is comprised of sequences specifically recognized by tumor associated proteases such as tissue plasminogen activator, urokinase type plasminogen activator or... 

Urokinase-type plasminogen activator (uPA, urokinase) is synthesized by endothelial and tumor cells as a single-chain glycoprotein (scuPA) without catalytic activity. When it is converted to a two-chain protein (tcuPA) by plasmin, an active serine protease center develops, which activates plasminogen. Thus, uPA (55 kDa) results in the amplification of fibrinolysis. 

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