Urokinase inhibitors

Human urokinase From a biased fragment library five novel binders were identified. By combining the best fragment (K = 56pM) with a known inhibitor, a potent urokinase inhibitor (Kj = 370nM) with improved oral bioavallability was produced. i65... 

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). 

Fig. 26.21 Structure-based discovery of novel urokinase inhibitors. For explanation, see text.

Amidines of the thieno[3,2-6]thiophene series 318 were patented as urokinase inhibitors (94EUP568289). 

Hyperfibrinogenemia and antiplasmin activity have sometimes been incriminated in the development of cerebral and coronary thrombosis. A 28-year-old patient experienced spontaneous thrombotic accidents for 18 years the condition can be ascribed to increased blood levels of fibrinogen, absence of fibrinolytic activities, and high levels of streptokinase and urokinase inhibitor. 

Factor Xlla converts prekallikrein to kallikrein and kallikrein cleaves HK to generate bradykinin. There is also an important positive feedback in the system in which the kallikrein generated rapidly converts unactivated factor XII to activated factor XII, and the rate of this reaction is hundreds of times faster than the rate of autoactivation [11]. Therefore, much of the unactivated factor XII can be cleaved and activated by kallikrein. Cl inhibitor inhibits all functions of factor Xlla and it is one of two major plasma kallikrein inhibitors. Thus all functions of kallikrein are also inhibited, including the feedback activation of factor XII, the cleavage of HK, and the activation of plasma pro-urokinase [66] to lead to plasmin formation. Cl inhibitor also inhibits the fibrinolytic enzyme plasmin, although it is a relatively minor inhibitor compared to a2-antiplasmin or a2-macroglobulin. 

Figure 51-7. Scheme of sites of action of streptokinase, tissue plasminogen activator (t-PA), urokinase, plasminogen activator inhibitor, and Kj-antiplasmin (the last two proteins exert inhibitory actions). Streptokinase forms a complex with plasminogen, which exhibits proteolytic activity this cleaves some plasminogen to plasmin, initiating fibrinolysis. 

The data for the use of GP Ilb/Illa inhibitors in conjunction with lAT are even more scant, and are limited to case reports. Intravenous abciximab has been successfully used as adjunctive therapy to lA rt-PA or UK in cases of acute stroke. Desh-mukh et al. reported on 21 patients with large vessel occlusion refractory to lAT with rt-PA who were treated with IV and/or lA abciximab, eptifibatide, or tirofiban. Twelve patients also received IV rt-PA and 18 patients underwent balloon angioplasty. Complete or partial recanalization was achieved in 17 of 21 patients. Three patients (14%) had asymptomatic ICH, but there were no cases of symptomatic ICH. Mangiafico et al. described 21 stroke patients treated with an intravenous bolus of tirofiban and heparin followed by lA urokinase. Nineteen of these patients also underwent balloon angioplasty. TIMI 2-3 flow was achieved in 17 of 21 patients. ICH occurred in 5 of 21 patients (3 symptomatic ICH and 2 SAH), and was fatal in 3... 

Mangiafico S, Cellerini M, Nencini P, Gensini G, Inzitari D. Intravenous glycoprotein nb/nia inhibitor (tirofihan) followed by intra-arterial urokinase and mechanical thrombolysis in stroke. Am J Neuroradiol 2005 26 2595-2601. 

Plasminogen activator inhibitors have been shown to be present in a large variety of different cells and tissues. These inhibitors are thought to play an important role in regulating tissue fibrinolysis. One of these inhibitors has been purified from cultured bovine aortic epithelial cells. This inhibitor has been shown to be a serine protease inhibitor and inhibits the function of two proteolytic enzymes urokinase and tissue plasminogen activator, both of which cleave and activate plasminogen. The mechanism by which this inhibitor functions is very similar to that described above with a-l-PI. Thus, the inhibitor forms a binary complex with the proteolytic enzyme and thereby inhibits its activity. Again in a situation comparable to that with a-l-PI, it was found that when the purified bovine aortic epithelial inhibitor was exposed to Al-chlorosuccinimide,... 

After the nucleophilic attack by the hydroxyl function of the active serine on the carbonyl group of the lactone, the formation of the acyl-enzyme unmasks a reactive hydroxybenzyl derivative and then the corresponding QM. The cyclic structure of the inhibitor prevents the QM from rapidly diffusing out of the active center. Substitution of a second nucleophile leads to an irreversible inhibition. The second nucleophile was shown to be a histidine residue in a-chymotrypsin28 and in urokinase.39 Thus, the action of a functionalized dihydrocoumarin results in the cross-linking of two of the most important residues of the protease catalytic triad. 

A plasmin inhibitor such as aprotinin used for blood collection, while effective in inhibiting activation of plasminogen by urokinase, is ineffective against the ac-... 

The hypothesis that stress can modulate MMP expression is also supported by studies in mice. Using social isolation as a stressor, the mRNA levels of MMP-2, MMP-9, matrix-type matrix metalloproteinase-1 (MT1-MMP), and urokinase-type plasminogen activator were higher in the tumor and liver tissues of the isolated mice than in control mice.91 Furthermore, a recent study has shown that restraint stress causes an increase in expression of the plasminogen activator inhibitor-1, another key player in the plas-minogen/plasmin enzyme system in mice.92 As these enzymes have been described to have functions besides their role in ECM remodeling,93 studies on stress-related effects on MMP/TIMP balance have implications in the relationship between stress and cancer initiation and progression.. 

G5. Grondahl-Hansen, J., Christensen, 1., Rosenquist, C., Brunner, N., Mouridsen, H. T., Dano, K., and Blichert-Toft, M., High levels of urokinase-type plasminogen activator and its inhibitor PA1-I in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res. 53, 2513-2521 (1993). 

J2. Janicke, F., Schmitt, M., Pache, L., Ulm, K., Harbeck, N., Hoffer, H., and Graeff, H., Urokinase plasminogen activator (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancer. Breast Cancer Res. Treat. 24, 195-208 (1993). 

Webb, C. P., C. D. Hose, S. Koochekpour, M. Jeffers, M. Oskarsson, E. Sausville, A. Monks, and G. F. Vande Woude. The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network. Cancer Res. 60 342-9.2000. 

Tincture of the dried seed, on agar plate at a concentration of 30 p,L/disc, was inactive on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Extract of 10 g plant material in 100 mL ethanol was used b Anticoagulation activity. Serpin BSZx (an inhibitor of trypsin and chemotrypsin) inhibited thrombin, plasma kallikrein, factor Vlla/tissue factor, and factor Xa at heparin-independent association rates. Only factor Xa turned a significant fraction of BSZx over as substrate. Activated protein C and leukocyte elastase were slowly inhibited by BSZx, whereas factor Xlla, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein, and elastase were not or only weakly affected. Trypsin from Fusarium was not inhibited, while interaction with subtilisin Carlsberg and Novo was rapid, but most BSZx was cleaved as a substrate L... 

Regulation of the fibrinolytic system is useful in therapeutics. Increased fibrinolysis is effective therapy for thrombotic disease. Tissue plasminogen activator, urokinase, and streptokinase all activate the fibrinolytic system (Figure 34-3). Conversely, decreased fibrinolysis protects clots from lysis and reduces the bleeding of hemostatic failure. Aminocaproic acid is a clinically useful inhibitor of fibrinolysis. Heparin and the oral anticoagulant drugs do not affect the fibrinolytic mechanism. 

Streptokinase is a protein (but not an enzyme in itself) synthesized by streptococci that combines with the proactivator plasminogen. This enzymatic complex catalyzes the conversion of inactive plasminogen to active plasmin. Urokinase is a human enzyme synthesized by the kidney that directly converts plasminogen to active plasmin. Plasmin itself cannot be used because naturally occurring inhibitors in plasma prevent its effects. However, the absence of inhibitors for urokinase and the streptokinase-proactivator complex permits their use clinically. Plasmin formed inside a thrombus by these activators is protected from plasma antiplasmins, which allows it to lyse the thrombus from within. 

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