Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When single-chain pro-UK is activated by plasmin or kallikrein, the Lys158-Ile159 bond is cleaved, leaving a C-terminal lysine on the A-chain (Lys-UK). Two-chain, high molecular weight urokinase (UK) purified from urine, however, has been shown to contain a phenylalanine residue as the C-terminal of the A-chain (Phe-UK). Since C-terminal lysine residues have a strong binding affinity for plasminogen that may promote its activation, we undertook kinetic studies comparing plasminogen activation by Lys- and Phe-UK. A two-stage method was employed in order to minimize factors known to interfere with plasminogen activation and plasmin determination. The Lys-UK was prepared by plasmin activation of pro-UK purified from human fetal kidney cell culture medium. The Phe-UK was prepared by carboxypeptidase B (CpB) treatment of Lys-UK. Removal of the C-terminal lysine of Lys-UK by CpB produced small but significant increases in the Michaelis constants for the activation of both Glu- and Lys-plasminogen. The apparent Michaelis constants for Glu-plasminogen activation by Lys- and Phe-UK were 3.7 microM +/- .36 microM and 5.9 microM +/- .70 microM, respectively and the Michaelis constants for Lys-plasminogen activation by Lys- and Phe-UK were 5.4 microM +/- .72 microM and 15.2 microM +/- 1.4 microM, respectively. The catalytic efficiency (kcat/Km) of Lys-UK was approximately 2-fold greater than that of Phe-UK for the activation of either Glu- or Lys-plasminogen. When the fibrinolytic activities of Lys- and Phe-UK were compared in a plasma milieu no significant differences were detected. In conclusion, the findings indicate that the C terminal lysine on the A-chain of UK significantly promotes the catalysis of plasminogen in a purified system. However, the higher catalytic efficiency of Lys-UK was not found to induce significant acceleration of clot lysis at pharmacological concentrations in plasma.
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PMID:The effect of the carboxy-terminal lysine of urokinase on the catalysis of plasminogen activation. 177 40

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.
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PMID:Binding of tissue plasminogen activator to human monocytes and monocytoid cells. 193 47

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.
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PMID:Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained. 296 31

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.
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PMID:Chemotherapeutic potential of methotrexate peptides. 307 29

Prokallikrein was activated by trypsin and by alpha-chymotrypsin, but not by proteases, such as plasmin, thrombin, urokinase, carboxypeptidase B, papain, elastase, pepsin, and cathepsin D. Moreover, rat fresh serum did not activate prokallikrein. Maximum activation of prokallikrein by trypsin was obtained at the concentration of 10 micrograms to 1 mg per ml in PBS and that by alpha-chymotrypsin was at the concentration of 5 mg per ml. The enzymic properties of trypsin-activated and alpha-chymotrypsin-activated kallikreins were identical with those of active kallikrein in the kidney.
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PMID:Activation of prokallikrein in the rat kidney by proteases. 637 43

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A-chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90-fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro-UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb-UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.
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PMID:The kinetics of plasminogen activation by thrombin-cleaved pro-urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator. 842 4

We have observed that a murine IgG1 monoclonal antibody directed against human urokinase-type plasminogen activator (uPA) greatly potentiates pro-uPA-mediated plasminogen activation. This effect was dependent on the interaction between the immunoglobulin and the kringle domain of pro-uPA and could be competed efficiently by kringle-containing proteolytic fragments of uPA. In addition, the potentiation could also be competed by the lysine analog 6-aminohexanoic acid, an antagonist of plasminogen binding. This unexpected plasminogen binding dependence was found to be due to a carboxyl-terminal lysine residue on the immunoglobulin gamma chain, which by analogy with other proteins represents a potential binding site for plasminogen. Removal of this residue with carboxypeptidase B resulted in a complete abolition of the potentiation. It appears therefore that the potentiatory effect involves a novel mechanism with the antibody acting to provide a specific template for the assembly of a ternary complex involving pro-uPA/uPA and plasminogen, enabling them to interact in a catalytically favorable manner. This interpretation was confirmed by studying the kinetics of plasminogen activation by the complex between active, two-chain uPA and the antibody, which resulted in an overall 50-fold increase in reaction efficiency (kcat/Km), primarily due to a reduction in Km from 20 to 0.1 microM. Pro-uPA activation by plasmin was also accelerated, although to a lesser extent. The potentiation due to complex formation also provides a mechanism for the initiation of this system, dependent only on the low intrinsic proteolytic activity of the zymogen forms. The effects observed here, mediated by ternary complex formation, simulate the effects we have previously observed on assembly of the uPA receptor-mediated cellular plasminogen activation system and may therefore represent a mechanistic model for both its activity and initiation.
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PMID:Potentiation of plasminogen activation by an anti-urokinase monoclonal antibody due to ternary complex formation. A mechanistic model for receptor-mediated plasminogen activation. 844 57

Histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, is a major plasminogen-binding protein. In this work we characterized extensively the circumstances under which HPRG accelerates plasminogen activation and the specificity of this effect. Soluble HPRG did not significantly influence plasminogen activation. In contrast, native HPRG bound to hydrazide or nickel chelate surfaces strongly stimulated the activation of plasminogen by tissue plasminogen activator, but not by urokinase or streptokinase. The efficiency of activation on surface-bound HPRG was increased for Glu-plasminogen (41-fold), Lys-plasminogen (17-fold), and cross-linked Glu-plasminogen (11-fold) but not for mini-plasminogen, and was mainly due to a decrease in the apparent Km. A reduced susceptibility to inhibition by chloride ions contributed to the higher activation rate of Glu-plasminogen on an HPRG surface. The immobilized N- and C-terminal domains, but not the histidine-proline-rich domain of HPRG, also bound plasminogen and stimulated its activation. HPRG-enhanced plasminogen activation was proportional to the quantity of HPRG immobilized and was abolished by anti-HPRG antiserum, by low concentrations of epsilon-aminocaproic acid, by methylation of lysine residues in HPRG, and by treatment of HPRG with carboxypeptidase B. Soluble HPRG and a plasminogen fragment, kringle 1-2-3, acted as competitive inhibitors by binding to plasminogen and immobilized HPRG, respectively. The interaction of the conserved C-terminal lysine of HPRG with the high affinity lysine binding site of plasminogen is necessary and sufficient to accelerate plasminogen activation. Unlike other stimulators of plasminogen activation, the effect of HPRG on fibrinolysis is modulated by factors that influence the equilibrium between solution and surface-bound HPRG.
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PMID:Acceleration of plasminogen activation by tissue plasminogen activator on surface-bound histidine-proline-rich glycoprotein. 910 1

Thrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.
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PMID:Thrombomodulin in human plasma contributes to inhibit fibrinolysis through acceleration of thrombin-dependent activation of plasma procarboxypeptidase B. 949 93

The amino terminal fragment (ATF, Ser(1)-Lys(135)) of urokinase-type plasminogen activator (uPA) containing an epidermal growth factor-like (EGF) and kringle domain is critically involved in some important functions of uPA, such as receptor binding and chemotactic activity. In this report, the effect of ATF on single-chain uPA (sc-uPA) induced plasminogen activation was investigated. It was shown that sc-uPA-induced activation of Glu-plasminogen or Lys-plasminogen was significantly inhibited in the presence of ATF. In addition, sc-uPA activation to two-chain uPA (tc-uPA) by Lys-plasmin and plasminogen activation to plasmin by tc-uPA were both found to be inhibited by ATF. The inhibition of these activations was significantly attenuated but not diminished when ATF was pretreated with immobilized carboxypeptidase B (CPB), indicating that the C-terminal Lys(135) as well as internal Lys/Arg residue binding was involved in the mechanism. Kinetic analysis showed that sc-uPA activation by Lys-plasmin competitively inhibited by ATF and CPB pretreated ATF (CPB-ATF) with an inhibitory constant (K(i)) of 3.8+/-0.31 and 12.4 +/- 1.8 microM, respectively. In contrast to sc-uPA-induced Glu- or Lys-plasminogen activation, sc-uPA-induced mini-plasminogen activation, sc-uPA activation by mini-plasmin and mini-plasminogen activation by tc-uPA were not affected by ATF. These findings suggested that the inhibitory effects of ATF on sc-uPA activation by Lys-plasmin and Glu- or Lys-plasminogen activation by tc-uPA were related to the binding of ATF (by its C-terminal Lys(135) and internal Lys/Arg residue) with the kringle 1-4 of plasmin and plasminogen, respectively.
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PMID:Amino-terminal fragment of urokinase-type plasminogen activator inhibits its plasminogen activation. 1218 8


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