Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

beta2-Glycoprotein I (beta2GPI) is a highly glycosylated plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids. The most relevant physiological role of beta2GPI is supposed to be the regulation of the function of anionic phospholipids like cardiolipin (CL). beta2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V). In the previous study, we found that factor Xa can produce the nicked form by cleaving Lys 317-Thr 318, using recombinant human domain V (r-Domain V). However, the reaction was extremely slow. In the present paper, we found that plasmin can produce the nicked form of domain V, using recombinant domain V (r-Domain V) and beta2GPI from human plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, r-Domain V was rapidly cleaved into a nicked form by plasmin, very slowly by factor Xa, but not by thrombin, tissue-type plasminogen activator, urokinase, and tissue factor/factor VIIa. The cleavage site of r-Domain V and beta2GPI by plasmin was proved to be Lys 317-Thr 318 by amino acid sequence analysis of the digest and of the C-terminal peptide isolated by high-performance liquid chromatography. The cleavage was completely inhibited by plasmin inhibitor (alpha2PI). The nicked form was demonstrated to show reduced affinity for CL with a dissociation constant of one order of magnitude larger than that of the intact beta2GPI. To determine whether the specific cleavage of beta2GPI by plasmin can occur also in plasma, human plasma was first acid-treated to inactivate alpha2PI and then incubated with urokinase. About 12% of beta2GPI in plasma was nicked when alpha2PI activity decreased to 80%. The nicked form was not generated in plasminogen-depleted plasma. These results suggest that plasmin can produce the nicked form of beta2GPI with the reduced ability to bind phospholipids in vivo.
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PMID:Plasmin can reduce the function of human beta2 glycoprotein I by cleaving domain V into a nicked form. 959 64

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
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PMID:Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1. 969 48

An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plasminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.
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PMID:Proteolysis of tissue factor pathway inhibitor (TFPI) by plasmin: effect on TFPI activity. 975 22

A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.
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PMID:1,2-Benzisothiazol-3-one 1,1-dioxide inhibitors of human mast cell tryptase. 982 54

The trypsin-like serine proteinase superfamily contains a number of potential therapeutic targets, many of which are unsuitable for routine X-ray crystallographic studies. We have cocrystallized a selection of benzamidine-based inhibitors with bovine trypsin and solved their structures to a resolution of up to 1.7 A. Despite similar chemical formulas, the inhibitors exhibit a range of diverse binding modes that reflect their inhibitory spectra against the serine proteinases trypsin, thrombin, factor Xa, tissue-type plasminogen activator (tPA) and urokinase (uPA). In contrast to the compact folded conformations of thrombin inhibitors which allow optimal binding in the well-defined hydrophobic S2/S4 pocket of thrombin, those effective against factor Xa exhibit an extended conformation that allows occupation of the S3/S4 region, where hydrophobic and electrostatic interactions can stabilize the conformation. One group of inhibitors containing an N-terminal 2,4, 6-triisopropylphenylsulfonyl (TIPPS) moiety show little or no penetration into the S3/S4 subsites of trypsin. These latter sites are occluded in uPA, explaining why this class of compounds is effective against uPA. Despite presenting an extensive hydrophobic surface toward the solvent, the Ki values for TIPPS-containing compounds against trypsin is in the range 10(-7) to 10(-8) M. Comparison of the binding of a bis-benzamidine inhibitor in trypsin and tPA indicate that a shift in potency can be induced by relatively minor changes in binding mode. Implications for the inhibition of these proteinases are discussed.
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PMID:Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase. 987 14

The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.
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PMID:Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length. 993 Jun 74

Recently, human umbilical vein endothelial cells (HUVEC) have been shown to express functional high-affinity receptors for factor Xa, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. In this paper, we demonstrate that when added to cultured HUVEC, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. The same doses of factor Xa also increased intracellular free calcium levels and phosphoinositide turnover. When added to confluent HUVEC, factor Xa induced the expression of tissue factor and the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1 without affecting urokinase expression. Indirect (antithrombin-pentasaccharide) and direct (DX9065) inhibitors of factor Xa affected all these activities of factor Xa in a dose-dependent manner. Taken together, these data show that the activities induced by factor Xa on HUVEC were dependent on its catalytic activity and could be inhibited by both direct and indirect factor Xa inhibitors.
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PMID:Activation of human vascular endothelial cells by factor Xa: effect of specific inhibitors. 1003 44

During the course of the development of active center-directed plasmin inhibitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine-4-methoxycarb onylanilide inhibited plasma kallikrein more potently than other enzymes such as plasmin, urokinase, and thrombin, although the inhibitory activity was not as potent and enzyme selectivity not as high. Based on studies of structure-activity relationship, we designed and synthesized the plasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarbonyl)-phenylalanine-4-carboxy methyl- anilide (Tra-Phe-APAA). Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator, factor Xa, factor XIIa, and thrombin with Ki values of > 500, 390, 200, > 500, > 500 > 500, and > 500 microM, respectively. We designated Tra-Phe-APAA as PKSI-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycinehydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma.
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PMID:Development of plasma kallikrein selective inhibitors. 1038 Mar 51

The serine proteinase plasmin is, together with tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), involved in the dissolution of blood clots in a fibrin-dependent manner. Moreover, plasmin plays a key role in a variety of other activation cascades such as the activation of metalloproteinases, and has also been implicated in wound healing, pathogen invasion, cancer invasion and metastasis. The leech-derived (Hirudo medicinalis) antistasin-type inhibitor bdellastasin represents a specific inhibitor of trypsin and plasmin and thus offers a unique opportunity to evaluate the concept of plasmin inhibition. The complexes formed between bdellastasin and bovine as well as porcine beta-trypsin have been crystallised in a monoclinic and a tetragonal crystal form, containing six molecules and one molecule per asymmetric unit, respectively. Both structures have been solved and refined to 3.3 A and 2.8 A resolution. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure like the tissue kallikrein inhibitor hirustasin. The interaction between bdellastasin and trypsin is restricted to the C-terminal subdomain of bdellastasin, particularly to its primary binding loop, comprising residues Asp30-Glu38. The reactive site of bdellastasin differs from other antistasin-type inhibitors of trypsin-like proteinases, exhibiting a lysine residue instead of an arginine residue at P1. A model of the bdellastasin-microplasmin complex has been created based on the X-ray structures. Our modelling studies indicate that both trypsin and microplasmin recognise bdellastasin by interactions which are characteristic for canonically binding proteinase inhibitors. On the basis of our three-dimensional structures, and in comparison with the tissue-kallikrein-bound and free hirustasin and the antistasin structures, we postulate that the binding of the inhibitors toward trypsin and plasmin is accompanied by a switch of the primary binding loop segment P5-P3. Moreover, in the factor Xa inhibitor antistasin, the core of the molecule would prevent an equivalent rotation of the P3 residue, making exosite interactions of antistasin with factor Xa imperative. Furthermore, Arg32 of antistasin would clash with Arg175 of plasmin, thus impairing a favourable antistasin-plasmin interaction and explaining its specificity.
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PMID:Structure of the complex of the antistasin-type inhibitor bdellastasin with trypsin and modelling of the bdellastasin-microplasmin system. 1051 18

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.
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PMID:Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. 1086 34


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