Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tripeptide aldehydes such as Boc-D-Phe-Pro-Arg-H (51) exhibit potent direct inhibition of thrombin. This distinction offers important insight for the design of more potent and selective serine protease inhibitors which may be useful pharmacological tools and hold promise for development of clinically useful agents. The structure-activity relationships (SAR) on a series of anticoagulant peptides with high selectivity for the enzyme thrombin are discussed. The SAR is centered on a series of di- and tripeptide arginine aldehydes based on the structure of 51. The structural and conformational role of the amino acid residue in position 1 was investigated by substitution with conformationally restricted aromatic amino acids, aromatic acids, and a dipeptide isostere containing the psi[CH2N] amide bond replacement. Many of these peptides demonstrate potent antithrombotic activity along with selectivity toward thrombin, determined by comparison of in vitro inhibitory effects on trypsin, plasmin, factor Xa, and tissue plasminogen activator. Compound 5f, D-1-Tiq-Pro-Arg-H.sulfate is highly active and the most selective tripeptide aldehyde inhibitor of thrombin reported to date.
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PMID:Highly selective tripeptide thrombin inhibitors. 842 61

Current hypotheses suggest that the degradation of cervical collagen and elastin leads to cervical effacement and dilation during labor. The collagenolytic activity is thought to be initiated through the conversion of latent (pro)collagenase to active collagenase by the plasmin formed from plasminogen or by other proteases similarly formed from their inactive zymogens. We presently demonstrate that meperidine stimulates the activity of several enzymes in the proteolytic cascade leading toward proteolysis of connective tissue proteins. Meperidine in its therapeutic concentration range produces a 26% stimulation of urokinase activity on substrate S-2444, a 39% stimulation of plasmin activity on substrate S-2551, and a 33% stimulation of collagenase activity on 14C-labeled globin substrate. These direct effects on the enzyme activities are noted in vitro with the purified enzymes and were confirmed with several small molecular weight chromogenic substrates and with 14C-globin protein substrate. Oxytocin at levels found during active labor fails to stimulate the in vitro activity of purified urokinase, plasmin, collagenase, trypsin, or tissue-type plasminogen activator. The effect of meperidine on the proteolytic enzymes suggests that its ability to promote cervical effacement and distention during labor may be at least partially due to a meperidine-induced stimulation of cervical proteases.
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PMID:Direct stimulation of urokinase, plasmin, and collagenase by meperidine: a possible mechanism for the ability of meperidine to enhance cervical effacement and dilation. 847 75

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.
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PMID:New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue. 849 23

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.
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PMID:Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. 855 84

Several H-N-Me-D-Phe-Pro-Lysyl-alpha-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by alpha-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The alpha-keto carbonyl inhibitors bound thrombin with a second order rate constant (k1 1-4 microM-1s-1) that was 10-100-fold slower than that expected for a diffusion-controlled reaction. Certain alpha-keto carbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10-19%) in rats demonstrated for three of the alpha-keto carbonyl thrombin inhibitors suggests the possibility that alpha-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.
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PMID:Inhibition of thrombin by peptides containing Lysyl-alpha-keto carbonyl derivatives. 856 Apr 21

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.
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PMID:Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. 860 1

A critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1). An increase in the plasma concentration of PAI-1 has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50: 5 to 80 muM). In contrast, other serpin-serine protease interactions, including alpha 1-antitrypsin-trypsin, alpha 2-antiplasmin- plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 +/- 5.1% SEM (n = 25, p < 0.01), 36.7 +/- 3.5% SEM (n = 36, p < 0.01) and 60.0 +/- 2.8% SEM (n = 17, p < 0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.
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PMID:Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. 872 28

Neuropeptide Y and calcitonin gene-related peptide are abundant neuropeptides in the mammalian central and peripheral nervous systems. Their enzymatic degradation by cultivated neurons, astrocytes, and microglia, as well as by purified urokinase-type plasminogen activator, plasmin, thrombin, and trypsin, was investigated in an in vitro approach to elucidate the role of matrix-degrading serine proteinases for inactivation of neuropeptides, especially those of higher amino acid chain length, in the brain. Astrocytes were almost unable to catabolize the peptides. Cultivated neurons and microglia digested neuropeptide Y through cleavage after Arg19, Arg25, Arg33, and Arg35, calcitonin gene-related peptide was cleaved after Arg11 and Arg18. The same cleavage pattern was observed, when neuropeptide Y and calcitonin gene-related peptide were degraded by purified urokinase-type plasminogen activator, plasmin, thrombin, and trypsin. For further characterization of the neuropeptide-degrading serine proteinase activities from cell cultures, urokinase-type plasminogen activator was identified on microglia by immunostaining, whereas tissue-type plasminogen activator mRNA occurred in neurons and astrocytes, but not in microglia. The data are consistent with the possibility that the neuropeptide-degrading serine proteinase activity on neurons and microglia is due to a mixture of plasmin and plasminogen activator activities.
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PMID:Metabolism of neuropeptide Y and calcitonin gene-related peptide by cultivated neurons and glial cells. 873 50

Artificially induced rat decidual tissue expresses plasminogen activator inhibitor (PAI). This PAI, isolated and purified employing chromatographic techniques, is a low molecular weight protein unlike the known PAIs. The final purified preparation resolves into a single band following SDS-PAGE and has an approximate molecular weight of 29 kDa. The properties studied include specificity for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, binding to conA and heparin, inhibition of thrombin, plasmin and trypsin. Decidual PAI is immunogenic in rabbit and a monospecific antiserum raised against the decidual inhibitor cross reacts with an extract of human placenta.
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PMID:A molecular variant of plasminogen activator inhibitor of rat decidual tissue. 874 33

The conversion of plasminogen to plasmin catalyzed by tissue-type plasminogen activator is accelerated in the presence of fibrin, leading to an increased dissolution of fibrin clots. This rate enhancement is mimicked by a dodecapeptide segment of the fibrin molecule containing the sequence A alpha 149-160 (RLEVDIDIKIRS). At low concentrations of the dodecapeptide, the potentiation increases with concentration, but at high concentrations, the stimulation effect diminishes, giving rise to a bell-shaped curve. The maximum rate enhancement of about 10 fold is achieved at a concentration of 85 micrograms/mL. This concentration dependent phenomenon is also observed for two synthetic peptide analogues, GLEVDIDIKIRS and RGGGGGGGKIRS, although the acceleration potential is less. These results indicate that the N-terminal amino acids are not critical for the rate enhancement. The bell-shaped activity-concentration curve suggests that the dodecapeptide may bind to both plasminogen and t-PA. This speculation is further supported by the modification of the potentiator. When the A alpha 149-160 dodecapeptide is pretreated with trypsin or phenyl glyoxal, the potentiation activity is eliminated. We speculate that the acceleration of the plasminogen-to-plasmin reaction catalyzed by t-PA is achieved through the action of the stimulator to bring the enzyme and its substrate together as a bi-dentate cross-linker. This effect increases the apparent concentration of the substrate at the enzyme active site, and is reflected as a decrease in Michaelis-Menten constant.
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PMID:Mechanism of potentiation of tissue-type plasminogen activator. 874 37


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