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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Site-directed mutagenesis has been used to construct a cDNA that encodes a recombinant variant human plasminogen (hPg) containing a Pro-611-->Ile mutation (MrhPg). The mutein was expressed in recombinant baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and purified. After activation of this zymogen to its corresponding form of the serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spatial relationships of the active-site His-603 to its partners of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic activity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant kcat of the enzyme. The serine protease inhibitor, diisopropylphosphofluoridate, when preincubated with MrhPm, did not inhibit restoration of its amidolytic activity with imidazole, whereas diisopropylphosphofluoridate did inhibit the amidolytic activity of MrhPm in the presence of imidazole. This result implies that His-603 directly influences the nucleophilic character of Ser-741. When imidazole as pretreated with alpha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, suggesting that N alpha-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffective as an His-603 substitute. These results indicate that the conformational reorientation of His-603 results in a malfunctional catalytic triad in the serine protease MrhPm, thus leading to an inactive enzyme despite the presence of all three essential amino acids of the catalytic triad. Addition of extramolecular imidazole restores a portion of the amidolytic activity of this mutant enzyme. These data also argue for an enzyme mechanism in which the active-center His-603 residue directly influences the nucleophilicity of the active-site Ser 741 residue.
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PMID:Breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of His-603 strongly inhibits the amidolytic activity of human plasmin. 850 86

TNF-stimulated gene 6 (tsg6), encoding a 35-kDa secretory glycoprotein (TSG-6), is induced in fibroblasts, chondrocytes, synovial cells, and mononuclear cells by the proinflammatory cytokines TNF-alpha and IL-1, or by LPS. Large amounts of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis. TSG-6 protein forms a stable complex with components of the serine protease inhibitor, inter-alpha-inhibitor (I alpha I). In this work, we show that TSG-6 potentiates the inhibitory effect of l alpha l on the protease activity of plasmin. The plasmin/plasminogen activator system is important in the protease network associated with inflammation. To test the hypothesis that through their cooperative inhibitory effect on plasmin TSG-6 and l alpha l can modulate the protease network and thus inhibit inflammation, we examined the effect of TSG-6 on experimentally induced inflammation. Human recombinant TSG-6 protein showed a potent anti-inflammatory activity in the murine air pouch model of carrageenan- or IL-1-induced acute inflammation. The inhibitory effect of locally administered TSG-6 on the IL-1-induced cellular infiltration was comparable with that of systemic dexamethasone treatment. Two mutant TSG-6 proteins with single amino acid substitutions close to the N terminus showed a complete or partial loss of anti-inflammatory activity. The anti-inflammatory effect of the TNF/IL-1-inducible TSG-6 protein, along with its ability to inhibit protease action through interaction with l alpha l, suggests that TSG-6 production during inflammation is part of a negative feedback loop operating through the protease network.
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PMID:TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo. 856 67

Plasmin and matrix metalloproteinases (MMPs) both participate in extracellular matrix remodeling. This study examined the effects of tumor necrosis factor-alpha (TNF-alpha) and plasminogen on collagenase, stromelysin, and plasminogen activator inhibitor-1 (PAI-1) synthesis of collagenase and stromelysin, which remained predominantly in proenzyme forms, as determined by Western analysis of culture media. In contrast, plasminogen and plasmin not only increased secretion of MMPs but also induced cleavage to their active forms. The serine protease inhibitor aprotinin inhibited this activation of MMPs by plasminogen and plasmin. TNF-alpha reduced plasminogen-induced activation of MMPs, suggesting induction of an inhibitor or plasmin generation, such as PAI-1. Enzyme-linked immunosorbent assay of culture media showed that TNF-alpha (10 ng/mL) increased PAI-1 secretion by 4.2 fold compared with control (105.5 +/- 9.6) versus 24.9 +/- 1.7 ng/mL, n = 3). Surprisingly plasminogen also increased PAI-1 secretion by vascular SMCs (3.6-fold over control). These results demonstrate coordination of cytokines and serine proteases in regulating MMP secretion and activation. In addition, the induction of PAI-1 by TNF-alpha and plasminogen suggests a negative feedback mechanisms limit both plasmin-mediated and MMP-mediated matrix degradation.
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PMID:Regulation of matrix metalloproteinases and plasminogen activator inhibitor-1 synthesis by plasminogen in cultured human vascular smooth muscle cells. 860 4

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily that functions to negatively regulate the plasmin-based pericellular proteolytic cascade, was induced early after exposure of growth-arrested normal rat kidney (NRK) cells to serum-containing medium. Increased PAI-1 transcription was rapid (evident within 10 min of serum addition) and involved immediate-early response kinetics. [3H]Thymidine autoradiography was used to map the time frame of PAI-1 expression during a synchronous growth cycle. PAI-1 transcript accumulation peaked in mid-G1 phase (approx. 4-6 h post-stimulation) and declined prior to, or concomitant with, the onset of DNA synthetic phase. Serum increased PAI-1 expression in NRK cells in agarose suspension, as well as monolayer, culture; induction in suspended cells (similar to monolayer culture conditions) also occurred in the presence of cyclohexamide or puromycin. The serum-inductive pathway leading to PAI-1 gene activation is thus functional regardless of adhesive conditions or capacity for de novo protein synthesis. The amplitude of induction and maintenance of expression in later stages of G1, however, were subject to adhesive influences. PAI-1 transcript accumulation at 4 and 8 h post-stimulation in newly adherent cells, moreover, was blocked by puromycin, indicating that both immediate-early and secondary mechanisms regulate PAI-1 mRNA levels during progression of NRK cells through an 'activated' G1 growth phase.
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PMID:Complex regulation of plasminogen activator inhibitor type-1 (PAI-1) gene expression by serum and substrate adhesion. 861 56

Insulin-like growth factor-binding proteins (IGFBPs) modulate IGF action at cellular level, through either inhibition or potentiation, and they also have intrinsic activity that is independent of their binding to IGFs. In prostate carcinoma (PC-3) cells, which are capable of growth for several days in serum-free medium, non-glycosylated recombinant human IGFBP-3 (rhIGFBP-3) had a biphasic mitogenic effect, stimulation being dose-dependent up to 20 ng/ml, followed by progressive depression down to zero stimulation at 150-200 ng/ml. This mitogenic effect was not intrinsic activity, but involved IGF-II secreted by the cells, since stimulation was abolished in the presence of anti-type 1 IGF receptor antibody (alpha IR-3). Western ligand- and immunoblot analysis of the culture media revealed several IGFBP species, in particular IGFBP-3 which exhibited an electrophoretic profile characteristic of limited proteolysis. The amounts of the proteolytic fragments increased in parallel with the concentrations of added rhIGFBP-3, but a large amount of intact protein remained at the highest concentrations added. When a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulphonyl fluoride (Pefabloc SC), was added at concentrations demonstrated to be non-toxic to the cells, IGFBP-3 proteolysis was diminished and rhIGFBP-3-induced stimulation of proliferation was suppressed. Conversely, in the presence of plasminogen transformed to plasmin by urokinase secreted by the cells, proliferation stimulated by rhIGFBP-3 and its proteolysis were enhanced. Our results suggest that the biphasic mitogenic effect of rhIGFBP-3 on PC-3 cells reflects changes in the availability to the cells of the IGF-II they secrete. This availability depends on the extent of IGFBP-3 proteolysis (which promotes release of bound IGF-II) and on the proportion of intact forms (which sequestrate secreted IGF-II).
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PMID:Recombinant human insulin-like growth factor (IGF) binding protein-3 stimulates prostate carcinoma cell proliferation via an IGF-dependent mechanism. Role of serine proteases. 889 45

Confluent cultures of two renal collecting duct cell lines (M-1 and mIMCD-K2 cells derived from cortical and inner medullary collecting ducts, respectively) express endothelin1 (ET1), transforming growth factor-beta (TGF beta; both TGF beta 1 and TGF beta 2), and both types of the TGF beta receptor. Experiments were performed to test whether endogenous TGF beta may be a paracrine modulator of ET1 expression in these cells. Treatment of M-1 and mIMCD-K2 cells with TGF beta 2 antisense oligodeoxynucleotides (ODN) significantly reduced ET1 messenger RNA (mRNA) and ET secretion (as well as TGF beta 2 mRNA) in a concentration-dependent manner, whereas control ODN were without significant effects. To produce ET inhibition, antisense ODN had to be present in the basolateral medium, whereas its sole presence in the apical medium was without effect. In addition, a pan-specific TGF beta antibody caused a significant reduction of ET1 mRNA expression and ET1 secretion. M-1 cells were found to express high levels of the mRNA for plasminogen activator of both tissue and urokinase types. Addition of the nonspecific serine protease inhibitor aprotinin (50 micrograms/ml) to the medium for 24 h significantly reduced the secretion of ET1. These results suggest that secretion of endogenous TGF beta, at least in part activated by the plasminogen/plasmin system, participates in the regulation of ET1 synthesis and secretion by collecting duct cell lines.
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PMID:Regulation of endothelin production and secretion in cultured collecting duct cells by endogenous transforming growth factor-beta. 889 74

During cardiopulmonary bypass (CPB), contact-phase activation of factor XII, prekallikrein, and high molecular weight kininogen initiates the intrinsic pathway of coagulation. To prevent gross clot formation during CPB, heparin is commonly used as an anticoagulant. There is a wide variability in the sensitivity of individual patients to the actions of heparin. We did not find a significant correlation between plasma heparin levels and concentrations of D-dimers, thrombin-antithrombin III complexes (TAT), and prothrombin fragments F1+2 as markers of fibrinolysis and coagulation activation. In addition, heparin cannot completely inhibit thrombin formation and action and may play a central role in the coagulation disorders associated with CPB. F1+2 and TAT rise throughout the course of CPB and fibrin monomers are generated. Attempts to improve anti-coagulation using heparin-coated bypass circuits and specific inhibitors of thrombin have not thus far proven successful. The serine protease inhibitor aprotinin can inhibit contact-phase activation, as evidenced by generation of significantly fewer prothrombin fragments F1+2, thrombin-antithrombin III complexes, fibrinopeptide A, and fibrin monomers in aprotinin-treated patients undergoing cardiac surgery. Studies performed with a simulated CPB system have shown attenuation of plasma kallikrein C1 inhibitor complex (PKC1 I) with aprotinin and the recombinant Arg 15 aprotinin. This action of aprotinin to inhibit contact-phase activation may influence the degree of anticoagulation with heparin. Patients treated with aprotinin require approximately 20% less heparin to achieve an activated clotting time (ACT) of 400 s than control patients. Despite lower plasma concentrations of heparin, aprotinin-treated patients had significantly lower concentrations of the markers of coagulation activation (thrombin-antithrombin III complex, fibrin monomers, and antiplasmin-plasmin complex). We have also investigated the role of aprotinin in contact-phase [correction of contact phase] activation of fibrinolysis. Patients treated with aprotinin showed higher concentrations of single-chain urinary type plasminogen activator (scuPA) at the end of CPB compared with control patients, indicating reduced contact- phase [correction of contact phase] activation.
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PMID:Reducing thrombin formation during cardiopulmonary bypass: is there a benefit of the additional anticoagulant action of aprotinin? 893 84

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.
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PMID:Characterization of placental bikunin, a novel human serine protease inhibitor. 911 95

An unimpeded circulation of blood depends on the concerted activation of coagulation and fibrinolytic factors. The latter entails the controlled, localised conversion of plasma zymogen plasminogen to the active enzyme plasmin mediated by tissue-type plasminogen activator (tPA). Bulk of tPA activity is in the proximity of the endogenous plasminogen activator inhibitor (PAI) as an active complex. The advent of molecular biology techniques has enabled isolation of cDNA for the inhibitors PAI-1, PAI-2 and PAI-3 and data indicate that these belong to the serine protease inhibitor (Serpine) family with arginine as its active site but immunologically distinct from each other. Enhanced tPA or PAI-1 forms one of the risk factors related to cardiac diseases and thrombotic disorders. A line of therapy entails lowering of PAIs with concomitant increase in tPA levels leading to net enhancement in fibrinolytic activity. In as much as plasminogen activators exert their action extracellularly, they are accessible to inhibitors and therefore PAIs could have a therapeutic potential and serve as prognostic indicators in cancer. Documented findings related to the biochemical characteristics and therapeutic potential of PAIs are presented and discussed in the review.
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PMID:Clot lysis: role of plasminogen activator inhibitors in haemostasis and therapy. 935 56

Neuroserpin is a serine protease inhibitor of the serpin family that has been identified as an axonally secreted glycoprotein in neuronal cultures of chicken dorsal root ganglia. To obtain an indication for possible functions of neuroserpin, we analyzed its expression in the developing and the adult CNS of the mouse. In the adult CNS, neuroserpin was most strongly expressed in the neocortex, the hippocampal formation, the olfactory bulb, and the amygdala. In contrast, most thalamic nuclei, the caudate putamen, and the cerebellar granule cells were devoid of neuroserpin mRNA. During embryonic development, neuroserpin mRNA was not detectable in neuroepithelia, but it was expressed in the differentiating fields of most CNS regions concurrent with their appearance. In the cerebellum, the granule cells and a subgroup of Purkinje cells were neuroserpin-positive during postnatal development. As a further step toward the elucidation of neuroserpin function, we performed a study to identify potential target proteases. In vitro, neuroserpin formed SDS-stable complexes and inhibited the amidolytic activity of tissue plasminogen activator, urokinase, and plasmin. In contrast, no complex formation with or inhibition of thrombin was found. Expression pattern and inhibitory specificity implicate neuroserpin as a candidate regulator of plasminogen activators, which have been suggested to participate in the modulation or reorganization of synaptic connections in the adult. During development, neuroserpin may attenuate extracellular proteolysis related to processes such as neuronal migration, axogenesis, or the formation of mature synaptic connections.
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PMID:Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. 936 46


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