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)

The primary amino acid sequence of the carboxyl-terminal portion of the alpha 3 chain of chicken type VI collagen (K-VI) presents a 58-residue motif with a high degree of homology with members of the Kunitz serine-proteinase inhibitors family. This module was cloned, expressed in E. coli, purified and compared to the bovine pancreatic trypsin inhibitor (BPTI) in an inhibition profile assay of two serine proteases, trypsin and plasmin. We found that recombinant K-VI is not endowed with inhibitory activity but it slightly activates both plasmin and trypsin, differently from other members of the family. Moreover, the ability to inhibit the serine protease activity is also lacking in the intact type VI collagen molecule.
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PMID:Expression, purification and functional characterisation of a Kunitz-type module from chicken type VI collagen. 748 30

We have analyzed the proteolytic activity of a recombinant form of apolipoprotein(a) [r-apo-(a)]. A mutant 17-kringle from of r-apo(a) was engineered that contained a serine to arginine substitution which reinstates the tissue-type plasminogen activator (tPA) as determined by SDS-PAGE and fluorography and by Western blot analysis. However, tPA cleavage did not result in an active protease as both wildtype r-apo(a) and the mutant, either free or incorporated into r-Lp(a) particles, were uniformly inactive against a variety of chromogenic serine protease tripeptide substrates. To assess whether the large number of kringle IV repeats present in apo(a) inhibits proteolytic activity, we generated truncated forms of the Ser-->Arg proteolytic activity, we generated truncated forms of the Ser-->Arg mutant containing one or 10 kringle IV repeats. These truncated versions of r-apo(a) were susceptible to cleavage by tPA but were inactive against the plasmin substrate S-2251. Treatment of the Ser-->Arg mutant of the 17-kringle r-apo(a) with tPA and diisopropylflurophosphate (DFP) did not result in modification of the mutant protease domain by DFP. Finally, we incubated r-apo(a) or r-Lp(a) particles formed in vitro with purified human LDL; no degradation of LDL was observed after 16 h at 37 degrees C. The results of this study suggest that one or more of the substitutions present in the protease domain of apo(a), in addition to the Arg-->Ser substitution, render apo(a) proteolytically inactive.
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PMID:Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a). 749 9

The insulin-like growth factors, IGF-I and IGF-II, are proteins that promote cellular growth and differentiation of various organs, including the kidney. These peptides interact with high affinity cell surface receptors and bind to a family of IGF-binding proteins (IGFBPs). Altered serum and urinary IGFBP patterns in children with chronic renal failure have been previously described. In this study, we evaluated serum and urinary IGFBP profiles in acute renal failure patients (ARF; n = 10) and chronic renal failure patients (n = 10), using Western ligand blots. Most patients with acute or chronic renal failure showed decreased intact serum IGFBP-3 and increased serum IGFBP-2. Both groups displayed marked urinary IGFBP alterations, including increased urinary IGFBP-1 and totally absent urinary IGFBP-3, as detected by Western ligand blot. To evaluate altered IGFBP profiles, we performed IGFBP-3 protease assays with sera and urine from renal failure patients and normal controls. Although control urine had only minor protease activity (defined by the ability to degrade [125I]IGFBP-3), significant protease activity was found in urine from renal failure patients. The proteolytic pattern and susceptibility to protease inhibitors in most renal failure urine samples were the same as those seen in normal urine and with plasmin. Protease activity was completely inhibited by serine protease inhibitors. We speculate that urinary protease activity is mediated primarily by a serine protease(s), which may be involved in the modulation of renal IGF activity in health and disease.
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PMID:Alteration in insulin-like growth factor-binding proteins (IGFBPs) and IGFBP-3 protease activity in serum and urine from acute and chronic renal failure. 752 35

Limited proteolysis in vivo of insulin-like growth factor-binding protein-3 (IGFBP-3) by as yet unidentified serine proteases plays a key role in controlling the bioavailability of IGFBP-3-associated insulin-like growth factors (IGFs). Both the IGF system and the system of plasminogen activators (PAs) and their inhibitors (PAIs) are involved in bone remodeling, and plasmin has been shown to provoke dissociation of IGFBP-IGF complexes in cultured MG-63 human osteoblasts. The aim of this work was to investigate interactions between IGF-I and the PA/PAI system and their influence on IGFBP-3 production and proteolysis in this cell model. At confluency, MG-63 cells maintained for 3 days in serum-free medium constitutively secreted IGFBP-2 and small amounts of IGFBP-3 and IGFBP-4. As shown by Western ligand and immunoblot analyses of the culture medium and Northern blot analysis of IGFBP-3 messenger RNA, production of these IGFBPs, and of IGFBP-3 in particular, was dose dependently stimulated by the addition of 12.5-100 ng/ml recombinant human (rh) IGF-I. Increasing concentrations of plasminogen (0.05-3.5 micrograms/ml) added during the final 12 h of culture reduced the amounts of IGFBP detectable by Western ligand blotting, especially IGFBP-3. This reduction reflected proteolysis, as shown by immunoblotting, which revealed 30-, 20-, and 16-kilodalton fragments of IGFBP-3. In the presence of 25 ng/ml IGF-I, which stimulated IGFBP-3 production, proteolysis was reduced by approximately half. Incubation of glycosylated [125I]rh-IGFBP-3 as substrate in cell-free conditioned medium gave the same results. Addition of 50 ng/ml rhIGF-I to conditioned medium (to promote IGFBP-3-rhIGF-I complex formation) failed to diminish plasmin-induced proteolysis of IGFBP-3. Urokinase PA activity in the conditioned medium decreased significantly when the cells were cultured with rhIGF-I, indicating a direct action of IGF-I on urokinase PA and/or PAI production. Our results support the notion of a regulation loop whereby IGF-I controls its own bioavailability via its action on both IGFBP-3 production and the PA/PAI system, which regulates IGFBP-3 proteolysis. The proteolytic cleavages of IGFBP-3 caused by plasmin were the same as those caused in vivo by serine protease acting on this IGFBP.
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PMID:Interactions between insulin-like growth factor-I (IGF-I) and the system of plasminogen activators and their inhibitors in the control of IGF-binding protein-3 production and proteolysis in human osteosarcoma cells. 752 30

The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.
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PMID:Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor. 755 14

The expression of the urokinase-type plasminogen activator, which plays a crucial role in tissue remodeling by controlling the synthesis of the broadly acting plasmin serine protease, is regulated by several tyrosine kinases. Since the actions of these tyrosine kinases is dependent on the activation of ras proteins, we undertook a study to identify signaling events downstream of ras responsible for the stimulation of urokinase promoter activity. Transient expression of an activated c-Ha-ras in OVCAR-3 cells, which do not harbor the mutated oncogene, led to a dose-dependent trans-activation of the urokinase promoter. A sequence residing between -2109 and -1964 was critical for the stimulation of the urokinase promoter by c-Ha-ras. Mutation of an AP-1 and a PEA3 site at -1967 and -1973, respectively, or the co-expression of a transactivation domain-lacking c-jun substantially impaired the ability of c-Ha-ras to stimulate urokinase promoter activity. The induction of the urokinase promoter by ras was completely blocked by expression of a dominant negative c-raf expression vector and substantially reduced in cells made to co-express a catalytically inactive mitogen-activated protein kinase kinase. Further, the expression of an ERK1/ERK2-inactivating phosphatase (CL100) abrogated the stimulation of the urokinase promoter by c-Ha-ras. These data argue for a role of a mitogen-activated protein kinase-dependent signaling pathway in the regulation of urokinase promoter activity by ras.
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PMID:Involvement of a mitogen-activated protein kinase signaling pathway in the regulation of urokinase promoter activity by c-Ha-ras. 755 39

Neuronal degeneration in the hippocampus, a region of the brain important for acquisition of memory in humans, occurs in various pathological conditions, including Alzheimer's disease, brain ischaemia and epilepsy. When neuronal activity is stimulated in the adult rat and mouse hippocampus, tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to the active protease plasmin, is transcriptionally induced. The activity of tPA in neural tissue is correlated with neurite outgrowth, regeneration and migration, suggesting that it might be involved in neuronal plasticity. Here we show that tPA is produced primarily by microglia in the hippocampus. Using excitotoxins to induce neuronal cell loss, we demonstrate that tPA-deficient mice are resistant to neuronal degeneration. These mice are also less susceptible to pharmacologically induced seizures than wild-type mice. These findings identify a role for tPA in neuronal degeneration and seizure.
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PMID:Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. 756 88

The receptor for urokinase-type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase-type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1-284 of human uPAR) was expressed with an N-terminal histidine-tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Prior to immunization the N-termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.
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PMID:Expression of the human urokinase-type plasminogen activator receptor in E. coli and Chinese hamster ovary cells: purification of the recombinant proteins and generation of polyclonal antibodies in chicken. 758 68

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.
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PMID:Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. 759 8

The plasminogen activator (PA)/plasmin pathway has been implicated in a variety of physiologic and pathologic processes that require tissue remodeling and cell motility. The pathway is highly regulated and results in the generation of the broad spectrum serine protease, plasmin, from the zymogen plasminogen. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are produced by osteoblasts, as are the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) and a cellular receptor for uPA. Little is known about regulation of the receptor, but the other 3 components of the pathway are regulated differentially by various osteotropic hormones and local factors. Several roles have been proposed for this pathway in bone. These involve proteolytic activation of procollagenase and latent growth factors, such as transforming growth factor beta (TGF beta) and insulin-like growth factor (IGF), as well as cell motility as a result of pericellular proteolysis. The roles of this pathway in bone remodeling may not be confined to proteolytic events, because uPA has been reported to act as a mitogen on osteoblast-like cells. This effect is independent of proteolytic activity but requires the growth factor domain of uPA to bind to uPA cellular receptors. If the plasminogen activator/plasmin pathway encompasses these roles, it would serve to couple formation and resorption of bone in a highly regulated manner.
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PMID:The plasminogen activator inhibitor system in bone cell function. 764 98


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