Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.
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PMID:Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain. 210 67

Fibrinolysis is regulated in part by the interaction between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor of the serpin family). It is known from our earlier work that deletion of a loop of amino acids (residues 296-302) from the serine protease domain of t-PA suppresses the interaction between the two proteins without altering the reactivity of t-PA towards its substrate, plasminogen. To define more precisely the role of individual residues within this loop, we have used site-directed mutagenesis to replace Lys-296, Arg-298, and Arg-299 with negatively charged glutamic residues. Replacement of all three positively charged amino acids generates a variant of t-PA that associates inefficiently with PAI-1 and is highly resistant to inhibition by the serpin. Two t-PAs with point mutations (Arg-298----Glu and Arg-299----Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296----Glu, His-297----Glu, and Pro-301----Gly) do not detectably affect the interaction of t-PA with PAI-1. None of these substitutions has a significant effect on the rate of catalysis by t-PA or on the affinity of the enzyme for its substrate, plasminogen. On the basis of these results, we propose a model in which positively charged residues located in a surface loop near the active site of t-PA form ionic bonds with complementary negatively charged residues C-terminal to the reactive center of PAI-1.
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PMID:Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. 211 Mar 66

Tissue-type plasminogen activator (t-PA) is a serine protease that converts a zymogen plasminogen into an active serine protease, namely, plasmin. Plasmin is the proteolytic enzyme that degrades fibrin. In the absence of fibrin, e.g., in circulating plasma, t-PA activates plasminogen at a very slow rate. However, when fibrin is present, this activity is enhanced two to three orders of magnitude. As a consequence of these kinetic characteristics, plasmin is predominantly generated on the fibrin surface. This in turn results in a relative sparing of circulating fibrinogen and other plasma proteins to plasmin--mediated degradation. Following the demonstration of the potential of natural t-PA as a thrombolytic agent, an intensive effort was launched to enhance its production by recombinant DNA technology. The pharmacological action and the clinical efficacy of t-PA has been tested by several Authors in the treatment of acute myocardial infarction (AMI), and more recently, of pulmonary embolism, a condition for which this drug seems to be very promising: from this point of view this short article provides evidence that the various thrombolytic agents are of equal ability in mediating the rapid lysis of a coronary thrombus after i.v. administration when given appropriately and at the proper time; clinical experience provides little support for the contention of the superiority of t-PA over other thrombolytic agents, particularly for coronary thrombolysis. We are waiting for the results that will come from the GISSI-2 study, that is comparing streptokinase (SK) vs. t-PA in AMI's patients.
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PMID:[Tissue-type plasminogen activator]. 211 66

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.
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PMID:Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis. 211 30

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.
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PMID:Thrombospondin forms complexes with single-chain and two-chain forms of urokinase. 214 8

Tissue-type plasminogen activator (tPA) is a glycosylated serine protease which is an effective thrombolytic agent. Native single-chain tPA (sc-tPA) is converted to two-chain tPA (tc-tPA) by plasmin, the product of the reaction of plasminogen with tPA. Native sc-tPA occurs as two glycoforms. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. The rates at which type I and type II human melanoma sc-tPA were converted to type I and type II tc-tPA by plasmin were determined by two different methods. In each case, the second-order rate constant (kcat/Km) for type II sc-tPA (approximately 8 microM-1 s-1) was about twice that for type I sc-tPA (approximately 4 microM-1 s-1). These results indicate that glycosylation at Asn-184 hinders the conversion of sc-tPA to tc-tPA and suggest that under physiological conditions type I sc-tPA may persist in the single-chain form longer than type II sc-tPA. Previous studies have shown that type I tc-tPA has a lower activity than type II tc-tPA and that sc-tPA has a lower activity and susceptibility to inhibition when compared to tc-tPA. The present work provides further evidence that tPA glycosylation serves to modulate activity. The two major glycoforms may represent more persistent but slow acting (type I) and less persistent but faster acting (type II) variants of tPA.
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PMID:Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin. 214 93

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.
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PMID:Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells. 216 6

Little is known about the functions of alveolar macrophages during the later resolving phases of pulmonary inflammation. We have used an animal model of resolving pulmonary inflammation to obtain inflammatory macrophages (IMs) and have compared several IM functions with those of resident macrophages (RMs). IMs were frequently peroxidase positive and contained large amounts of myeloperoxidase activity. IMs also contained significant amounts of a serine protease type of elastase. The procoagulant activity of IMs was less than that of RMs, and IMs exhibited increased plasminogen activator activity when incubated on fibrin matrices. IMs also degraded fibrin directly, without plasminogen, and this activity was due to two different enzymes of molecular weights 39 and 63 kD that were present in IM granules and plasma membranes. These results suggest that, in vivo, IMs take up PMN enzymes and alter their procoagulant and fibrinolytic activity to maximize fibrin removal. These IM functions may be important for successful resolution of inflammatory injury.
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PMID:Characteristics of alveolar macrophages in an animal model of resolving pulmonary inflammation. 216 24

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.
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PMID:Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. 237 37

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.
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PMID:Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells. 245 88


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