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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Localization of the plasminogen binding sites on fibrin has been difficult since these interactions occur on polymerizing fibrin, and studies with fragments can be misleading because of multiple carboxyl-terminal lysines that may bind to plasminogen. A hetero-functional photoaffinity cross-linker was used to study these interactions. Following attachment of the cross-linker to plasminogen in the dark, a clot was formed by addition of fibrinogen or fragment X and thrombin, and then the plasminogen was cross-linked to adjacent parts of fibrin by exposure to light. There was more Glu1-plasminogen bound to fibrin than to fibrinogen and more to fragment X polymer than to fibrin. Electron microscopy of rotary shadowed individual molecules reveals that Glu1-plasminogen appears to be more compact than Lys78-plasminogen or Glu1-plasminogen with 6-aminohexanoic acid. Cross-linked complexes from the dissolved clot observed by electron microscopy reveal plasminogen bound to the end of fibrin or bridging the ends of two fibrin molecules; larger complexes were also observed. Analysis of changes in the appearance of negatively contrasted fibers with plasminogen bound also indicates the probable locations of binding sites, yielding results consistent with the cross-linking studies. The photoaffinity probe was also used to study interactions between plasminogen and fibrin or its derivatives in the course of tissue plasminogen activator-mediated fibrinolysis. Samples cross-linked at various times indicate that complexes with fragment X are particularly dominant during the rapid phase of plasminogen activation. In conclusion, these studies indicate that plasminogen binds to the pocket at the end-to-end junction between two fibrin or fragment X molecules in the protofibril; from this position, it can reach all of the sites that are cleaved during fibrinolysis.
J Mol Biol 1994 Jan 21
PMID:Interactions of plasminogen with polymerizing fibrin and its derivatives, monitored with a photoaffinity cross-linker and electron microscopy. 828 11

A method for determining the plasminogen activation rate by urokinase via a cascade enzymatic reaction system is presented. A procedure of parameter estimation has been proposed for the determination of the activity of urokinase and the kinetic constants. Urokinase from urine has been successfully assayed through use of a plasminogen concentration lower than its saturating level. The methodology presented in this work may be adopted for the analysis of other cascade enzymatic reaction systems.
Biochem Mol Biol Int 1993 Apr
PMID:Plasminogen activation activity of urokinase determined via a cascade enzymatic reaction system. 833 12

We have previously reported the purification of a 37 KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura and have shown that it is present in a subset of TTP patients, but absent in normal subjects. In this study, we would like to report some of the physico-chemical and immunological properties of this protein. The native molecular weight of PAP p37 from gel filtration was found to be 36,000, which is in agreement with denatured molecular weight (37,000), determined by SDS--polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. The values of Stoke's radius (25A), diffusion coefficient (8.59 x 10(-7)cm2/s) and frictional ratio (1.13), determined by molecular sieve chromatography, suggest that the native protein is compact and globular. The purified protein has an S20,w of 3.5s. Preliminary carbohydrate analysis suggested that p37 is a glycoprotein and contained about 11% neutral sugars and 6.6% sialic acid. Amino acid analysis indicated that the protein is relatively rich in aspartate and serine and has low cysteine, methionine and tryptophan contents. In dot immunobinding ELISA assay, PAP p37 did not react with antibodies to thrombospondin, fibrinogen, fibronectin, plasminogen and von Willebrand factor. Our results suggest that PAP p37 is a single polypeptide compact and globular glycoprotein and is immunologically not related to the aforementioned proteins.
Biochem Mol Biol Int 1993 Jun
PMID:Characterization of platelet agglutinating protein p37 purified from the plasma of a patient with thrombotic thrombocytopenic purpura. 836 16

We have previously identified an endothelial cell membrane protein of M(r) 45 kDa that binds plasminogen in a kringle-dependent, specific and reversible manner (Dudani et. al. (1991) Mol. Cell. Biochem. 108: 133-139). In this study, we have developed and optimized a protocol for the isolation of the 45 kDa plasminogen receptor from venous endothelial cells using a four step procedure consisting of lysis and detergent extraction followed by ligand affinity chromatography and preparative polyacrylamide gel electrophoresis. Control experiments were carried out using BSA-Sepharose instead of plasminogen-Sepharose as the affinity matrix. No plasminogen binding proteins were recovered from the former columns. However, a 45 kDa protein was recovered from lysine eluates of plasminogen-Sepharose. This material was then purified to homogeneity using preoperative electrophoresis. Analyses of proteins at various steps in the purification by SDS-PAGE showed enrichment of a band of 45 kDa which superimposed with the observed binding activity of plasminogen in ligand blots. The above binding could be inhibited by excess lysine. The 45 kDa protein could be distinguished from alpha-enolase which also binds plasminogen by: (i) significant differences in the profile of retention times of CNBr-degradation fragments on reversed phase HPLC; and (ii) partial peptide sequencing of one of the CNBr-degradation fragments of the 45 kDa protein. Moreover, the derived sequence did not show any significant homology to any protein in the Swiss Prot (release 20) database. We thus propose that the 45 kDa protein represents a novel plasminogen receptor on human venous endothelial cells.
 ...
PMID:Isolation of a novel 45 kDa plasminogen receptor from human endothelial cells. 838 63

Elevated levels of plasma lipoprotein(a) [Lp(a)] have been correlated with the development of atherosclerosis in human populations. Apolipoprotein(a) [apo(a); the distinguishing protein component of Lp(a)] is characterized by multiple repeats of a sequence that closely resembles kringle IV of plasminogen. Variably-sized Lp(a) isoforms that are observed in the human population have been shown to occur as a result of differences in the numbers of the repeated kringle IV units in apo(a). Using PCR analysis of human liver mRNA, we have analyzed apo(a) from 10 unrelated individuals in order to determine the presence or absence of kringle IV repeat #1, and #30-#37. Based on the apo(a) cDNA sequence published for one individual, these kringles all differ to some degree in amino acid sequence from the major kringle IV repeat, which is present in a number of identically repeated copies. We found that sequences corresponding to apo(a) kringle IV repeat #1, and #30-#37 were present in all individuals studied. This suggests that the inverse relationship that has been observed between Lp(a) isoform size and plasma Lp(a) levels is mediated by different numbers of identical kringle IV repeats, by an as yet undetermined mechanism. During the course of this study, we identified a Met-->Thr polymorphism in the apo(a) kringle IV repeat #37. The calculated frequencies of the Met and Thr alleles were 0.58 and 0.42 respectively. We did not observe a correlation between the Met-->Thr substitution and either plasma Lp(a) levels, or apo(a) transcript size.
Hum Mol Genet 1993 Apr
PMID:The apolipoprotein(a) kringle IV repeats which differ from the major repeat kringle are present in variably-sized isoforms. 838 24

Severe deterioration of surfactant function is noted under conditions of plasma protein leakage into the alveolar space; moreover, fibrinogen has previously been reported to possess strong surfactant inhibitory capacity. Dissolution of alveolar deposits of fibrinogen and fibrin (e.g., hyaline membranes) requires enzymatic degradation by the plasminogen/plasmin system or by leukocyte-derived proteases. We investigated the surfactant inhibitory properties of differently prepared sets of fibrinogen cleavage products. Proteolysis was performed with plasmin, with predominant split products D (mol wt 85,000) and E (mol wt 50,000). In addition, fibrinogen was cleaved by leukocyte elastase and trypsin, with fragments ranging mainly between mol wt of 30,000 and 50,000. To provide split products of even lower molecular weight, fibrinogen was incubated sequentially with trypsin and endoproteinase (split products < mol wt 25,000). Natural surfactant extracts used in clinical replacement studies (CLSE, Alveofact, Curosurf, Survanta) as well as an apoprotein-based phospholipid mixture (PLM-C/B; DPPC:PG:PA = 68.5:22.5:9 with 2% [wt/wt] nonpalmitoylated recombinant human SP-C and 1% [wt/wt] natural bovine SP-B) were employed. Experiments were performed in a pulsating bubble surfactometer (standard phospholipid concentration 2 mg/ml) with assessment of surfactant activity measuring adsorption and dynamic surface tension. Fibrinogen caused dose-dependent, severe deterioration of the surface activities of Curosurf and Survanta, whereas CLSE, Alveofact, and PLM-C/B were only moderately affected up to protein-surfactant ratios of 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Sep
PMID:Proteolytic cleavage of fibrinogen: amplification of its surfactant inhibitory capacity. 839 60

Short-term co-cultivation of blastemal cells from 12-day-old mouse limb buds and human rheumatoid synovial fluid cells in high density cultures (Trowell culture system) resulted, depending on when co-cultivation started, either in (1) an inhibition of chondrogenesis (co-cultivation right from the start) or in (2) an extensive breakdown of cartilaginous matrix (co-cultivation after formation of embryonic cartilage). These synovial effects were markedly impeded if Avarol (a dioxygenase inhibitor) was applied singly or in combination with PAI-2 (a u-PA-inhibitor). PAI-2 alone, however, had no effect on the synovial-induced inhibition of chondrogenesis, but produced a pronounced inhibitory effect on matrix breakdown. The effects of both inhibitors were studied electron microscopically and biochemically (determination of sulfated-glycosaminoglycans in the high density cultures by Alcian Blue binding assay). The results of this study are consistent with the presumption that rheumatoid synovial cells are capable of inhibiting chondrogenesis and enhancing the breakdown of the cartilaginous matrix. Amongst others, the possible mediators involved are prostaglandins and plasminogen activators. The response to the inhibitors Avarol and PAI-2 is compatible with their mode of action. The chondroprotective action of these substances may be useful in developing potential antirheumatic drugs.
Virchows Arch B Cell Pathol Incl Mol Pathol 1993
PMID:Inhibition of the effects of rheumatoid synovial fluid cells on chondrogenesis and cartilage breakdown in vitro: possible therapeutical conclusions. A morphological--biochemical study. 840 16

We established an immortalized cell line from endothelial cells derived from a human coronary artery, isolated at autopsy from 76-year-old male, by transfecting the cells with origin-minus simian virus 40 DNA. These cells showed SV40 T antigen in the nuclei and Ulex europaeus I agglutinin and factor VIII-related antigen, as endothelial cell markers, in their cytoplasm. This cell line synthesized prostacyclin, tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) as well as produced the proform of matrix metalloproteinase 1, which was activated by cultivating the cells with plasminogen. These findings reveal that this immortalized endothelial cell line retains characteristics of human coronary endothelial cells, indicating that this cell line is useful for studying atherogenesis of the coronary artery.
Biochem Mol Biol Int 1995 Jul
PMID:Establishment and characterization of immortalized human coronary endothelial cells. 852 34

Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.
Mol Biochem Parasitol 1995 Jul
PMID:Characterization of a plasminogen activator produced by Acanthamoeba castellanii. 857 23

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.
J Mol Biol 1996 Apr 26
PMID:The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator. 861 82


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