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: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of gynaecological surgery on the fibrinolytic and inhibitor mechanisms were followed up for 24 h post-operatively in patients receiving a single dose of ketorolac infusion (n = 18) as compared with those not receiving ketorolac infusion (n = 11). A pre-operative state of lower mean t-PA activity and higher PAI-1 levels with increased platelet activation than that reported in normal subjects were observed in both groups of patients. Increased t-PA activity upon anaesthetic induction together with a decreased level at 24 h post-operation was seen in both groups. However, fibrinolytic 'shut-down' was not evident as significant increase in D-dimer levels was observed post-operatively, suggesting an enhanced lytic state concurrent with an enhanced activation of coagulation and diminished platelet activation although beta-TG remained above the normal level; plasmin from this enhanced lytic state affects platelet adhesion and cleaves platelet glycoprotein Ib thus inhibit release reaction. Ketorolac infusion elicited a significant response in PAI-1 activity within 24 h post-operation and this was not seen in the non-ketorolac group in spite of the rising trend by 24 h post-operation which did not achieve statistical significance. There were no statistical significant differences in blood loss and duration of surgery between the two groups of patients. Overall, both groups of patients showed similar haemostatic changes post-operatively for 24 h, a longer duration of post-operative study would have revealed any subtle changes in the molecular markers of thrombosis which was not the objective of this study.
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PMID:The effects of gynaecological surgery on coagulation activation, fibrinolysis and fibrinolytic inhibitor in patients with and without ketorolac infusion. 750 76

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.
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PMID:Localization of vitronectin binding domain in plasminogen activator inhibitor-1. 751 53

Post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAT) result from formation of alloantibodies to platelet membrane glycoprotein-associated antigens. The detection and identification of platelet-specific alloantibodies in patient sera is often complicated by the presence of co-existing HLA antibodies and/or more than one platelet specificity in the same serum. We describe a solid phase assay that specifically detects antibodies to platelet membrane associated alloantigens by measuring the ability of patient antisera to inhibit the binding of glycoprotein GPIIb or GPIIIa monoclonal antibodies to intact platelets. When tested in the GPIIIa assay against a panel of random platelet donors, the reactivities of two known PLAI antisera that also contained different HLA antibodies were highly correlated (r = 0.99) and allowed PLA phenotyping of the population. A standard direct binding platelet ELISA, on the other hand, was unable to accurately PLA phenotype the same population. The reactivities of two known Baka antisera (one containing additional anti-PLA2 and the other anti-Brb specificities) were highly correlated (r = 0.95) in the GPIIb assay, and Bak phenotype determination was similarly accomplished for a random platelet panel. Furthermore, a comparison of platelet phenotype results (using the monoclonal inhibition assay) and genotype results (using DNA analysis) for the PLA and Bak systems showed a concordance of 98% for 146 alleles tested. In conclusion, the platelet monoclonal antibody inhibition assay: (1) allows determination of platelet-specific alloantibodies in the presence of contaminating HLA antibodies and/or in sera containing multiple platelet alloantibodies; (2) allows accurate platelet phenotyping for the GPIIIa-associated PLA and GPIIb-associated Bak antigen systems; and (3) may be applicable to the detection of other known or even novel platelet glycoprotein-associated antigens.
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PMID:A platelet monoclonal antibody inhibition assay for detection of glycoprotein IIb/IIIa-related platelet alloantibodies. 765 19

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.
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PMID:A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. 773 Mar 29

Current prevention or treatment of coronary thrombosis relies on antiplatelet agents (aspirin), antithrombin agents (heparin), and plasminogen activators (t-PA). The purpose of this review is to describe novel antithrombotic agents in each of these classes and to discuss recent and future clinical trials with the new agents. Whereas aspirin is a cyclo-oxygenase inhibitor, the most promising new antiplatelets are directed at an integrin cell surface receptor--glycoprotein (GP) IIb/IIIa--which represents the final common pathway for platelet aggregation. The monoclonal F(ab) antibody c7E3, a chimeric murine-human immunoglobulin G (IgG) fragment, is the most intensively studied to date. c7E3 was assessed by the Evaluation of Platelet Monoclonal Antibody to Prevent Ischemic Complications (EPIC) trial in which 2,099 high-risk angioplasty patients were randomized to bolus (placebo) plus infusion (placebo), bolus (c7E3, 0.25 mg/kg) plus infusion (placebo), and bolus (c7E3, 0.25 mg/kg) plus infusion (c7E3, 10 micrograms/min; 12 hours). The overall event rate at 30 days was significantly decreased from 12.8% (placebo) to 8.3% (c7E3), a 36% relative reduction (p = 0.009). Integrelin is a cyclic heptapeptide with marked specificity for GP IIb/IIIa integrin. It was studied during the Integrelin to Manage Platelet Aggregation to Prevent Coronary Thrombosis (IMPACT) trial, which enrolled 150 routine coronary intervention patients. At endpoint, overall event rate was reduced from 11.9% (placebo) to 5.6% (integrelin). The much larger (4,010 patients) IMPACT-II trial has just completed enrollment to confirm and extend these encouraging results. Hirudin is the prototype of the direct antithrombins; it binds to the active catalytic site and the substrate recognition site (exosite) of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Novel antithrombotic approaches to coronary artery disease. 786 68

1. We studied DMP728, a non-peptide glycoprotein (GP) IIb/IIIa receptor antagonist, for prevention of coronary artery thrombosis or rethrombosis in a chronic canine model subjected to arterial injury. 2. In protocol I, DMP728 (1.0 mg kg-1, i.v., n = 8) or saline (n = 8) was administered and a 150 microA anodal current was applied to the intimal surface of the left circumflex coronary artery (LCX). Dogs were monitored for 6 h and again on each of 5 subsequent days. 3. Ex vivo platelet aggregation was inhibited but returned to baseline 1 day after drug administration. Thrombus weight was reduced (saline, 20.7 +/- 5.0 mg; DMP728 1.7 +/- 0.4 mg; P < 0.05), as was infarct size [saline, 27.5 +/- 4.3; DMP728, 1.6 +/- 0.7 (per cent left ventricle); P < 0.05]. All control animals died by day 3, while all but one of the treated dogs survived the entire protocol (P < 0.05). 4. In protocol II, an LCX thrombus was induced and thrombolytic therapy was initiated 30 min later. DMP728 (1.0 mg kg-1, i.v., n = 8) or saline (n = 8) was administered 5 min after recombinant tissue-type plasminogen activator infusion had begun. The incidence of reocclusion was reduced by DMP728 (saline, 4/8; DMP728, 1/8). One day after thrombolysis, 7/8 DMP728-treated animals were alive compared with 1/8 in the control group (P = 0.01). 5. DMP728 inhibited ex vivo platelet aggregation, prevented primary and secondary occlusive thrombus formation, reduced thrombus weight and infarct size and increased survival in a chronic canine model of coronary artery thrombus formation. DMP728 is an effective anti-platelet intervention when used as the singular adjunctive agent in association with thrombolytic therapy.
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PMID:Prevention of thrombosis and rethrombosis and enhancement of the thrombolytic actions of recombinant tissue-type plasminogen activator in the canine heart by DMP728, a glycoprotein IIb/IIIa antagonist. 788 89

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.
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PMID:Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex. 798 19

Mass spectrometric analysis of the recombinant hybrid plasminogen activator K2tu-PA at the native glycoprotein and corresponding protein level (high mass) is described. For gross structural characterization of the major glycotypes present, a combination of enzymatic degradation and matrix-assisted laser desorption mass spectrometric analysis of the products proved convenient. In this way, mono- and di-N-glycosylated one- and two-chain molecules, unresolved in the spectra of native material, were identified, with the one-chain type monoglycosylated at Asn247 representing the major component. Actual detection of resolved original glycoforms, or unresolved groups thereof when composed of isobaric species, and their assignments regarding antennicity and degree of sialylation were possible by electrospray mass spectrometry for the major monoglycosylated one-chain species. Desialation also allowed detection of the diglycosylated one-chain species as a minor constituent. The electrospray mass spectrometric results were correlated with structural and quantitative data available from a parallel high-performance liquid chromatographic and 1H nuclear magnetic resonance study performed at the glycan (low mass) level on liberated individual carbohydrate components. The results of the two studies showed full consistency in all respects amenable to evaluation, i.e. excellent agreement for the assignments of antennicity and degree of sialylation of the major glycan components, and good agreement for the determination of proportions in which these were present in the major monoglycosylated glycoprotein. This provided a sound basis for direct glycoform profiling by electrospray mass spectrometry. Typical applications to batch quality control and to structural characterization of non-standard material are shown.
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PMID:Characterization and direct glycoform profiling of a hybrid plasminogen activator by matrix-assisted laser desorption and electrospray mass spectrometry: correlation with high-performance liquid chromatographic and nuclear magnetic resonance analyses of the released glycans. 803 26

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.
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PMID:alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells. 811 82

This study was undertaken to determine the role of platelet glycoprotein (GP) IIb/IIIa receptors in the modulation of plasminogen activator type-1 (PAI-1) release from human platelets as compared to other platelet functions. To address this issue, the effect of various agonists on human platelet aggregation, [125I]fibrinogen binding and the release of PAI-1 was examined in normal and Glanzmann's thrombasthenic (GT) platelets. In control subjects, maximum platelet aggregation and PAI-1 secretion were observed within 5 min in response to the different agonists including thrombin, collagen, adenosine diphosphate (ADP), and arachidonic acid. Agonist-induced platelet GpIIb/IIIa receptor activation was confirmed by [125I]fibrinogen binding analysis. In contrast, platelets from GT subjects demonstrated a lack of fibrinogen binding and a lack of an aggregatory response to all agonists tested except to the GPIb- mediated aggregation induced by ristocetin. However, GT platelets demonstrated normal responsiveness in secreting PAI-1 in response to the various agonists. Similarly, when platelet GpIIb/IIIa receptors were blocked in normal platelets by the tripeptide Arg-Gly-Asp (RGD) or the tetrapeptide Arg-Gly-Asp-Ser (RGDS) at 10(-3) M, agonist-induced platelet aggregation and fibrinogen binding were blocked, but platelet PAI-1 release was not blocked. Furthermore, flow cytometric analysis using dual fluorescence markers for the platelet GPIIb/IIIa membrane receptors (FITC-labeled cyclic RGD analog, XL086) and for the alpha granule (PE-monoclonal antibody for P-selectin) demonstrated a dissociation between the platelet GPIIb/IIIa receptors and granular secretion. These results suggest a lack of a role for platelet GpIIb/IIIa receptors in the modulation of platelet PAI-1 release.
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PMID:Role of platelet GpIIb/IIIa receptors in the modulation of platelet plasminogen activator inhibitors type-1 (PAI-1) release. 815 39


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