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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10(6)independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA we identified 9 other recombinants encoding a protein with considerable similarity (74%) TO PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.
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PMID:Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family. 296 95

The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4-benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+)-induced calpain activation and a fusion process inhibited by annexin V.
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PMID:Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors. 754 94

Anti-annexin V (anti-ANXV) IgG and lupus anticoagulant (LAC) were both shown to be capable of inducing apoptosis in umbilical vein endothelial cells [Nakamura et al.: Biochem Biophys Res Commun 205:1488-1493, 1994]. In the present study, we have demonstrated that anti-ANXV IgG prolongs the activated partial thromboplastin time and has an affinity for phospholipids in enzyme-linked immunosorbent assay. This indicates overlapping of anti-ANXV and LAC activities, suggesting that ANXV may be involved in the autoimmune mechanism for LAC production.
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PMID:Antibody to annexin V has anti-phospholipid and lupus anticoagulant properties. 763 81

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.
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PMID:Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide. 774 Apr 53

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromatography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 micrograms ml-1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC50) in the low nanomolar range.
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PMID:Isolation and characterization of recombinant annexin V expressed in Saccharomyces cerevisiae. 776 33

Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation.
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PMID:Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation. 806 19

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of 125I-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (Kd 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (Kd 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of 125I-annexin V binding sites remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Annexin V as a probe of the contribution of anionic phospholipids to the procoagulant activity of tumour cell surfaces. 807 8

The effect of hyperlipidaemia on endothelial cell haemostatic properties was examined using ex vivo studies on aortic segments obtained from fat-fed Chinchilla rabbits, mounted in a template device which exposed the luminal surface. Exposure of arterial endothelium to lipids resulted in marked enhancement of externally exposed anionic phospholipids, detected using either fluorescence microscopy with the probe merocyanine 540 or by binding of 125I-polymyxin B and 125I-Annexin V. Consistent with the known procoagulant properties of anionic phospholipid, following the lipid and cholesterol-rich diet intake, intact endothelial cells demonstrated enhanced binding of radioiodinated factors IX/IXa and Xa, and enhanced factor IXa/VIII-dependent factor X activation and factor Xa-factor Va-mediated prothrombin activation. Both factor Xa and thrombin formation were blocked, in large part, by polymyxin B, suggesting dependence of the reaction on anionic phospholipids. Consistent with these results, evidence of increased activation of the coagulation mechanism in vivo was observed in hyperlipidaemic animals, as assessed by a three-fold increase in levels of circulating antithrombin-protease complexes, compared with normolipidaemic controls.
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PMID:Intrinsic procoagulant surface induced by hypercholesterolaemia on rabbit aortic endothelium. 829 24

Activation of factor IX in an umbilical vein model was established to result solely from factor VIIa/tissue factor (TF) activity generated within the umbilical vein wall, and the model was then used to study regulation of such extravascular factor VIIa-TF complexes. Vein segments were filled with a reaction mixture containing factor VIIa, Ca2+, a substrate, either [3H]factor IX or [3H]factor X, and a test material. Subsamples were assayed for activation peptide release. Test materials included defibrinated plasma or recombinant protein as a source of TF pathway inhibitor (TFPI), recombinant factor VIIa to 10 times plasma factor VII concentrations, and annexin V. A plasma concentration of TFPI inhibited but did not totally suppress factor VIIa/TF activity. Reducing the TFPI concentration by 50% markedly reduced the inhibition. A 10-fold increase in the factor VIIa concentration in reaction mixtures failed to accelerate factor Xa generation. Annexin V, in contrast to its inhibition of factor VIIa/TF formed with TF reconstituted into mixed phospholipid vesicles, failed to inhibit factor VIIa-TF complexes formed within the vessel wall. We conclude that 1) moderate variation in plasma TFPI concentration or activity may affect TFPI's ability to inhibit factor VIIa/TF activity during hemostasis, 2) a plasma concentration of factor VII suffices to saturate TF sites exposed in a vessel after tissue injury, and 3) the resistance of factor VIIa-TF complexes to inhibition by annexin V suggests that they are formed in the umbilical vein model primarily on cell surfaces.
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PMID:Regulation of factor VIIa/tissue factor functional activity in an umbilical vein model. 842 31

We compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely de-endothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p < 0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits. 847 45


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