UNIPROT:P43026 - FACTA Search

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Query: UNIPROT:P43026 (lipopolysaccharide)
62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We described in a foregoing report findings on serpin, a serine protease inhibitor, newly identified in horseshoe crab (Tachypleus tridentatus) hemocytes and we name it limulus intracellular coagulation inhibitor, LICI (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547). This serpin specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C. In ongoing studies on limulus serpin, we have found another inhibitor, LICI type-2 (LICI-2), which inhibits not only factor C (k1 = 7.1 x 10(4) M-1 S-1) but also limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 S-1). LICI-2 inhibits mammalian serine proteases, including alpha-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k1 = 1.2 x 10(6) M-1 S-1). The purified LICI-2 is a single chain glycoprotein with an apparent M(r) = 42,000. A cDNA for LICI-2 was isolated and the open reading frame coded for a mature protein of 386 amino acids, of which 160 residues were confirmed by peptide sequencing. Although LICI-2 shows significant sequence similarity to the previous limulus serpin, LICI-1 (42% identity), LICI-2 contains a unique putative reactive site, -Lys-Ser-, distinct from that of LICI-1 (-Arg-Ser-). Northern blotting revealed expression of LICI-2 mRNA only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. The immunoblot of large and small granule components with antiserum against purified LICI-2 suggests that LICI-2 is stored specifically in large granules, as in the case of LICI-1, and is released in response to external stimuli. We propose that the LICIs be classified into a new subfamily of intracellular serpins, regulated secretory serpins.
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PMID:A limulus intracellular coagulation inhibitor type 2. Purification, characterization, cDNA cloning, and tissue localization. 782 80

Endotoxin(lipopolysaccharide = LPS), cell wall component of gram-negative bacteria, activates monocytes and macrophages to release cytokines, reactive nitrogen intermediates (RNI), and to generate tissue factor(TF) which initiate coagulation. We have purified 7kDa and 18kDa cationic antibacterial proteins (CAP-7 and CAP-18) with LPS-binding and LPS-neutralizing activities from rabbit granulocytes using as an assay the agglutination of erythrocytes coated with Re-LPS. From protein sequencing, CAP-7 was identified as the C-terminal 37 amino acid fragment of CAP-18. Synthetic peptide #197 (identical sequence to CAP-7, Gly1-Try37) and #36-1 (a truncation of CAP consisting of 32 amino acid residues, Gly1-Ala32) showed LPS-binding activity. Each peptide inhibited LPS-induced tissue factor(TF) generation by murine peritoneal macrophages, even added 1-3 hours after stimulation of cells with LPS. C57BL/6 mice treated with #197 were significantly protected from lethal LPS challenge. Peptide #36 also blocked the LPS-induced lethality. These peptides had antibacterial activity to gram-negative bacteria, such as E.coli, S.typhimurium, K.pneumonia, Ps.aeruginosa and also to gram-positive S.aureus (Methicillin sensitive and resistant strains). Both peptides inhibited TF- and Xa-induced plasma clotting. Using synthetic chromotogenic substrates, both CAP7 peptides blocked the coagulation cascade at two sites, activation of factor X to Xa and conversion of Factor II (prothrombin) to factor IIa (thrombin). In vivo treatment of peptide #197 prevented acute lethality in mice injected with tissue factor (rabbit brain thromboplastin). Two other peptides, #32(Gly1-Phe9) and #50(Ile13-Typ37) failed to demonstrate LPS-binding, LPS-neutralizing, antibacterial and anticoagulant activities. The active peptides but not the inactive peptide maintain a putative heparin binding domain at their N-termini. This heparin binding domain is participate in the LPS-binding, LPS neutralizing, antibacterial and anticoagulant activities of CAP7. These active peptides may have a therapeutic potential for treatment for DIC due to sepsis and endotoxin shock.
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PMID:Endotoxin-binding synthetic peptides with endotoxin-neutralizing, antibacterial and anticoagulant activities. 783 55

Healthy endothelium is a metabolically active interface between the blood and extravascular tissues. Its intimal surface is anticoagulant and antithrombotic, and it secretes a variety of molecules involved in regulating platelet function and blood coagulation. The rapid interactions between platelets, their secreted components, or thrombin and endothelial cells at sites of vessel damage ensure the local secretion of mediators such as prostacyclin and nitric oxide that limit the intravascular growth of the haemostatic plug. There is considerable evidence that a decreased ability of endothelial cells to synthesize NO contributes to the pathogenesis of arterial disease. Local deficiency of PGI2 synthesis has also been implicated in the thrombotic problems in haemolytic uraemic syndrome. Endothelium is also the source of circulating von Willebrand factor, important for efficient platelet adhesion. Chronically elevated plasma levels of vWF in a series of diseases where there is vascular pathology apparently reflect endothelial cell damage or activation, and may contribute to the prothrombotic tendency they exhibit. They may be compounded by decreased levels of the surface anticoagulant thrombomodulin, if the increased concentrations of the soluble forms of thrombomodulin detected in the circulation under similar conditions are a reflection of loss from the endothelium. Further alterations of function in a procoagulant/prothrombotic direction take place when endothelial cells are exposed to certain cytokines or lipopolysaccharide. Tissue factor synthesis is induced, thrombomodulin expression is decreased, and there is enhanced sensitivity of vWF secretion. In addition, the balance of tissue-type plasminogen activator and plasminogen activator inhibitor type I secretion is changed in favour of the latter. These processes are each likely to contribute to the occurrence of disseminated intravascular coagulation which can accompany septic shock.
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PMID:Endothelial cell function and thrombosis. 784 94

The purpose of this study was to compare the ability of fresh and cryopreserved mononuclear cells to generate thrombin, induce fibrin formation and finally resolve the fibrin formed, when exposed to plasma. Peripheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diacetate and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were seeded, stimulated with lipopolysaccharide(LPS), and exposed to a standard heparinized overlay plasma. Plasma was harvested at intervals (0-7 days). Thrombin generation and fibrin formation were measured by quantification of prothrombin fragment (F1 + 2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin (ogen) degradation products (FbDP and FgDP). Recovery of cells after thawing was about 80%, and the viability of fresh and cryopreserved PBM was > 95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothombinase activity (F1 + 2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there were significant variations between the donors in all the parameters measured. We conclude that cryopreservation of human blood mononuclear cells is possible with maintainance of the potential of the cells to mediate coagulation in plasma upon LPS stimulation, whereas the fibrin resolving capacity apparently is reduced by the preservation procedure.
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PMID:Procoagulant and profibrinolytic activities of cryopreserved human monocytes. 787 96

The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor-coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein-sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.
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PMID:Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells. 794 70

The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. 799 88

An experimental disseminated intravascular coagulation (DIC) was induced in female CD rats by the intravenous administration of living bacteria (9.5 x 10(7) cfu Klebsiella pneumoniae), sublethal (5 mg/kg) or lethal (50 mg/kg) lipopolysaccharide (LPS), or tissue factor (1.5 micrograms/kg i.v. bolus or 0.4 micrograms/kg x hr i.v. infusion). We used a new fibrin monomer (FM) assay to follow the course of DIC. FM were detected by their ability to stimulate the tissue-type (t-PA) plasminogen activator dependent conversion of plasminogen to plasmin by a chromogenic assay. Miniplasminogen was used instead of plasminogen to avoid interference of the assay by alpha 2-antiplasmin. As a marker of DIC, elevated levels of FM were observed with all DIC-inducing agents (plasma levels were up to 90 micrograms/ml). The kinetics of FM formation were similar to the course of thrombin-antithrombin III (TAT) levels (maximal plasma levels 70 ng/ml); however, in the bacterial infection group, both parameters rose after a lag phase of about 1 hr. A 4 hr infusion of the highly specific thrombin inhibitor recombinant (rec.) hirudin (0.125 mg/kg x hr) resulted in a decrease of FM levels from 89.2 +/- 14.4 micrograms/ml in the LPS group (n = 10) to 27.4 +/- 11.2 micrograms/ml in the rec. hirudin group (n = 10; P < 0.001). The respective values for TAT levels were 73.1 +/- 19.7 micrograms/ml in the LPS group and 52.7 +/- 15.7 ng/ml in the rec. hirudin group (P < 0.001). Other coagulation parameters, such as platelets, fibrinogen, and fibrin(ogen) degradation products, were ameliorated accordingly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Formation of fibrin monomers in experimental disseminated intravascular coagulation and its inhibition by recombinant hirudin. 805 64

Cemented total hip replacement surgery is associated with intraoperative cardiorespiratory depression and postoperative proximal deep vein thrombosis which may be linked to an extreme intraoperative thrombin generation and local and systemic effects of monomethylmethacrylate (MMA) released into circulation from curing cement. This in vitro study demonstrates that MMA alone or in combination with thrombin have effects on monocytes and human umbilical vein endothelial cells (HUVEC) which modulate their procoagulant activities. Moderate doses of MMA had a slight tissue factor (TF) inducing effect on monocytes. Small doses of MMA (0.1-1 mg/ml) [corrected] markedly potentiated the TF inducing effect of thrombin or lipopolysaccharide (LPS) which was included as a reference stimulant. These TF modulating effects of MMA were not seen with HUVEC. However, the generation of fibrinopeptide A (FPA) in cell overlay plasma indicated enhanced procoagulant activity of HUVEC treated with moderate doses of MMA, probably reflecting MMA cytotoxicity leading to cell retraction and exposure of extracellular matrix. Furthermore, small doses of MMA had a slight enhancing effect on FPA generation when coincubated with thrombin. These findings indicate that MMA in concentrations found in central venous blood in vivo, alone or together with thrombin, directly or indirectly exert effects that contribute to activation of coagulation.
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PMID:Effect of monomethylmethacrylate on procoagulant activities of human monocytes and umbilical vein endothelial cells in vitro. 808 39

We investigated the effect of blood mononuclear cell-conditioned medium on prostacyclin (PGI2) production by human umbilical vein endothelial cells in culture (HUVEC), and compared the potency of the conditioned medium in PGI2 production with that of various cytokines and lipopolysaccharide (LPS). HUVEC which had been preincubated with LPS, interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor (TNF alpha), or interferon-gamma (IFN-gamma) produced more PGI2 than control cells in response to thrombin. However, the HUVEC preincubated with the conditioned medium made with mononuclear cells with or without LPS (LPS-Mo-CM, Mo-CM) produced more PGI2 than those preincubated with LPS, IL-1 alpha, IL-1 beta, TNF alpha, or IFN-gamma. Although the concentrations or IL-1 beta and TNF alpha in the post-culture medium of HUVEC treated with LPS-Mo-CM were much higher than those with Mo-CM, LPS-Mo-CM which was made with 13,000/ml of mononuclear cells and 1 microgram/ml of LPS did not significantly augment the subsequent PGI2 production by HUVEC as compared with Mo-CM made with the same numbers of mononuclear cells. PGI2 production by Mo-CM-treated HUVEC still exceeded that of control cells, even when an excess amount of antibody to TNF alpha and/or IL-1 alpha was added to the Mo-CM. It is possible that Mo-CM contains unknown cytokines besides IL-1 and TNF which stimulate the HUVEC to produce PGI2.
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PMID:Mononuclear cell-conditioned medium enhances thrombin-stimulated PGI2 production by human umbilical vein endothelial cells in culture. 824 72

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

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